Hypoxic exercise training promotes anti-tumor cytotoxicity of natural killer cells in men

Clinical Science (2011) Immediate Publication, doi:10.1042/CS20110032 Hypoxic exercise training promotes anti-tumor cytotoxicity of natural killer cells in menJong-Shyan Wang and Tzu-Pin WengGraduate Institute of Rehabilitation Science, Chang Gung University, Tao-Yuan 333, Taiwan. s5492@mail.cgu.edu.twThe cytotoxic functions of natural killer cells (NKs) are critical in enabling the immune system to cope efficiently with malignancy. This investigation compared how various exercise regimens with/without hypoxia influence phenotypic characteristics of NK subsets and cytotoxicity of NKs to nasopharyngeal carcinoma cells (NPCs). Sixty sedentary males were randomly divided into five groups. Each group (n=12) underwent one of five interventions: normoxic (21%O2) resting, hypoxic (15%O2) resting, normoxic exercise (50% maximal work-rate under 21%O2, N-E), hypoxic-relative exercise (50% maximal heart rate reserve under 15%O2, H-RE), or hypoxic-absolute exercise (50% maximal work-rate under 15%O2, H-AE) for 30 minutes/day, five days/week for four weeks. The results showed that hypoxic exercise regimens increased pulmonary ventilation and tissue oxygen utilization. Moreover, H-RE enhanced aerobic fitness at a less intensive training workload than H-AE. Before each intervention, strenuous exercise elevated NK perforin/granzyme B contents and promoted cytotoxicity of NKs to NPCs. However, the percentages of NKs-expressing homing (CD11a)/terminally differentiated (CD57)/inhibitory (KLRG1) molecules that entered the bloodstream from peripheral tissues increased following this exercise. After four weeks of interventions, both H-AE and H-RE up-regulated the memory (CD45RO)/activating (NKG2D) expressions and were accompanied by the decreases in the CD57/KLRG1 levels on NKs at rest and after strenuous exercise. Furthermore, the two regimens increased resting and exercise NK perforin/granzyme B contents and NK-induced phosphatidylserine exposure of NPCs. In contrast, no significant change in phenotypic characteristics of blood NK subsets or NK-induced NPC apoptosis was observed following the N-C, H-C, and N-E. Therefore, we conclude that 15%O2 exercise training reduces terminally differentiated NK subsets and up-regulates the expressions of activating molecules and cytotoxic granule proteins of NKs, thereby enhancing capacity of anti-NPC cytotoxicity by NKs. These findings can help to determine effective hypoxic exercise regimens for improving individual aerobic capacity and simultaneously promoting the natural cytotoxicity of NKs.

View the original article here

Exhaustion of tumor-specific CD8+ T cells in metastases from melanoma patients

J Clin Invest. doi:10.1172/JCI46102.
Copyright © 2011, The American Society for Clinical Investigation. Lukas Baitsch1, Petra Baumgaertner1, Estelle Devêvre1, Sunil K. Raghav2, Amandine Legat1, Leticia Barba1, Sébastien Wieckowski3, Hanifa Bouzourene3, Bart Deplancke2, Pedro Romero4, Nathalie Rufer1,3 and Daniel E. Speiser1

1Clinical Tumor Immune-Biology Unit, Ludwig Institute for Cancer Research, Lausanne, Switzerland.
2Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
3University Hospital Center and University of Lausanne, Lausanne, Switzerland.
4Translational Tumor Immunology Group, Ludwig Institute for Cancer Research, Lausanne, Switzerland.

Address correspondence to: Daniel Speiser, Ludwig Institute for Cancer Research, Hôpital Orthopédique, 05/1552, Av. P.-Decker 4, CH-1011 Lausanne, Switzerland. Phone: 41.21.314.0182; Fax: 41.21.314.7477; E-mail: d.e.speiser@gmail.com.

Published May 9, 2011
Received for publication December 14, 2010, and accepted in revised form March 16, 2011.

In chronic viral infections, CD8+ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8+ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8+ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1–specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

CD8+ T cell responses in acute viral diseases have been extensively characterized in mice and humans (1–6). While viruses multiply rapidly during the first week of infection, CD8+ T cells become activated and expand vigorously, reaching a peak of T cell effector function. In parallel with the consequent decline of viral antigen, the majority of CD8+ T cells undergo apoptosis (contraction phase). After pathogen clearance, memory T cells persist for years at low frequencies, ready for accelerated protective immune responses in case of reinfection.

When pathogens are not eliminated, T cells may persist in much larger numbers. They are composed of large numbers of effector cells and low percentages of memory cells. Essentially, there are 2 scenarios of long-term CD8+ T cell activity in viral infection: the first scenario is observed, e.g., in persistent herpesvirus infection (e.g., EBV, CMV) in healthy individuals, where T cells successfully contain the viruses and thus are protective even though they do not eliminate the viruses entirely. The second scenario is associated with viral spread and progressive tissue damage in the presence of large numbers of CD8+ T cells, e.g., in HIV-1, HBV, or HCV infection, and in the murine model of lymphocytic choriomeningitis virus clone 13 (LCMV clone 13) infection. These 2 scenarios are distinguished by a fundamentally different functional competence of CD8+ T cells. In the first scenario, such as in healthy donors infected with EBV or CMV, viral antigen-specific T cells are functionally competent and thus ready for immediate cytokine production and cytotoxicity (7). These cells contribute to rapid reduction of viral load and restoration of health by viral containment to small anatomical compartments (8–10). In contrast, CD8+ T cells in the second scenario (i.e., the failure of viral containment) are functionally impaired (11, 12). The murine infection with LCMV clone 13 is a prototype model of functional T cell impairment, called T cell exhaustion, with progressively reduced production of IL-2, TNF-a, and IFN-?, followed by incapacity to lyse (infected) target cells (13, 14). Analysis of such cells has led to significant discoveries, such as the identification of PD-1, a major inhibitory receptor involved in T cell function (15). Gene expression profiling of murine T cells allowed a global assessment, revealing that T cell exhaustion is associated with numerous molecular alterations, affecting genes regulating chemotaxis, adhesion, coreceptors, migration, metabolism, and energy (2). Hereafter, we call these multiple changes exhaustion profile.

In humans, functional deficits were found in HIV-1–, HCV-, and HBV-specific CD8+ T cells (11, 16, 17), and a recent gene expression study described an exhaustion profile in HIV-1 patients (18). In contrast to virus-specific T cells, only little is known of self- and tumor-specific CD8+ T cells. In humans and mice, it remains to be determined whether functional impairments of bona fide self-antigen–specific T cells represent exhaustion, anergy, or other functional states. In melanoma patients, there are substantial numbers of long-term persisting effector-memory CD8+ T cells, despite failures of immune protection from disease. Circulating human tumor-specific CD8+ T cells may be cytotoxic and produce cytokines in vivo (19–21), indicating that self- and tumor-specific human CD8+ T cells can reach functional competence after potent immunotherapy such as vaccination with peptide, incomplete Freund’s adjuvant (IFA), and CpG (19) or after adoptive transfer (22). In contrast to peripheral blood, T cells from metastasis are functionally deficient, with abnormally low cytokine production and upregulation of the inhibitory receptors PD-1, CTLA-4, and TIM-3 (20, 23–25). Functional deficiency is reversible, since T cells isolated from melanoma tissue can restore IFN-? production after short-term in vitro culture (20). However, it remains to be determined whether this functional impairment involves further molecular pathways, possibly resembling T cell exhaustion or anergy as defined in animal models (2, 26).

The identification of mechanisms responsible for functional impairment of self- and tumor-specific T cells may reveal targets for novel cancer therapies. Human CD8+ T cell responses specific for the melanoma antigen Melan-A/MART-1 represent a model wherein self-specific T cells can be studied in great detail. Furthermore, we took advantage of the strong immunogenicity of vaccination with peptide plus CpG (19). By direct ex vivo analysis, we compared Melan-A/MART-1–specific T cells (hereafter called tumor-specific T cells) with virus-specific T cells by microarray analysis, quantitative PCR (qPCR), and flow cytometry. Recent studies focused on circulating T cells (27), whereas T cells residing in tumor tissue remain poorly characterized. Therefore, we isolated T cells from both peripheral blood and metastases. We found that the former show molecular and functional features of effector cells, similar to circulating CMV-specific T cells, demonstrating that human self- and tumor-specific T cells have the potential to become competent effector cells. In marked contrast, the tumor-specific T cells isolated from metastatic tissue displayed an exhaustion profile, consisting of large numbers of molecular alterations.

Naive and virus-specific T cells show no significant differences between melanoma patients and healthy donors. Recently we demonstrated reproducibility of gene expression profiling of small numbers of (1,000) T cells (28). Applying this technique (Supplemental Figure 1, A–D; supplemental material available online with this article; doi: 10.1172/JCI46102DS1), we analyzed naive and antigen-specific T cells upon sorting of PBMC subsets by flow cytometry. We compared gene expression profiles of naive CD8+ T cells from melanoma patients and healthy donors and found no significant differences (Figure 1A), confirming previous studies (29). For the isolation of antigen-specific cells, we used tetramers and sorted T cells specific for the tumor antigen Melan-A/MART-1, the EBV antigen BMLF1, and the CMV antigen pp65. We compared EBV-specific T cells between healthy donors and patients and did not observe significant differences in gene expression (Figure 1B). In parallel, we found similar phenotypes and similar IFN-? production (Figure 1, C and D). Thus, many CD8+ T cells appeared relatively normal in our patients.

Figure 1 Naive and virus-specific T cells show no significant differences between melanoma patients and healthy donors. (A and B) Volcano plots for all gene probes on the microarray, showing expression differences and P values of naive T cells (healthy donors [HDs] versus patients; A) or EBV-specific T cells (healthy donors versus patients; B). Each point represents 1 gene probe. (C) Lymphocytes were stained with an A2/EBV BMLF1280–288 tetramer together with anti-CD8, anti-CD45RA, and anti-CCR7. The inset shows a dot plot distinguishing the phenotypes among total CD8+ T cells analyzed as controls: naive (N) (CD45RA+CCR7+), central memory (CM) (CD45RA–CCR7+), effector memory (EM) (CD45RA–CCR7–) and effector memory RA+ cells (EMRA) (CD45RA+CCR7–). Bar graph depicts the percentage (mean ± SD) of each phenotype of total CD8+ cells or total EBV tetramer-positive populations from healthy donors or patients. (D) IFN-? production by EBV-specific T cells upon 4-hour stimulation. Whiskers in box plots indicate maximum and minimum values measured. Cross indicates the mean, while line indicates the median.

