T-cell receptor Vbeta gene usage by T cells reactive with the tumor-rejection antigen SART-1 in oral squamous cell carcinoma

We recently described that the SART-1(690-698) peptide could induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs), which recognize the SART-1(259) (+) tumor cells from peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) cancer patients. In our study, in 5 of 14 HLA-A24(+) patients with oral squamous cell carcinomas (SCCs), CTLs could be induced with the SART-1(690-698) peptide from the PBMCs. In 2 of the patients from whom the highest CTL activities were induced, the T-cell receptor (TCR) Vbeta repertoire expressed by the SART-1(690-698)-specific CTLs was found to be restricted and multiple Vbeta families were predominantly expressed in each patient. Although the predominant Vbeta families were different between the 2 patients, Vbeta7 was highly and commonly predominant. The same predominant Vbeta families were also detected in the tumor-infiltrating lymphocytes (TILs) from each patient, and each Vbeta family contained one or more unique T-cell clonotypes. The unique T-cell clonotypes were found to be common between the TILs and SART-1(690-698)-specific CTLs from each patient, and especially 2 T-cell clonotypes with Vbeta7 were identical even in the 2 patients. One of the 2 T-cell clonotypes with Vbeta7 was detected in the TILs from 11 of 14 HLA-A24(+) patients and another was found in those from 8 of HLA-A24(+) patients, while none of 10 HLA-A24(-) patients demonstrated both T-cell clonotypes. These results strongly suggest that the T-cell clonotypes with Vbeta7 are major TCR Vbeta genes expressed by SART-1(690-698)-specific CTLs. Furthermore, autologous tumor cells from one of the HLA-A24(+) patients stimulated the PBMCs and regional lymph node cells (LNCs) to expand the same T-cell clonotypes as those in the SART-1(690-698)-specific CTLs. These results strongly suggest that the SART-1(690-698)-specific CTLs clearly accumulate in vivo, especially in the TILs, as a consequence of in situ antigenic stimulation by autologous tumor cells. The identification of the unique TCR Vbeta genes used by SART-1(259)-specific CTLs should help to improve the diagnosis of the specific immune response in patients with SART-1(259) (+) cancers, especially during anticancer immunotherapy.     

Clinical significance of decreased zeta chain expression in peripheral blood lymphocytes of patients with head and neck cancer.

Patients with squamous cell carcinoma of the head and neck (SCCHN) frequently have impaired immune responses. Alterations in T-cell receptor-associated signaling molecules in tumor-infiltrating as well as circulating lymphocytes have been reported in these patients. Using quantitative flow cytometry analysis, we have demonstrated that expression of the zeta chain is significantly decreased relative to normal controls in both CD8+ and CD4+ T cells as well as CD3- CD56+ CD16+ natural killer cells in the peripheral blood of patients with SCCHN who, as a result of previous therapies, have no evident disease. Patients with a more aggressive type of SCCHN and those who experienced a recurrence or had a second primary cancer within the last 2 years of the study had the lowest zeta chain expression. In addition, SCCHN patients showed a significantly greater spontaneous ex vivo apoptosis, as measured by a terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, in PBMCs, compared to normal controls. The observed decreased expression of zeta in T and natural killer cells coincided but did not directly correlate with significantly increased spontaneous apoptosis of lymphocytes obtained from treated patients with no evident disease. The results suggest that in patients with SCCHN, zeta chain defects and lymphocyte apoptosis are manifestations of long-lasting negative effects of tumor on the immune system.     

Development of T-cell lymphomas in Emu-IEX-1 mice

Inhibition of apoptosis or abnormal cell survival can result in tumorigenesis by facilitating the insurgence of various mutations. Immediate-early response gene X-1 (IEX-1), protects T cells from apoptosis induced by the ligation of Fas or the T-cell receptor (TCR)/CD3 complex in Emu-IEX-1 mice that direct the gene expression in both T and B cell lineages under the control of the Emu enhancer. Consistent with a biased effect of IEX-1 towards T cells, Emu-IEX-1 mice selectively developed T-cell lymphomas in the spleen, when they aged, which may be associated with increased levels of IEX-1 phosphorylation in T cells compared to B cells. The lymphomas were single positive (CD4+CD8-, CD4-CD8+), double positive (CD4+CD8+), or double negative (CD4-CD8-) T cells. They resulted from aberrantly clonal expansions of T cells expressing a specific TCR, as suggested by the TCR repertoire analysis using a panel of monoclonal antibodies recognizing TCR Vbeta chain, as well as by TCR beta gene rearrangements. The study provides, for the first time, unambiguous evidence of the oncogenic potential of IEX-1 in a cell-specific manner. The animal model may help our understanding of peripheral T-cell lymphoma development.     

Expression of pro- and antiapoptotic proteins in circulating CD8+ T cells of patients with squamous cell carcinoma of the head and neck.