Gene expression profiling of naive versus nonnaive T cells. Before analyzing tumor-specific T cells, we validated our approach using only 1,000 cells, by searching for the known molecular differences between naive and nonnaive CD8+ T cells (28, 30). We selected genes showing a 3-fold or greater change between naive and nonnaive CD8+ T cells, plus a P value adjusted for the false discovery rate (FDR) of less than 0.05 (Supplemental Figure 2A). With this strategy, we identified 409 upregulated and 364 downregulated genes in naive relative to nonnaive CD8+ T cells (Supplemental Table 1) and found that all naive T cell populations clustered together and apart from all nonnaive T cells (Figure 2A). We selected 8 genes for verification by qPCR. Without exception, they confirmed the microarray results, whereby qPCR detected quantitatively larger differences, owing to the higher sensitivity of qPCR (Supplemental Figure 2B). Additionally, the data for many of the differentially expressed genes (e.g., CCR7, LEF1, SELL, IFNG, GZMB, and HLADR; Supplemental Figure 2C) confirmed well-known differences between naive and nonnaive T cells.

Figure 2 Gene expression of naive and effector T cells from peripheral blood. (A) Two-way hierarchical clustering based on the identified 773 genes, separating all naive from nonnaive T cells. Red indicates overexpression and blue underexpression relative to the mean. Each row represents 1 gene and each column 1 1,000-cell sample from 1 patient or healthy donor. (B) Relative overexpression of GO terms associated with the identified genes, calculated as described in Methods. (C and D) GSEA of publicly available gene sets describing naive and effector T cells. Positions of selected example genes are indicated. Gene sets comprise genes enriched in naive T cells (C) or in effector cells (D). Genes to the left and right of the rank-ordered list are enriched in naive T cells and nonnaive T cells, respectively.

We assessed biological classification of the 773 differentially expressed genes by assigning them to 9 Gene Ontology (GO) terms and then determined whether any of these GO terms were overrepresented in our list compared with the predicted frequency in a random gene list. Not surprisingly, we found about twice as many immune response genes as the number predicted from a random gene test (Figure 2B). Additionally, the GO terms for translation, cell death, and apoptosis were overrepresented in nonnaive cells, whereas genes involved in DNA repair were underrepresented.

In 2005, Willinger et al. made a thorough gene expression analysis of human CD8+ T cells from healthy donors without distinction of antigen specificity (31). They determined large differences between naive and total effector cells, providing gene sets characteristic for the distinction of the 2 populations. From these data, we used 2 gene sets, one which is up- and one which is downregulated in effector CD8+ T cells. Furthermore, in 2007, Wherry et al. defined gene sets that were up- or downregulated in antigen-specific memory, effector, and exhausted CD8+ T cells from LCMV-infected mice (2). While the gene sets from Willinger et al. described long-term effects of effector differentiation (analysis of total human CD8+ T cell subsets in steady state), the gene sets from Wherry et al. described shorter-term changes of gene expression (model of acute and chronic infection). With Gene Set Enrichment Analysis (GSEA), we determined whether gene sets were enriched in our rank-ordered list of differentially expressed genes. Our naive T cells showed upregulation of the 2 gene sets downregulated in effector cells as identified by Wherry et al. (ref. 2 and Figure 2C) and by Willinger et al. (ref. 31 and Figure 2C). Conversely, the gene sets enriched in our nonnaive T cells were those upregulated in effector cells as defined by Wherry et al. (Figure 2D) and by Willinger et al. (Figure 2D). Together, these data confirm the reproducibility of microarray analysis of highly purified cells, validating our approach of ex vivo analysis of antigen-specific T cells with small cell numbers.

Different gene expression profiles between circulating tumor- and virus-specific T cells. A major aim of our study was to determine whether tumor-specific CD8+ T cells were similar to or different from virus-specific T cells. By applying the same selection criteria as above (i.e., fold change = 3, adjusted P < 0.05), we found 390 genes that were differentially expressed between tumor- and EBV-specific T cells (259 upregulated and 131 downregulated) (Figure 3A and Supplemental Table 2), while only 184 genes (72 upregulated and 112 downregulated) were differentially expressed when compared with CMV-specific T cells (Figure 3B and Supplemental Table 3). Therefore, the differences between CMV- and tumor-specific T cells were smaller than between EBV- and tumor-specific T cells. A 2-way hierarchical clustering with these probes showed clear distinction between tumor- and EBV-specific (Figure 3C) and tumor- and CMV-specific T cell populations (Figure 3D) from the individual patients and healthy donors despite the high genetic heterogeneity between individuals and the similarity of surface markers of these T cell populations (Supplemental Figure 3A). Microarray data were confirmed through the analysis of a series of genes by qPCR, among them several inhibitory receptors (Figure 3, E and G). As compared with both EBV- and CMV-specific cells, TIM3 and CTLA4 were upregulated in tumor-specific T cells, while CD160 was upregulated in virus-specific T cells (Figure 3, E and G). 2B4 was upregulated in CMV-specific T cells. Interestingly, as compared with EBV-specific T cells, tumor-specific T cells expressed more mRNA encoding granzyme B (GZMB) and granulysin (GNLY), but less XCL1 (lymphotactin). XCL1 was also upregulated in CMV-specific T cells. Finally, we performed a GO term analysis and found that the differences between tumor- and the 2 virus-specific T cell populations were smaller (Figure 3, F and H) than the differences of naive versus nonnaive T cells (Figure 2B). Remarkably, immune response genes were not specifically overrepresented relative to a random gene list, suggesting overall similar expression of immune genes in effector T cells specific for EBV, CMV, and Melan-A/MART-1, despite the differences found for inhibitory receptors.

Figure 3 Gene expression of circulating CD8+ T cells depending on antigen specificity. (A and B) Volcano plots for all gene probes, showing differential expression and P values of the comparison of tumor- versus EBV-specific T cells (A) or tumor- versus CMV-specific T cells (B); diagramming is similar to that in Figure 1. (C and D) Two-way hierarchical clustering based on the identified gene probes separating all tumor-specific T cells from EBV- (C, 405 gene probes corresponding to 390 genes) and from CMV-specific T cells (D, 187 gene probes corresponding to 184 genes). Red indicates overexpression and blue underexpression relative to the mean. Each row represents 1 gene and each column 1 1,000-cell sample from 1 patient (tumor-, EBV- and CMV-specific cells) or healthy donor (EBV-specific cells, n = 4). (E and G) Log fold changes between tumor- and EBV- (E) or tumor- and CMV-specific T cells (G) of data from microarrays (blue bars) and qPCR (red bars). Positive and negative values indicate overexpression in tumor- and in virus-specific T cells, respectively. Data are represented as mean ± SEM. (F and H) Relative overexpression of GO terms associated with the identified 390 genes (Melan-A/MART-1 versus EBV; F) or with the identified 184 genes (Melan-A/MART-1 versus CMV; H), calculated as described in Methods.

The gene expression profile of circulating tumor-specific CD8+ T cells corresponds to late-differentiated effector cells. EBV- and CMV-specific T cells are recognized as prototypes of early- and late-differentiated effector cells, respectively (7). This distinction fits with the phenotypes of these 2 populations (Supplemental Figure 3A). We created rank-ordered gene lists to compare tumor-specific with the 2 virus-specific CD8+ T cell populations. The gene sets defined as upregulated in effector cells by Wherry et al. (ref. 2 and Figure 4A) and Willinger et al. (ref. 31 and Figure 4A) were enriched in tumor-specific cells, as compared with their EBV-specific counterparts. In contrast, the only gene set enriched in EBV-specific T cells compared with tumor-specific T cells was the small gene set containing genes specifically upregulated in memory cells when compared with naive CD8+ T cells as defined by Wherry et al. (Figure 4B). This is likely due to the lower degree of effector differentiation of EBV-specific T cells (which are nevertheless predominantly effector rather than memory cells; Supplemental Figure 3A). When we compared tumor- with CMV-specific CD8+ T cells, we could not find enrichment for any gene set (Figure 4C), confirming the late differentiation stage of tumor-specific T cells. To verify the differential expression of granzyme B and perforin ex vivo on the protein level, we performed intracellular staining. As expected, the tumor- and CMV-specific CD8+ T cells expressed more granzyme B and perforin than the EBV-specific CD8+ T cells (Figure 4D). However, all 3 antigen-experienced T cells produced high levels of IFN-? after 4 hours triggering with peptide-loaded T2 cells (Supplemental Figure 3B). Together, our results demonstrate that tumor- and CMV-specific CD8+ T cells resembled each other closely, while EBV-specific CD8+ T cells were in earlier stages of effector differentiation.

Figure 4 Circulating tumor-specific T cells are late-differentiated effector cells, resembling CMV-specific T cells. (A) Gene set enrichment of genes describing effector cells (see Figure 2D). Genes to the left and right of the rank-ordered list are enriched in tumor- and EBV-specific T cells, respectively. (B) Gene set enrichment of genes describing memory cells (2). Genes to the left and right of the rank-ordered list are enriched in tumor- and EBV-specific T cells, respectively. (C) No differences were found between Melan-A/MART-1– and CMV-specific T cells, demonstrated by a gene set defining effector cell–related genes (31). (D) Intracellular staining of naive and antigen-specific T cells. Top panels show 1 representative example; below are the combined results of all samples (EBV and CMV, n = 5; Melan-A, n = 15; naive, n = 25). Data of EBV- and CMV-specific T cells are from healthy donors, while data of tumor-specific T cells are from patients. ***P < 0.001. Whiskers in box plots indicate maximum and minimum values measured. Line indicates the median.

In contrast to circulating T cells, tumor-specific T cells from tumor-infiltrated lymph nodes show an exhaustion profile. Previous studies indicated that functional impairment of tumor-specific T cells may occur primarily in situ (20, 24), which was also the case after strong systemic T cell activation by CpG-based vaccination (25). Therefore, we established a clinical investigation protocol to recover large numbers of live cells from tumor-infiltrated lymph nodes (TILN). This enabled us to perform functional studies and gene expression analysis ex vivo from tumor-specific T cells from TILN, in comparison with circulating T cells. Tumor-specific T cells from metastases showed highly insufficient IFN-? production upon 4-hour peptide triggering (Figure 5A), as published previously (20, 24). Microarray analysis allowed the identification of 332 genes (201 up- and 131 downregulated in TILN; Supplemental Table 4) that were differentially expressed between tumor-specific CD8+ T cells from PBMC versus TILN, using the same criteria as before (Figure 5B). Hierarchical clustering using these genes divided the 13 samples into 2 groups only, one for blood and the other for TILN-derived tumor-specific T cells (Figure 5C). qPCR performed for a selection of genes allowed proper validation (Figure 5D). Among the genes upregulated in tumor-specific cells from TILN were the lymph node retention receptor CRTAM, the chemokines XCL1 and XCL2, the activation marker TNFRSF9, and the inhibitory receptor CTLA4. CXCL13, a B cell chemoattractant usually found in the B cell compartment of lymph nodes, was one of the most highly overexpressed genes. Among the genes downregulated in TILN cells were the cell-growth–regulating protein LYAR and the inhibitory receptor KLRG1. When classifying the differentially expressed genes into broad GO terms, we found that genes involved in cell death and apoptosis and in the immune response were overrepresented compared with a randomly selected gene list (Figure 5E). To obtain a more general overview of the differences of tumor-specific CD8+ T cells from blood versus TILN, we studied gene sets specific for effector cells, naive cells, memory cells, and exhausted cells, as described above. Remarkably, the gene set described for exhausted T cells (2) was significantly enriched in tumor-specific cells from TILN, in contrast with the gene sets characteristic for naive, memory, and effector T cells (Figure 5F). These large-scale data demonstrate an impressive exhaustion profile, with extended molecular alterations of multiple pathways in tumor-specific CD8+ T cells from metastases.

Figure 5 Exhaustion profile of tumor-specific T cells in situ. (A) IFN-? production by tumor-specific T cells from the circulation (blood; n = 6) or TILN (n = 8) after 4-hour antigen stimulation. Whiskers in box plots indicate maximum and minimum values measured. Cross indicates the mean, while line indicates the median. **P < 0.01. (B) Differential gene expression by tumor-specific T cells isolated from blood versus TILN, as illustrated by a volcano plot for all gene probes. (C) Two-way hierarchical clustering based on the identified 346 genes separating all blood-derived tumor-specific T cells from their TILN counterparts. Red indicates overexpression and blue underexpression relative to the mean. Each row represents 1 gene and each column 1 1,000-cell sample from 1 patient. (D) Log fold change between tumor-specific T cells from blood versus TILN; data from microarrays (blue bars) and qPCR (red bars). Positive and negative values indicate overexpression in tumor-specific T cells from TILN and from blood, respectively. Mean ± SEM. (E) Relative overexpression of GO terms associated with the identified genes, calculated as described in Methods. (F) Enrichment of the gene set described for exhausted T cells (2) in TILN-derived tumor-specific T cells, relative to their blood-derived counterparts. The positions of inhibitory receptors found in this gene set on the rank-ordered gene list are indicated. A position to the left indicates enrichment in TILN-derived cells, a position to the right enrichment in blood-derived cells.

Enhanced expression of inhibitory receptors, such as CTLA4 and LAG3, was observed in T cell exhaustion (2, 23, 32–34). Interestingly, their expression was enriched in TILN cells, with the notable exceptions of PTGER2 and KLRG1 (Figure 5F). However, KLRG1 was described as more strongly expressed in functionally competent effector cells than in exhausted T cells (2), compatible with our data. The absolute expression values of selected inhibitory receptors are detailed in Table 1. Although it seems likely that the tumor microenvironment plays a role, the reasons for the observed enhanced expression of inhibitory receptors remain to be elucidated.

Table 1 Expression of selected inhibitory receptors by tumor-specific T cells

Differential protein expression of multiple inhibitory receptors by tumor- and virus-specific CD8+ T cells. To determine expression of inhibitory receptors at the protein level, we produced tetramers labeled with (multiple) different fluorochromes and used them in combination with several monoclonal antibodies (multi-tetramer staining; Figure 6A). Compatible with mRNA data, CD160 and 2B4 were more frequently expressed by both EBV- and CMV-specific T cells than by tumor-specific T cells from peripheral blood (Figure 6B), in agreement with a study reporting that most CD160+ cells coexpressed 2B4 (35). In contrast, circulating tumor-specific T cells expressed more TIM-3 and more PD-1 than the 2 virus-specific T cell populations (Figure 6B), in line with 2 recent reports of TIM-3+PD-1+ cells among tumor-specific T cells (23, 36). Large percentages of PD-1+ tumor-specific T cells coexpressed TIM-3 and/or KLRG-1. Similar results were obtained when we analyzed the mean fluorescence intensity (Supplemental Figure 4). Our technique allowed analyzing simultaneous coexpression of multiple inhibitory receptors, for CD160, KLRG-1, PD-1, and TIM-3, or for 2B4, LAG-3, and CTLA-4. We found a pronounced increase in inhibitory receptor coexpression from naive to central memory, effector memory, and effector memory RA+ cells (data not shown). On antigen-specific T cells, there were various combinations of inhibitory receptors. Melan-A–specific T cells from TILN expressed more CTLA-4, LAG-3, and TIM-3, but less KRLG-1 than their counterparts from peripheral blood (Figure 6C), confirming the results obtained by the microarray analysis. These data reveal a high level of heterogeneity, with multiple antigen-specific T cell subpopulations expressing different combinations of inhibitory receptors. It is likely that many of these subpopulations are effector memory cells and effector memory RA+, as they make up the vast majority of Melan-A–specific T cells (Supplemental Figure 3A). Naive and central memory cells were infrequent, but may nevertheless contribute to this heterogeneity. Furthermore, extended studies are necessary to determine the functional impact of coexpressed inhibitory receptors. Finally, the marked differences between tumor-, CMV-, and EBV-specific T cells suggest different roles of inhibitory receptors in viral infection versus cancer.

Figure 6 Multi-tetramer staining assessing coexpression of inhibitory receptors. (A) Staining with tetramers binding to EBV- (PE–Texas Red), Melan-A/MART-1– (APC–eFluor 780), or CMV- (PE–Texas Red and APC–eFluor 780) specific T cells (labeling tetramers with 2 instead of 1 fluorochrome identifies larger numbers of epitope-specific T cell populations than the number of fluorescence channels used). T cells were analyzed for coexpression of 7 inhibitory receptors: KLRG-1 (Alexa Fluor 488), TIM-3 (PE), PD-1 (PerCP-eFluor710), and CD160 (Alexa Fluor 647), or LAG-3 (FITC), 2B4 (PE-Cy5.5), and CTLA-4 (APC). (B) Expression of 7 different inhibitory receptors. Histograms of a representative sample are gated on CD8+ tetramer+ cells. Box plots summarize the data of all patients analyzed (EBV, n = 16; CMV, n = 6; Melan-A blood, n = 10, except for CTLA-4, n = 3; Melan-A TILN, n = 8–9). Whiskers in box plots indicate the maximum and minimum values measured. Cross indicates the mean, while line indicates the median. *P < 0.05; #P < 0.01; §P < 0.001. (C) Coexpression of 0 to 4 and 0 to 3 inhibitory receptors was analyzed with SPICE (48).

In peripheral blood, tumor-specific T cells induced by vaccination showed an effector cell profile (Supplemental Figure 5), similar to CMV-specific T cells and similar to the murine counterpart of CD8+ T cells in acute LCMV Armstrong infection (2). Differentiation of EBV-specific CD8+ T cells was less pronounced, but they nevertheless resembled effector cells. In contrast to these 4 effector cell populations, tumor-specific T cells in situ displayed an exhaustion profile, with significant similarity to murine T cells in chronic infection with LCMV clone 13 (2).

Tumor-infiltrating T cells are functionally deficient (20, 23, 24, 34), which is likely coresponsible for the limited efficacy of immunotherapy. However, the underlying mechanisms remain poorly characterized, in contrast with chronic infectious diseases (1, 2). Our finding of T cell exhaustion in melanoma metastases results from what we believe is the first comprehensive molecular characterization of self- and tumor-specific T cells, providing explanations for their functional impairment. Tumor-specific T cells from metastases showed considerable molecular alterations, with surprisingly strong overexpression of many genes regulating various cell functions. This included genes involved in immune responses, cell death and apoptosis, and cell cycle and DNA repair. Thus, the data point to enhanced immune activation and apoptosis, and problems in maintaining DNA integrity and sustaining cell cycling in T cells of metastases.

We did not find significant correlations between our T cell data and clinical results (e.g., patient survival). However, phase I studies such as the present trial of immunotherapy are not suited for clinical outcome analysis. Rather, they are designed for providing enhanced biological insight. Indeed, we identified specific molecular alterations potentially representing molecular targets for improved therapy. Nevertheless, further studies are required to determine which of these targets are most promising for evaluation in large-scale phase III clinical trials.

Based on the available evidence for functional T cell impairment in HIV-1, HBV, and HCV infections (11, 16–18, 37), it will be useful to perform comparative molecular profiling of T cells in different infections and malignancies in order to identify similarities and differences, providing the rational basis for therapy optimization. Very recently, HIV-1–specific T cells have been profiled, with identification of T cell exhaustion and BATF upregulation by PD-1 in patients failing to control HIV infection (18). Even though we did not find enhanced BATF expression in tumor-specific T cells from TILN, we observed similarities in gene expression signatures and upregulation of multiple inhibitory receptors on tumor-specific T cells also at the protein level.

Besides analysis of tumor-specific T cells after vaccination, it would be interesting to profile spontaneously arising T cell responses and naive tumor-specific T cells from patients and healthy donors, with the aim of identifying disease mechanisms responsible for altered T cell function. We expect that tumor-specific T cells from healthy donors would show an expression profile similar to total naive CD8+ T cells, while spontaneously responding T cells may show some degree of effector cell differentiation. However, such studies are technically challenging, since tumor-specific T cells in healthy donors and early stages of cancer are rare and difficult to isolate for ex vivo analysis. Therefore, laboratory techniques must be optimized for comprehensive characterization of even smaller cell numbers, ultimately down to the single cell level.

T cell tolerance to self and tumor antigens is assured by negative selection in the thymus and through anergy induction and T cell deletion in the periphery. Anergy has been characterized in at least 9 different experimental settings, most of them in vitro models and/or CD4+ T cell models (26). Unfortunately, no comprehensive gene expression data are available. Therefore we could not systematically evaluate anergy in our study. Nevertheless, we made an attempt by evaluating 29 anergy-related genes described in a model of ionomycin-induced anergy and a model of deletional tolerance (38, 39). We found that some of these genes (e.g., CBLB and CTLA4) were enriched in tumor-specific T cells from metastases (Supplemental Table 4), but most of the described genes (e.g., ITCH, EGR2, and DGKZ) were not enriched (not shown).

Our study was performed in patients with advanced stage III–IV melanoma. It has been hypothesized that late cancer stages may be associated with T cell exhaustion (1), whereas anergy and tolerance would be induced already at early stages of tumorigenesis (40, 41). Possibly, self- and tumor-specific T cells may show discrete alterations already at the naive stage and/or after spontaneous activation. Perhaps anergy mechanisms are functional even at later disease stages. The elucidation of these points requires further methodological progress. For the time being, our data support the conclusion that exhaustion likely contributes to the functional deficiencies, but does not rule out the involvement of further mechanisms such as anergy or self tolerance.

In circulating tumor-specific T cells, we found effector cell signatures compatible with their ample production of granzyme B and perforin (Figure 4) and efficient expression of IFN-? upon 4-hour triggering with antigen (Supplemental Figure 3B and refs. 20, 21). Due to the high efficacy of CpG 7909 as adjuvant, the circulating tumor-specific T cells studied here were more strongly activated (19) than in most other cancer vaccine studies with their lower frequencies and less pronounced effector cell differentiation. Thus, our data of circulating cells are not representative for the latter, but nevertheless demonstrate that self- and tumor-specific T cells have the potential to become effector cells. Despite the high efficacy of the adjuvant used, we could not observe significant bystander effects on circulating T cells with specificities other than for the vaccine (Figure 1).

One could argue that vaccination should have activated the tumor-specific T cells to an even higher degree than CMV-specific T cells in healthy donors (which was actually the case in some of our melanoma patients; our unpublished observations). Protection from latent CMV is likely less demanding for T cells than protection from acute viral disease. Possibly, even more strongly activated T cells may be required for protection from cancer progression. Indeed, adoptive transfer therapy has shown that tumor-specific T cells at much higher frequency and strong activation can eliminate large melanoma metastases (22). Molecular profiling of these cells in comparison with T cells during acute viral infections may reveal eventual differences from our data. Alternatively, therapeutic success and protection from disease may be primarily achieved due to high numbers of T cells with molecular properties similar to those described here. However, patients with acute viral infections are rarely accessible for clinicians and researchers. Moreover, antitumor vaccines rarely induce T cell responses comparable to acute viral infections. In contrast to vaccines consisting of synthetic molecules and inactivated pathogens, live vaccines (essentially vaccinia virus and yellow fever vaccine) can induce high T cell frequencies (42, 43) and efficient protection. Comprehensive profiling of these T cells is feasible and may likely contribute to identifying protective mechanisms of human T cells.

Differentiation from naive to effector T cells introduces large changes in expression of not only immune response genes, but also of genes involved in translation, in cell death and apoptosis, and in cell migration. These changes result in increased production of effector molecules, migration to pathologic tissue, and cell survival. Based on GO terms, we compared EBV- with circulating tumor-specific T cells and found that the latter overexpressed genes involved in translation, cell death, and apoptosis, likely reflecting the fact that the tumor-specific T cells were more advanced in effector cell differentiation. Compared with CMV-specific T cells, circulating tumor-specific T cells expressed slightly more genes related to transcription, but fewer genes involved in cell migration. Despite these distinctions, the 3 effector cell populations from peripheral blood were relatively similar.

Inhibitory receptors were prominent among the differentially expressed genes. This group of genes is attracting increasing attention, also because of its importance in T cell exhaustion and therapeutic potential (2). Nevertheless, the circulating tumor-specific T cells expressed granzyme B and perforin at high levels and were functionally competent (19). Apparently, effector cells can express inhibitory receptors but nevertheless maintain functional competence. Our study demonstrates coexistence of functional cells in circulation and exhausted cells in metastases. We have preliminary data indicating that this may occur even within individual T cell clonotypes (our unpublished observations). It appears that migration of T cells into the tumor tissue is associated with downregulation of cytokine production and exhaustion as a consequence of encountering inhibitory receptor ligands expressed in the tumor tissue, in conjunction with antigen recognition. Thus, exhaustion of T cells in metastases but not in peripheral blood may be linked to the frequent and strong expression of these ligands in the tumor microenvironment. This interpretation is compatible with our earlier findings that the functional deficiency of tumor-residing T cells is readily reversible, since T cells from metastases regain function after 1 to 2 days culture in vitro (20, 44).

HCV-specific T cells may coexpress up to 4 of the inhibitory receptors KLRG1, 2B4, CD160, and PD-1, correlating with CD127 downregulation and functional impairment (32). Even though many tumor-specific T cells expressed KLRG1, 2B4, PD-1, and TIM-3, they did not express CD160. This difference may be functionally relevant for HCV- versus tumor-specific T cells. Moreover, expression of inhibitory receptors was more abundant in T cells from TILN as opposed to blood. The differential coexpression of multiple inhibitory receptors in viral infection versus cancer, and depending on antigen specificity/differentiation status and anatomical localization, suggests that the functional regulation of antigen-specific T cells is more complex than previously thought.

In summary, our study provides comprehensive molecular profiles of human CD8+ T cells. Although tumor-specific T cells can acquire substantial effector cell properties, they display an exhaustion profile in metastases. With modern technologies applied to small cell numbers, it becomes increasingly possible to determine whether functional impairment and molecular exhaustion of tumor-specific T cells are due to their specificity for self antigen, and/or immune suppression in situ.

Healthy donors, melanoma patients, lymphocyte isolation, and flow cytometry. Blood from 4 A2+ healthy donors was obtained from the university blood transfusion center of Lausanne, Switzerland. Peripheral blood and surgery specimens were obtained from A*0201+ patients with stage III/IV metastatic melanoma. Patients had received multiple monthly low-dose vaccinations s.c. with 100 µg Melan-A/MART-1 peptide and CpG (500 µg PF-3512676/7909; provided by Pfizer/Coley Pharmaceutical Group), emulsified in IFA (300–600 µl Montanide ISA-51; provided by Seppic) as described previously (19). Analysis of circulating tumor-specific T cells was done after 11 ± 5 monthly vaccinations; the last was at a mean of 96 days before blood withdrawal. Tumor-specific T cells from TILN were prepared after finely mincing surgery specimens, which were obtained after 7 ± 2 monthly vaccinations, the last at a mean of 79 days before surgery. Vaccinations were done in the context of Ludwig Institute for Cancer Research trials (19, 45) and approved by the Ludwig Institute for Cancer Research protocol review committee as well as by the medical and ethical committees of the University Hospital (Lausanne). Blood and tissue were obtained upon informed patient consent, and the study was performed according to the relevant regulatory standards. Mononuclear cells were purified by density gradient using Lymphoprep (Axis-Shield) and immediately cryopreserved in RPMI 1640 supplemented with 40% FCS and 10% DMSO.

For microarray analysis, 1,000 cells from each sample were sorted using a Vantage SE directly into lysis and storage buffer provided by Miltenyi Biotec as shown in Supplemental Figure 1. CD8+ T cells were enriched using magnetic bead sorting (Miltenyi Biotec). Cells were stained on ice and diluted at one million cells/ml. Cells were stained with CD8-specific antibody, the dead cell marker DAPI, and either with lineage markers (CD4, CD14, CD16, CD19) together with A2/EBV BMLF1280–288 (GLCTLVAML), A2/CMV pp65495–503 (NLVPMVATV), or A2/Melan-A/MART-126–35A27L (ELAGIGILTV) tetramers binding to high- and low-affinity T cell receptors (46) or with CD45RA-, CCR7-, CD28-, and CD27-specific antibodies. Naive T cells were defined as CD8+CD45RA+CCR7+CD27+CD28+. The sorting strategy is shown in Supplemental Figure 1. Manipulations were done at 4°C, avoiding gene expression alteration due to staining and sorting. Sorting purity was high, as determined by analyzing aliquots before and after FACS sorting. Representative examples are shown in Supplemental Figure 1, B–D. Among CD8+ T cells, percentages for A2/EBV tetramer+ cells were 1.00 ± 0.89 (4 healthy donors and 12 patients); for A2/CMV tetramer+ cells, 1.53 ± 1.08 (7 patients); and for A2/Melan-A/MART-1 tetramer+ cells, 1.43 ± 1.31 in blood (11 patients) and 3.35 ± 3.35 in TILN (7 patients). After sorting, lysed cells were incubated for 10 minutes at 45°C and then directly frozen at –80°C.

Intracellular antibody staining was performed as previously described (27). In brief, cells from the CD8+ fraction were first stained with PE-labeled tetramers, followed by anti–CD8–Pacific Blue antibody. After washing in PBS, cells were incubated with LIVE/DEAD-Fixable-Aqua (Invitrogen) for dead cell exclusion, and fixed at room temperature (RT) during 30 minutes (1% formaldehyde buffer). Cells were washed and stained with mAbs anti–perforin-FITC or anti–granzyme B–FITC (BD) in FACS buffer with 0.1% saponin for 30 minutes at 4°C. For the staining of IFN-?, CD8+ cells were stimulated with peptide-loaded T2 cells for 4 hours in the presence of Brefeldin-A (Sigma-Aldrich) prior to antibody staining with anti–IFN-?–PE-Cy7 (BD Pharmingen). Data of IFN-?–production from tumor-specific T cells were previously published (20).

For antibody staining of multiple inhibitory receptors, samples were purified and enriched as described above and then stained using tetramers detecting the same EBV, CMV, or Melan-A/MART-1 epitopes as described above. Melan-A–specific tetramers were labeled with APC–eFluor 780 (eBioscience), EBV-specific tetramers were labeled with PE–Texas Red (BD Pharmingen), and CMV-specific tetramers were labeled with both APC–eFluor 780 and PE–Texas Red, allowing for individual analysis of T cells specific for the 3 epitopes in a single sample (multi-tetramer staining technique; ref. 47). After 45 minutes at 4°C, cells were washed and surface staining was performed for CD8, CCR7, CD45RA and (a) LAG-3 (Alexis Biochemicals) and 2B4 (BioLegend) or (b) KLRG-1 (gift from H.-P. Pircher, Department of Immunology, University of Freiburg, Freiburg, Germany), TIM-3 (R&D Systems), PD-1 (eBioscience), and CD160 (eBioscience). Samples (a) were fixed at room temperature for 30 minutes (1% formaldehyde buffer) and then stained for CTLA-4 (BD Biosciences — Pharmingen) in FACS buffer with 0.1% saponin for 30 minutes at 4°C. LIVE/DEAD-Fixable-Aqua (Invitrogen) was used as a dead cell exclusion marker, and appropriate isotype controls were used to define negative populations. Data were acquired on a Gallios Flow Cytometer (Beckman Coulter) and analyzed using FlowJo 9.1 (TreeStar). Analysis of coexpression of inhibitory receptors used SPICE version 5.1 (48).

Microarray and qPCR. Gene expression profiling was done in 2 experiments. The first experiment included samples from blood-derived naive, EBV-, and tumor-specific T cells. The second experiment included tumor-specific T cells from blood and metastasis, and CMV-specific T cells from blood. Frozen samples were sent to Miltenyi Biotec and processed according to the vendor-recommended protocol for gene expression analysis. Samples were hybridized to Agilent Whole Human Genome Oligo Microarrays 4x44K and scanned using the Agilent microarray scanner system (Agilent). The Agilent Feature Extraction Software was used for readout and processing of image files. Background correction, filtering of data, and quantile normalization were done using the Agi4x44PreProcess software package as described in the package manual. The Limma software package was used to identify the differentially expressed genes and creation of rank-ordered lists. We analyzed eventual contaminations from B cells, monocytes, and dendritic cells, and found that expression levels of IGHG1, CD19, TLRs, and CD1 were between 0.28% and 2.72% of the respective expression of CD3E, confirming the high purity of our samples. We also evaluated intra-group variability possibly leading to high background. For this, we randomly split the data from 13 naive CD8+ T cell samples into 3 pairs of 2 groups of 6 and 7 samples each and analyzed differences between the groups. We found that none of the gene probes were different in any of the pairings, demonstrating that the background was low (data not shown). For nonnaive cells (Figure 2), the data from EBV- and tumor-specific CD8+ T cells were pooled. Genes were assigned to broad GO terms using the GO Term Mapper ( http://go.princeton.edu/cgi-bin/GOTermMapper), yielding both the percentage of submitted genes attributed to a given GO term versus the percentage of all annotated genes attributed to that GO term. Relative overrepresentation was calculated by dividing the percentage of submitted genes attributed to a GO term by the percentage of all available genes annotated with this GO term. Rank-ordered gene lists (ranked according to the B value) were analyzed with GSEA ( www.broadinstitute.org/gsea; ref. 49). Enrichment was considered significant if P was less than 0.05 and FDR was less than 0.25 as suggested in the online tool.

qPCR was performed to validate the enriched genes observed in microarray experiments. Custom-ordered oligos (Microsynth) were designed using the online tool from Universal Roche Library Assay Design Centre (Supplemental Table 5). Reaction mix used was Power Sybr Green Master Mix (Applied Biosystems), and amplification was monitored with Applied Biosystems 7900HT Fast Real-Time PCR System (15-minute enzyme activation and 40 cycles of 15 seconds 95°C, 1 minute 60°C). A Hamilton Liquid Handling Robotic System was used to assemble the 384-well plates. Amplified cDNA samples used for microarray analysis were diluted (1:50) and used for qPCR after confirming the linear and single product amplification by the primers. Samples were measured in triplicate. GAPDH was used as a housekeeping gene to calculate relative expression values.

Statistics. For quantitative comparisons, Student’s t test (2-sample 2-tailed comparison) or 1-way ANOVA with Tukey post-test (multiple-sample comparison) was performed with Prism 5.0; P < 0.05 was considered as significant. P values and FDRs for GSEA were calculated with 1,000 permutations in the online tool. Microarray analysis was done with relatively restrictive criteria, i.e., by considering gene probes as significant if the P value, corrected for a FDR of 0.05, was P = 0.05 and the fold change was = 3.

Accession numbers. The gene-expression data described in this paper have been deposited in the NCBI Gene Expression Omnibus and are accessible through the GEO accession number GSE24536.

View Supplemental data

We are obliged to the patients for their dedicated collaboration. We gratefully acknowledge M. Delorenzi, F. Schütz, H.-P. Pircher, M. Etzrodt, M. Pittet, M. Matter, O. Michielin, L.J. Old, J. O’Donnell-Tormey, E.W. Hoffman, and A. Krieg for essential contributions; D. Zehn, P. Ohashi, H.R. MacDonald, J. Skipper, and H.F. Oettgen for support; and P. Schneider, L. Derre, M. Braun, C. Christiansen-Jucht, C. Jandus, J.-P. Rivals, T. Lövgren, and M. Iancu for collaboration and advice. We thank P. Guillaume and I. Luescher for tetramers, and Pfizer and Coley Pharmaceutical Group (USA) for providing CpG 7909 (PF-3512676). This work was supported by the Ludwig Institute for Cancer Research, the Cancer Research Institute (USA), the Cancer Vaccine Collaborative, Atlantic Philanthropies (USA), the Wilhelm Sander-Foundation (Germany), the Swiss Cancer League (grant 02279-08-2008), the Swiss National Science Foundation, and the Swiss National Center of Competence in Research (NCCR) Molecular Oncology.

Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article: J Clin Invest doi:10.1172/JCI46102.

Kim PS, Ahmed R. Features of responding T cells in cancer and chronic infection. Curr Opin Immunol. 2010;22(2):223–230. Wherry EJ, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 2007;27(4):670–684. Turner SJ, Kedzierska K, La Gruta NL, Webby R, Doherty PC. Characterization of CD8+ T cell repertoire diversity and persistence in the influenza A virus model of localized, transient infection. Semin Immunol. 2004;16(3):179–184. Gallimore A, Hengartner H, Zinkernagel R. Hierarchies of antigen-specific cytotoxic T cell responses. Immunol Rev. 1998;164:29–36. Yewdell JW, Bennink JR. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol. 1999;17:51–88. Appay V, Douek DC, Price DA. CD8+ T cell efficacy in vaccination and disease. Nat Med. 2008;14(6):623–628. Appay V, Rowland-Jones SL. Lessons from the study of T cell differentiation in persistent human virus infection. Semin Immunol. 2004;16(3):205–212. Makedonas G, et al. Perforin and IL-2 upregulation define qualitative differences among highly functional virus-specific human CD8+ T cells. PLoS Pathog. 2010;6(3):e1000798. Chen SF, et al. Antiviral CD8+ T cells in the control of primary human cytomegalovirus infection in early childhood. J Infect Dis. 2004;189(9):1619–1627. Guerreiro M, et al. Human peripheral blood and bone marrow Epstein-Barr virus-specific T cell repertoire in latent infection reveals distinct memory T cell subsets. Eur J Immunol. 2010;40(6):1566–1576. Zajac AJ, et al. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998;188(12):2205–2213. Moskophidis D, Lechner F, Pircher H, Zinkernagel RM. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature. 1993;362(6422):758–761. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persistence alters CD8+ T cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol. 2003;77(8):4911–4927. Yi JS, Cox MA, Zajac AJ. T cell exhaustion: characteristics, causes and conversion. Immunology. 2010;129(4):474–481. Barber DL, et al. Restoring function in exhausted CD8+ T cells during chronic viral infection. Nature. 2006;439(7077):682–687. Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol. 2005;5(3):215–229. Letvin NL, Walker BD. Immunopathogenesis and immunotherapy in AIDS virus infections. Nat Med. 2003;9(7):861–866. Quigley M, et al. Transcriptional analysis of HIV-specific CD8(+) T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med. 2010;16(10):1147–1151. Speiser DE, et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest. 2005;115(3):739–746. Zippelius A, et al. Effector function of human tumor-specific CD8+ T cells in melanoma lesions: a state of local functional tolerance. Cancer Res. 2004;64(8):2865–2873. Speiser DE, et al. Memory and effector CD8+ T cell responses after nanoparticle vaccination of melanoma patients. J Immunother. 2010;33(8):848–858. Rosenberg SA, Dudley ME. Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol. 2009;21(2):233–240. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 2010;207(10):2187–2194. Ahmadzadeh M, et al. Tumor antigen-specific CD8+ T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009;114(8):1537–1544. Appay V, et al. New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site. J Immunol. 2006;177(3):1670–1678. Choi S, Schwartz RH. Molecular mechanisms for adaptive tolerance and other T cell anergy models. Semin Immunol. 2007;19(3):140–152. Derre L, et al. BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination. J Clin Invest. 2010;120(1):157–167. Appay V, et al. Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages. J Immunol. 2007;179(11):7406–7414. Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma. PLoS Med. 2007;4(5):e176. Cham CM, Xu H, O’Keefe JP, Rivas FV, Zagouras P, Gajewski TF. Gene array and protein expression profiles suggest post-transcriptional regulation during CD8+ T cell differentiation. J Biol Chem. 2003;278(19):17044–17052. Willinger T, Freeman T, Hasegawa H, McMichael AJ, Callan MF. Molecular signatures distinguish human central memory from effector memory CD8+ T cell subsets. J Immunol. 2005;175(9):5895–5903. Bengsch B, et al. Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation. PLoS Pathog. 2010;6(6):e1000947. Blackburn SD, et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol. 2009;10(1):29–37. Jin HT, et al. Cooperation of Tim-3 and PD-1 in CD8+ T cell exhaustion during chronic viral infection. Proc Natl Acad Sci U S A. 2010;107(33):14733–14738. Rey J, et al. The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function. Eur J Immunol. 2006;36(9):2359–2366. Fourcade J, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010;207(10):2175–2186. McMahan RH, et al. Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J Clin Invest. 2010;120(12):4546–4557. Macian F, Garcia-Cozar F, Im SH, Horton HF, Byrne MC, Rao A. Transcriptional mechanisms underlying lymphocyte tolerance. Cell. 2002;109(6):719–731. Parish IA, et al. The molecular signature of CD8+ T cells undergoing deletional tolerance. Blood. 2009;113(19):4575–4585. Willimsky G, Blankenstein T. Sporadic immunogenic tumours avoid destruction by inducing T cell tolerance. Nature. 2005;437(7055):141–146. Willimsky G, et al. Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness. J Exp Med. 2008;205(7):1687–1700. Miller JD, et al. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity. 2008;28(5):710–722. Gaucher D, et al. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med. 2008;205(13):3119–3131. Barbey C, et al. IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells. J Immunol. 2007;178(6):3566–3574. Lienard D, et al. Ex vivo detectable activation of Melan-A-specific T cells correlating with inflammatory skin reactions in melanoma patients vaccinated with peptides in IFA. Cancer Immun. 2004;4:4. Romero P, et al. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J Exp Med. 1998;188(9):1641–1650. Hadrup SR, et al. Parallel detection of antigen-specific T cell responses by multidimensional encoding of MHC multimers. Nat Methods. 2009;6(7):520–526. Roederer M, Nozzi JL, Nason MC. SPICE: Exploration and analysis of post-cytometric complex multivariate datasets. Cytometry A. 2011;79(2):167–174. Subramanian A, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–15550.

View the original article here

New Gene Therapy Technique On Induced Pluripotent Stem Cells Holds Promise In Treating Immune System Disease

Researchers have developed an effective technique that uses gene therapy on stem cells to correct chronic granulomatous disease (CGD) in cell culture, which could eventually serve as a treatment for this rare, inherited immune disorder, according to a study published in Blood, the Journal of the American Society of Hematology.

CGD prevents neutrophils, a type of white blood cell of the immune system, from making hydrogen peroxide, an essential defense against life-threatening bacterial and fungal infections. Most cases of CGD are a result of a mutation on the X chromosome, a type of CGD that is called "X-linked" (X-CGD).

While antibiotics can treat infections caused by X-CGD, they do not cure the disease itself. Patients with X-CGD can be cured with a hematopoietic stem cell (HSC) transplant from healthy bone marrow; however, finding a compatible donor is difficult. Even with a suitable donor, patients are at risk of developing graft-versus-host disease (GVHD), a serious and often deadly post-transplant complication that occurs when newly transplanted donor cells recognize a recipient's own cells as foreign and attack the patient's body.

Another treatment option under development for X-CGD is gene therapy, a technique for correcting defective genes responsible for disease development that involves manipulation of genetic material within an individual's blood-forming stem cells using genetically engineered viruses. However, this gene therapy has so far proved to be inefficient at correcting X-CGD. In addition, these engineered viruses insert new genetic material at random locations in the blood-forming stem cell genome, putting patients at significantly higher risk for developing genetic mutations that may eventually lead to serious blood disorders, including blood cancer.

In order to develop a more effective and safer gene therapy for X-CGD, researchers from the National Institute of Allergy and Infectious Disease (NIAID) at the National Institutes of Health (NIH) and The Johns Hopkins University School of Medicine embarked on a study using a more precise method for performing gene therapy that did not use viruses for the gene correction. Researchers removed adult stem cells from the bone marrow of a patient with X-CGD and genetically reprogrammed them to become induced pluripotent stem cells (iPS cells). Like embryonic stem cells, these patient-specific iPS cells can be grown and manipulated indefinitely in culture while retaining their capacity to differentiate into any cell type of the body, including HSCs.

"HSCs that are derived from gene corrected iPS cells are tissue-compatible with the patient and may create a way for the patient's own cells to be used in a transplant to cure the disease, removing the risk of GVHD or the need to find a compatible donor," said Harry L. Malech, MD, senior study author, Chief of the Laboratory of Host Defenses and Head of the Genetic Immunotherapy Section of NIAID at the NIH. "However, turning iPS cells into a large number of HSCs that are efficently transplantable remains technically difficult; therefore, our study aimed at demonstrating that it is possible to differentiate gene corrected iPS cells into a large number of corrected neutrophils. These corrected neutrophils, grown in culture, are tissue-compatible with the patient and may be used to manage the life-threatening infections that are caused by the disease."

Typically, iPS cells from a patient with an inherited disorder do not express disease traits, despite the fact that the iPS cell genome contains the expected mutation. The researchers were able to prove, in culture, that iPS cells from a patient with X-CGD could be differentiated into mature neutrophils that failed to produce hydrogen peroxide, thus expressing the disease trait. This is the first study in which the disease phenotype has been reproduced in neutrophils differentiated from X-CGD patient-specific iPS cells.

After discovering that the disease could be reproduced in cell culture, the researchers then sought to correct the disease and produce healthy neutrophils in culture. They used synthetic proteins called zinc finger nucleases (ZFNs) to target a corrective gene at a specifically defined location in the genome of the X-CGD iPS cells. The iPS cells were then carefully screened to identify those containing a single copy of the corrective gene properly inserted only at the safe site. The researchers observed that some of the gene-corrected iPS cells could differentiate into neutrophils that produced normal levels of hydrogen peroxide, effectively "correcting" the disease.

"This is the first study that uses ZFNs in specific targeting gene transfer to correct X-CGD," said Dr. Malech. "Demonstrating that this approach to gene therapy works with a single-gene disease such as X-CGD means that the results from our study offer not only a potential treatment for this disease, but more importantly, a technique by which other single-gene diseases can be corrected using specifically targeted gene therapy on iPS cells."

Source:
American Society of Hematology

Scientists Create Stable, Self-Renewing Neural Stem Cells

In a paper published in the April 25 early online edition of the Proceedings of the National Academy of Sciences, researchers at the University of California, San Diego School of Medicine, the Gladstone Institutes in San Francisco and colleagues report a game-changing advance in stem cell science: the creation of long-term, self-renewing, primitive neural precursor cells from human embryonic stem cells (hESCs) that can be directed to become many types of neuron without increased risk of tumor formation.

"It's a big step forward," said Kang Zhang, MD, PhD, professor of ophthalmology and human genetics at Shiley Eye Center and director of the Institute for Genomic Medicine, both at UC San Diego. "It means we can generate stable, renewable neural stem cells or downstream products quickly, in great quantities and in a clinical grade millions in less than a week that can be used for clinical trials and, eventually, for clinical treatments. Until now, that has not been possible."

Human embryonic stem cells hold great promise in regenerative medicine due to their ability to become any kind of cell needed to repair and restore damaged tissues. But the potential of hESCs has been constrained by a number of practical problems, not least among them the difficulty of growing sufficient quantities of stable, usable cells and the risk that some of these cells might form tumors.

To produce the neural stem cells, Zhang, with co-senior author Sheng Ding, PhD, a former professor of chemistry at The Scripps Research Institute and now at the Gladstone Institutes, and colleagues added small molecules in a chemically defined culture condition that induces hESCs to become primitive neural precursor cells, but then halts the further differentiation process.

"And because it doesn't use any gene transfer technologies or exogenous cell products, there's minimal risk of introducing mutations or outside contamination," Zhang said. Assays of these neural precursor cells found no evidence of tumor formation when introduced into laboratory mice.

By adding other chemicals, the scientists are able to then direct the precursor cells to differentiate into different types of mature neurons, "which means you can explore potential clinical applications for a wide range of neurodegenerative diseases," said Zhang. "You can generate neurons for specific conditions like amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), Parkinson's disease or, in the case of my particular research area, eye-specific neurons that are lost in macular degeneration, retinitis pigmentosa or glaucoma."

The new process promises to have broad applications in stem cell research. The same method can be used to push induce pluripotent stem cells (stem cells artificially derived from adult, differentiated mature cells) to become neural stem cells, Zhang said. "And in principle, by altering the combination of small molecules, you may be able to create other types of stem cells capable of becoming heart, pancreas, or muscle cells, to name a few."

The next step, according to Zhang, is to use these stem cells to treat different types of neurodegenerative diseases, such as macular degeneration or glaucoma in animal models.

Source: University of California

Autologous Induced Pluripotent Stem Cells And Gene Repair Therapy For Treatment Of Familial Hypercholesterolemia

Study shows, for the first time, the successful reprogramming of diseased human hepatocytes into induced pluripotent stem cells (iPSC).1

Results also found differentiation into mature hepatocytes was more efficient than that with fibroblast-derived iPSCs.

The generation of diseased hepatocyte-derived human iPSC lines provides a good basis for the study of liver disease pathogenesis.

Such technology could give a potentially unlimited reservoir of cells for the treatment of human liver diseases: generating genetically corrected liver cells via auto-transplantation of genetically modified hepatocytes, thus avoiding liver transplant and lifelong immunosuppression.

References:
1 Bosman, A. et al. Progress toward the clinical application of autologus induced pluripotent stem cells and gene repair therapy for treatment of familial hypercholesterolemia. Abstract presented at The International Liver CongressTM 2011.

Source:
Travis Taylor
European Association for the Study of the Liver

Versatility Of Stem Cells Controlled By Alliances, Competitions Of Proteins

to make, stem cells have a process to "decide" whether to transform into a specific cell type or to stay flexible, a state that biologists call "pluripotency." Using a technology he invented, Brown researcher William Fairbrother and colleagues have discovered new molecular interactions in the process that will help regenerative medicine researchers better understand pluripotency.

In a paper published in advance online in the journal Genome Research, Fairbrother's team showed that different proteins called transcription factors compete and cooperate in the cells to produce complex bindings along crucial sequences of DNA. This game of molecular "capture the flag," played in teams and amid shifting alliances, appears to be a necessary part of what determines whether stem cells retain their pluripotency and whether specialized, or differentiated, cells can regain it.

In recent years scientists have reported spectacular successes in turning fully differentiated cells back into pluripotent stem cells, a process called reprogramming. But the animals derived from these cells often suffer higher rates of tumors and other problems, Fairbrother said. The reason may be because the complex details of the reprogramming process haven't been fully understood. He said there are many misconceptions about how reprogramming transcription factors interact with DNA.

"Most people think of a protein binding to DNA as a single, surgical thing where you have this isolated binding event," Fairbrother said. "But in fact we show that sometimes these binding events occur over hundreds of nucleotides so they seem more like great greasy globs of proteins that are forming. In addition, the proteins interact with each other, diversifying their function by appearing in complexes with with different partners at different places."

By employing a high-throughput, high-resolution binding assay that he's dubbed MEGAShift, Fairbrother and his colleagues, who include pathology researchers from the University of Utah School of Medicine, were able to analyze the interactions of several key transcription factors in a region of 316,000 letters of DNA with a resolution as low as 10 base pairs. Through hundreds of thousands of array measurements, lead authors Luciana Ferraris and Allan Stewart, Fairbrother, Alec DeSimone, and the other authors learned previously unspotted patterns of protein interactions.

"How do stem cells stay in the state where they can keep their options open?" Fairbrother said. "A key player is POU5F1. But what are the key players that could interact with it and modulate its function? We've developed technology to look at that question."

One of several findings in the paper concerned POU5F1 and its archrival, POU2F1, which binds to exactly the same eight-letter DNA sequence. Which protein binds to the sequence first influences whether a stem cell specializes or remains pluripotent. Experiments showed that a determining factor was a third protein called SOX2. SOX2 helped both proteins bind, but it helps POU2F1 more than POU5F1. In contrast, the team found that another player, NANOG, exclusively helps POU5F1.

"Who binds next to a protein is a determinant of who ends up binding to a sequence," Fairbrother said.

With support from the National Institutes of Health, Fairbrother's group is also applying MEGAShift to other questions, including how protein-protein interactions affect the formation of RNA-protein complexes, which can be even more complicated than binding DNA.

They will also look at the problem of narrowing the field of hundreds of genomic sequence variations that exist naturally in the population down to the real genetic "causal variants" of disease risk. MEGAShift can sort through which variants associated with disease result in an altered binding event that results in a clinical manifestation, such as diabetes or lupus.

Notes:

In addition to Fairbrother, DeSimone, Ferraris and Stewart, other authors on the paper Matthew Gemberling at Brown, Dean Tantin at the University of Utah, and Jinsuk Kang also at the University of Utah.

The research was funded by the National Human Genome Research Institute.

Source:
David Orenstein
Brown University

ACT Files European Clinical Trial Application For Phase 1/2 Study Using Embryonic Stem Cells To Treat Macular Degeneration

Advanced Cell Technology, Inc. ("ACT"; OTCBB: ACTC), a leader in the field of regenerative medicine, announced today that it has filed a clinical trial application (CTA) with the European Medicines and Healthcare products Regulatory Agency (MHRA) seeking clearance to initiate its Phase 1/2 clinical trial using retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) to treat patients with Stargardt's Macular Dystrophy (SMD).

"With this filing, our initiatives in Europe are really starting to gain momentum," said Gary Rabin, interim chairman and CEO of ACT. "Through data from this proposed trial, and the two trials we are preparing to commence in the United States, we are eagerly anticipating beginning to assess the capabilities of our RPE cells to repair and regenerate the retina. As in the US, we also intend to file in Europe for clinical trials involving Dry Age-Related Macular Degeneration (Dry AMD) and other degenerative diseases of the retina, concurrently targeting the two largest pharmaceutical markets in the world."

The proposed clinical trial will be a prospective, open-label study that is designed to determine the safety and tolerability of the RPE cells following sub-retinal transplantation to patients with advanced SMD, similar to the FDA-cleared U.S. trial which is set to commence in the first half of this year. During the CTA review process, which requires a minimum of 60 days, the reviewers decide if an applicant is permitted to proceed with its proposed clinical trial. Additional information may be requested from the applicant, which could extend the review period.

"We are very excited about this European filing, because our preclinical data from various animal models with hESC-derived RPE cells have been tremendously encouraging," said Robert Lanza, M.D., chief scientific officer at ACT. "In rats we have seen 100 percent improvement in visual performance over untreated animals without any adverse effects. Near-normal function was also achieved in a mouse model of Stargardt's disease."

In 2010, the US Food and Drug Administration (FDA) granted Orphan Drug designation for ACT's RPE cells for treating SMD, and earlier this year the company received a positive opinion from the Committee for Orphan Medicinal Products (COMP) of the European Medicines Agency (EMA) towards designation of this product as an orphan medicinal product for the treatment of Stargardt's disease. ACT anticipates adoption of the EMA's recommendation by the European Commission in coming weeks.

About Stargardt's Macular Dystrophy and Degenerative Diseases of the Retina

Stargardt's Macular Dystrophy (SMD) is one of the most common forms of macular degeneration in the world. SMD causes progressive vision loss, usually starting in children between 10 to 20 years of age. Eventually, blindness results from photoreceptor loss associated with degeneration in the pigmented layer of the retina, called the retinal pigment epithelium or RPE cell layer.

Degenerative diseases of the retina are among the most common causes of untreatable blindness in the world. As many as thirty million people in the United States and Europe suffer from macular degeneration, which represents a $25-30 billion worldwide market that has yet to be effectively addressed. Approximately 10% of people ages 66 to 74 will have symptoms of macular degeneration, the vast majority the "dry" form of AMD which is currently untreatable. The prevalence increases to 30% in patients 75 to 85 years of age.

Source: Advanced Cell Technology, Inc

Note: Any medical information published on this website is not intended as a substitute for informed medical advice and you should not take any action before consulting with a health care professional. For more information, please read our terms and conditions.

Please note that we publish your name, but we do not publish your email address. It is only used to let you know when your message is published. We do not use it for any other purpose. Please see our privacy policy for more information.

If you write about specific medications or operations, please do not name health care professionals by name.

All opinions are moderated before being included (to stop spam)

Contact Our News Editors

For any corrections of factual information, or to contact the editors please use our feedback form.

Please send any medical news or health news press releases to:

Privacy Policy | Terms and Conditions

MediLexicon International Ltd
Bexhill-on-Sea, UK
MediLexicon International Ltd © 2004-2011 All rights reserved.

'Universal' Virus-Free Method Developed By Scientists To Turn Blood Cells Into 'Beating' Heart Cells

Johns Hopkins scientists have developed a simplified, cheaper, all-purpose method they say can be used by scientists around the globe to more safely turn blood cells into heart cells. The method is virus-free and produces heart cells that beat with nearly 100 percent efficiency, they claim.

"We took the recipe for this process from a complex minestrone to a simple miso soup," says Elias Zambidis, M.D., Ph.D., assistant professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center.

Zambidis says, "many scientists previously thought that a nonviral method of inducing blood cells to turn into highly functioning cardiac cells was not within reach, but "we've found a way to do it very efficiently and we want other scientists to test the method in their own labs." However, he cautions that the cells are not yet ready for human testing.

To get stem cells taken from one source (such as blood) and develop them into a cell of another type (such as heart), scientists generally use viruses to deliver a package of genes into cells to, first, get them to turn into stem cells. However, viruses can mutate genes and initiate cancers in newly transformed cells. To insert the genes without using a virus, Zambidis' team turned to plasmids, rings of DNA that replicate briefly inside cells and eventually degrade.

Adding to the complexity of coaxing stem cells into other cell types is the expensive and varied recipe of growth factors, nutrients and conditions that bathe stem cells during their transformation. The recipe of this "broth" differs from lab to lab and cell line to cell line.

Reporting in the April 8 issue of Public Library of Science ONE (PLoS ONE), Zambidis' team described what he called a "painstaking, two-year process" to simplify the recipe and environmental conditions that house cells undergoing transformation into heart cells. They found that their recipe worked consistently for at least 11 different stem cell lines tested and worked equally well for the more controversial embryonic stem cells, as well as stem cell lines generated from adult blood stem cells, their main focus.

The process began with Johns Hopkins postdoctoral scientist Paul Burridge, Ph.D., who studied some 30 papers on techniques to create cardiac cells. He drew charts of 48 different variables used to create heart cells, including buffers, enzymes, growth factors, timing, and the size of compartments in cell culture plates. After testing hundreds of combinations of these variables, Burridge narrowed the choices down to between four to nine essential ingredients at each of three stages of cardiac development.

Beyond simplification, an added benefit is reduced cost. Burridge used a cheaper growth media that is one-tenth the price of standard media for these cells at $250 per bottle lasting about one week.

Zambidis says that he wants other scientists to test the method on their stem cell lines, but also notes that the growth "soup" is still a work in progress. "We have recently optimized the conditions for complete removal of the fetal bovine serum from one brief step of the procedure - it's made from an animal product and could introduce unwanted viruses," he says.

In their experiments with the new growth medium, the Hopkins team began with cord blood stem cells and a plasmid to transfer seven genes into the stem cells. They delivered an electric pulse to the cells, making tiny holes in the surface through which plasmids can slip inside. Once inside, the plasmids trigger the cells to revert to a more primitive cell state that can be coaxed into various cell types. At this stage, the cells are called induced pluripotent stem cells (iPSC).

Burridge then bathed the newly formed iPSCs in the now simplified recipe of growth media, which they named "universal cardiac differentiation system." The growth media recipe is specific to creating cardiac cells from any iPSC line.

Finally, they incubated the cells in containers that removed oxygen down to a quarter of ordinary atmospheric levels. "The idea is to recreate conditions experienced by an embryo when these primitive cells are developing into different cell types," says Burridge. They also added a chemical called PVA, which works like glue to make cells stick together.

Nine days later, the nonviral iPSCs turned into functional, beating cardiac cells, each the size of a needlepoint.

Burridge manually counted how often iPSCs formed into cardiac cells in petri dishes by peering into a microscope and identifying each beating cluster of cells. In each of 11 cell lines tested, each plate of cells had an average of 94.5 percent beating heart cells. "Most scientists get 10 percent efficiency for IPSC lines if they're lucky," says Zambidis.

Zambidis and Burridge also worked with Johns Hopkins University bioengineering experts to apply a miniversion of an electrocardiograph to the cells, which tests how cardiac cells use calcium and transmit a voltage. The resulting rhythm showed characteristic pulses seen in a normal human heart.

Virus-free, iPSC-derived cardiac cells could be used in laboratories to test drugs that treat arrhythmia and other conditions. Eventually, bioengineers could develop grafts of the cells that are implanted into patients who suffered heart attacks.

Zambidis' team has recently developed similar techniques for turning these blood-derived iPSC lines into retinal, neural and vascular cells.

Notes:

The research was funded by the Maryland Stem Cell Research Fund and the National Institutes of Health.

Research participants include Susan Thompson, Michal Millrod, Seth Weinberg, Xuan Yuan, Ann Peters, Vasiliki Mahairaki, Vassilis E. Koliatsos, and Leslie Tung at Johns Hopkins.

Source:
Vanessa Wasta
Johns Hopkins Medical Institutions

 

Study Of Umbilical Cord Blood-Derived Stem Cells For Lupus Therapy

Main Category: Lupus
Also Included In: Stem Cell Research
Article Date: 12 Apr 2011 - 4:00 PDT window.fbAsyncInit = function() { FB.init({ appId: 'aa16a4bf93f23f07eb33109d5f1134d3', status: true, cookie: true, xfbml: true, channelUrl: 'http://www.medicalnewstoday.com/scripts/facebooklike.html'}); }; (function() { var e = document.createElement('script'); e.async = true; e.src = document.location.protocol + '//connect.facebook.net/en_US/all.js'; document.getElementById('fb-root').appendChild(e); }()); email to a friend   printer friendly   opinions  

Human umbilical cord blood-derived mensenchymal stem cells (uMSCs) have been found to offer benefits for treating lupus nephritis (LN) when transplanted into mouse models of systemic lupus erythematosus (SLE). SLE is an autoimmune disease with "myriad immune system aberrations" characterized by diverse clinical conditions, including LN, a leading cause of morbidity and mortality for patients with SLE.

The beneficial results were reported in a study by Taiwanese researchers published in the current issue of Cell Transplantation (20:2), freely available on-line here.

According to corresponding author Dr. Oscar K. Lee of the National Yang-Ming University School of Medicine, MSCs have been shown to possess immune-modulatory capabilities and can alleviate immune responses by inhibiting inflammation as well as the function of mature and immature immune system T cells. Seeking to explore the therapeutic effects of uMSCs in treating LN, their study transplanted umbilical cord blood-derived stem cells into mice modeled with systemic immune diseases closely resembling SLE in humans.

"We found that uMSC transplantation markedly delayed the deterioration of renal function, reduced certain antibody levels, alleviated changes in renal pathology and the development of proteinuria - the presence of excess protein serum in the urine and a sign of renal damage," said Dr. Lee.

The positive difference in survival rate for mice treated at two months of age compared with mice treated at six months of age, led the researchers to conclude that early uMSC transplantation may be most efficacious. The researchers also deduced that their findings favored the use of allogenic (other-donated) rather than autologous (self-donated) MSCs for SLE treatment, which would make sense with an autoimmune disorder.

"The therapeutic effects demonstrated in this pre-clinical study support further exploration of the possibility of using uMSCs from mismatched donors in LN treatment," concluded Dr. Lee.

"The ability of uMSCs to reduce inflammation means that they are likely to be of use in the treatment of autoimmune disorders and this study supports that reasoning and, in this case, also advocates the use of non-self cells," said Dr. David Eve, associate editor of Cell Transplantation and an instructor at the University of South Florida Center of Excellence for Aging and Brain Repair.

Citation:
Chang, J-W.; Hung , S-P.; Wu, H-H.; Wu, W-M.; Yang, A-H.; Tsai, H-L.; Yang, L-Y.; Lee, O. K. Therapeutic Effects of Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation in Experimental Lupus Nephritis. Cell Transplant. 20(2):245-257; 2011.

Source:
David Eve
Cell Transplantation Center of Excellence for Aging and Brain Repair

Note: Any medical information published on this website is not intended as a substitute for informed medical advice and you should not take any action before consulting with a health care professional. For more information, please read our terms and conditions.

Please note that we publish your name, but we do not publish your email address. It is only used to let you know when your message is published. We do not use it for any other purpose. Please see our privacy policy for more information.

If you write about specific medications or operations, please do not name health care professionals by name.

All opinions are moderated before being included (to stop spam)

Contact Our News Editors

For any corrections of factual information, or to contact the editors please use our feedback form.

Please send any medical news or health news press releases to:

Privacy Policy | Terms and Conditions

MediLexicon International Ltd
Bexhill-on-Sea, UK
MediLexicon International Ltd © 2004-2011 All rights reserved.

View the original article here

<

To Reprogram Stem Cells Penn Study Eliminates The Use Of Transcription Factors And Increases Efficiency 100-Fold

Researchers at the University of Pennsylvania School of Medicine have devised a totally new and far more efficient way of generating induced pluripotent stem cells (iPSCs), immature cells that are able to develop into several different types of cells or tissues in the body. The researchers used fibroblast cells, which are easily obtained from skin biopsies, and could be used to generate patient-specific iPSCs for drug screening and tissue regeneration.

iPSCs are typically generated from adult non-reproductive cells by expressing four different genes called transcription factors. The generation of iPSCs was first reported in 2006 by Shinya Yamanaka, and multiple groups have since reported the ability to generate these cells using some variations on the same four transcription factors.

The promise of this line of research is to one day efficiently generate patient-specific stem cells in order to study human disease as well as create a cellular "storehouse" to regenerate a person's own cells, for example heart or liver cells. Despite this promise, generation of iPSCs is hampered by low efficiency, especially when using human cells.

"It's a game changer," says Edward Morrisey, PhD, professor in the Departments of Medicine and Cell and Developmental Biology and Scientific Director at the Penn Institute for Regenerative Medicine. "This is the first time we've been able to make induced pluripotent stem cells without the four transcription factors and increase the efficiency by 100-fold." Morrisey led the study published this week in Cell Stem Cell.

"Generating induced pluripotent stem cells efficiently is paramount for their potential therapeutic use," noted James Kiley, PhD, director of the National Heart, Lung, and Blood Institute's Division of Lung Diseases. "This novel study is an important step forward in that direction and it will also advance research on stem cell biology in general."

Before this procedure, which uses microRNAs instead of the four key transcription factor genes, for every 100,000 adult cells re-programmed, researchers were able to get a small handful of iPSCs, usually less than 20. Using the microRNA-mediated method, they have been able to generate approximately 10,000 induced pluripotent stem cells from every 100,000 adult human cells that they start with. MicroRNAs (miRNAs) are short RNA molecules that bind to complementary sequences on messenger RNAs to silence gene expression.

The Morrisey lab discovered this new approach through studies focusing on the role of microRNAs in lung development. This lab was working on a microRNA cluster called miR302/367, which plays an important role in lung endoderm progenitor development. This same microRNA cluster was reported to be expressed at high levels in embryonic stem cells, and iPSCs and microRNAs have been shown to alter cell phenotypes.

The investigators performed a simple experiment and expressed the microRNAs in mouse fibroblasts and were surprised to observe colonies that looked just like iPSCs. "We were very surprised that this worked the very first time we did the experiment," says Morrisey. "We were also surprised that it worked much more efficiently than the transcription factor approach pioneered by Dr. Yamanaka."

Since microRNAs act as repressors of protein expression, it seems likely that they repress the repressors of the four transcription factors and other factors important for maintaining the pluripotent-stem-cell state. However, exactly how the miRNAs work differently compared to the transcription factors in creating iPSCs will require further investigation.

The iPSCs generated by the microRNA method in the Morrisey lab are able to generate most, if not all, tissues in the developing mouse, including germ cells, eggs and sperm. The group is currently working with several collaborators to redifferentiate these iPSCs into cardiomyocytes, hematopoietic cells, and liver hepatocytes.

"We think this method will be very valuable in generating iPSCs from patient samples in a high-throughput manner" says Morrisey. microRNAs can also be introduced into cells using synthetically generated versions of miRNAs called mimics or precursors. These mimics can be easily introduced into cells at high levels, which should allow for a non-genetic method for efficiently generating iPSCs.

"The upshot is that we hope to be able to produce synthetic microRNAs to transform adult cells into induced pluripotent stem cells, which could eventually then be redifferentiated into other cell types, for example, liver, heart muscle or nerve cells" says Morrisey.

Other authors of the study include Frederick Anokye-Danso, Chinmay M. Trivedi, and Jonathan A. Epstein, all from Penn. These studies were funded by the National Heart, Lung and Blood Institute Progenitor Cell Biology Consortium and Division of Lung Disease and the American Heart Association Jon DeHaan Myogenesis Center Award.

Source:
Karen Kreeger
University of Pennsylvania School of Medicine

 

Scientists Identify A Surprising New Source Of Cancer Stem Cells

Whitehead Institute researchers have discovered that a differentiated cell type found in breast tissue can spontaneously convert to a stem-cell-like state, the first time such behavior has been observed in mammalian cells. These results refute scientific dogma, which states that differentiation is a one-way path; once cells specialize, they cannot return to the flexible stem-cell state on their own.

This surprising finding, published online this week in the Proceedings of the National Academy of Sciences (PNAS), may have implications for the development of cancer therapeutics, particularly those aimed at eradicating cancer stem cells.

"It may be that if one eliminates the cancer stem cells within a tumor through some targeted agent, some of the surviving non-stem tumor cells will generate new cancer stem cells through spontaneous de-differentiation," says Whitehead Founding Member Robert Weinberg. Cancer stem cells are uniquely capable of reseeding tumors at both primary and distant sites in the body.

During differentiation, less-specialized stem cells mature into many different cell types with defined functions. These differentiated cells work together to form tissues and organs. In breast tissue, for example, differentiated basal cells and luminal cells combine to form milk ducts.

While analyzing cells from human breast tissue, Christine Chaffer, who is a postdoctoral researcher in the Weinberg lab and first author of the PNAS paper, observed a small number of living basal cells floating freely in the tissue culture medium.

Intrigued by the cells' unusual behavior, Chaffer conducted further targeted investigations, including injection of the floating basal cells into mice. After 12 weeks she found that the injected basal cells gave rise to milk duct-like structures containing both basal and luminal cells a clear indication that the floating cells had de-differentiated into stem-like cells.

Until now, no one has shown that differentiated mammalian cells, like these basal cells, have the ability to spontaneously revert to the stem-like state (a behavior described as plasticity).

To see if basal cells could become cancer stem cells, Chaffer inserted cancer-causing genes into the cells. When these transformed cells were injected into mice, the resulting tumors were found to include a cancer stem cell population that descended from the original injected basal (more differentiated) cells. These results indicate that basal cells in breast cancer tumors can serve as a previously unidentified source of cancer stem cells.

As research for new cancer therapies has recently focused on eliminating cancer stem cells, Weinberg cautions that the plasticity seen in these basal cells suggests a more complicated scenario than previously thought.

"Future drug therapies that are targeted against cancer will need to eliminate the cancer stem cells and, in addition, get rid of the non-stem cells in tumors both populations must be removed," says Weinberg, who is also a professor of biology at MIT. "Knocking out one or the other is unlikely to suffice to generate a durable clinical response."

Chaffer is now focusing on what actually prompts these flexible cells to de-differentiate, and in the case of cancer cells, how to stop the cells from converting into cancer stem cells.

"This plasticity can occur naturally, and it seems that the trigger may be a physiological mechanism for restoring a pool of stem cells," says Chaffer. "We believe that certain cells are more susceptible to such a trigger and therefore to conversion from a differentiated to a stem-like state, and that this process occurs more frequently in cancerous cells."

In the case of normal epithelial cells, the observed behavior may also allow patient specific adult stem cells to be derived without genetic manipulation, holding promise for degenerative disease therapy.

This research was supported by the National Health and Medical Research Council of Australia, National Institutes of Health, MIT's Ludwig Center for Molecular Oncology, the Breast Cancer Research Foundation, and a Department of Defense Breast Cancer Research Program Idea Award.

Source: Whitehead Institute for Biomedical Research