OBJECTIVE: Apoptosis of T lymphocytes in the circulation of patients with squamous cell carcinoma of the head and neck (SCCHN) was shown to target effector CD8+ rather than CD4+ T cells. This study evaluates the contribution of pro- and antiapoptotic components of the mitochondria-dependent pathway to apoptosis of circulating CD8+ T cells in these patients. EXPERIMENTAL DESIGN: Blood samples were obtained from 77 patients with SCCHN and 51 normal control(s) (NC). Percentages of CD8+Annexin V+ (ANX+) and CD8+CD95+ cells, changes in mitochondrial membrane potential and levels of expression of Bcl-2, Bcl-XL, and Bax in CD8+ T lymphocytes were measured by quantitative flow cytometry. RESULTS: Elevated percentages (P < 0.001) of early apo-ptotic (CD8+ANX+ CD95+) T cells in the circulation distinguish SCCHN patients from NCs but not patients with no evidence of disease (NED) from those with active disease (AD). Circulating CD8+ but not CD4+ T cells in patients were found to contain higher levels of proapoptotic Bax and antiapoptotic Bcl-XL (P < 0.01) than NC cells. The Bax/Bcl-2 ratio was elevated in CD8+ T cells of patients relative to NCs (P < 0.01), and it correlated with the percentage of ANX+CD8+ T cells (P = 0.007). The Bax/Bcl-XL ratio discriminated AD from NED patients. CONCLUSION: Apoptosis of circulating CD8+T cells is found in SCCHN patients with AD or NED. Up-regulated Bax and Bcl-XL expression, the elevated Bax/Bcl-2 ratio and its association with ANX binding implicate the mitochondrial pathway in death of CD8+ T cells of patients with SCCHN. Understanding of molecular mechanisms of T-cell death and survival is essential for the development of more effective biotherapies for SCCHN.     

Impaired lymphocyte responses and their restoration in oral cancer patients expressing distinct TCR variable region

We have analyzed TCR Vbeta gene usage and clonality of T cells in the peripheral blood of patients with oral cancer and healthy individuals. A large repertoire of clonal TCR Vbeta was observed in the peripheral blood of cancer patients, and this clonal expansion was dominantly represented in the CD8+ T cells. A marked decrease in the lymphocyte proliferative responses to mitogen and anti-CD3 MAb and spontaneous apoptosis was observed in lymphocytes. Appropriate co-stimulation of lymphocytes (anti-TCR Vbeta MAb and anti-CD28 MAb) restored the lymphocyte proliferative responses and CD3-zeta chain expression in these patients.     

Simultaneous generation of CD8+ and CD4+ melanoma-reactive T cells by retroviral-mediated transfer of a single T-cell receptor

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen-reactive T cells for transfer into cancer patients. Genes encoding tumor antigen-specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor-specific for a class I MHC (HLA-A2)-restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4(+) tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor-transduced T cells secreted various cytokines when cocultured with tyrosinase peptide-loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8(+) and CD4(+) transduced T cells could recognize HLA-A2(+) melanoma cells, giving us the possibility of engineering class I MHC-restricted effector and T helper cells against melanoma. The ability to confer class I MHC-restricted tumor cell recognition to CD4(+) T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer-based immunotherapy.     

B cell lymphomas of C57L/J mice; the role of natural killer cells and T helper cells in lymphoma development and growth

The Hodgkin's-like Type B neoplasms which arise spontaneously in aging C57L mice (25% incidence at 21 months of age) were first reported over 40 years ago, but since then relatively little has been published about these lymphomas. Based on previous studies in SJL mice, we investigated the phenotypic and functional properties of C57L-derived lymphomas in relation to Mtv29-encoded vSAg expression by the tumor cells, and their ability to stimulate TCR Vbeta-restricted T cells. The cell surface phenotype of the C57L lymphomas indicates a B cell origin (sIg(+), MHC II(+)). These B lymphoma cells also express co-stimulatory molecules [B7-1 (CD80) and HSA (CD24)], and stimulate marked proliferation of syngeneic CD4(+) T cells. C57L B lymphoma cells exhibit Mtv-encoded mRNA by northern analysis, and also stimulate IL-2 production from Vbeta16(+) T cell hybrids, suggesting a role for Mtv 29 in this syngeneic T cell response. After transfer to syngeneic recipients, primary C57L lymphomas grow slowly, if at all. However, tumor growth is greatly accelerated by pretreatment of C57L recipients with anti-asialo GM1 antibody (but not anti-CD8 mAb), suggesting that NK cells play a major role in inhibiting lymphoma growth. If, in addition to anti-asialo GM1, the mice are also pretreated with anti-CD4 mAb, tumor growth is markedly inhibited, indicating that the lymphoma-responsive syngeneic CD4(+) T cells promote tumor growth. Therefore, although the vSAg-induced response stimulated by vSAg29 expressing lymphoma cells in syngeneic TCR Vbeta-restricted CD4(+) T cells is an important etiologic factor in this type of B cell neoplasm both in C57L and in SJL mice, the final outcome of the spontaneous neoplastic process appears strongly influenced by endogenous NK activity in aging mice.     

The T-cell receptor repertoire of tumor-infiltrating regulatory T lymphocytes is skewed toward public sequences

The accumulation of CD4(+) T regulatory cells (Treg) in tumor tissue is a widely described phenomenon in mouse models and in human cancer patients. Understanding the mechanisms by which Treg migrate and accumulate in tumors is important because they strongly influence the potential efficacy of many immunotherapies. In this study, we used immunoscope technology to analyze the T-cell receptor (TCR) repertoire of tumor-infiltrating T cells in non-TCR transgenic mice. Both tumor-infiltrating Tregs and T effector cells (Teff) displayed sequence profiles in the CDR3 region that were characteristic of biased repertoires seen during clonal cell expansions, implying that strong T-cell responses have occurred within the tumor tissue. By comparing the TCR sequences of tumor-infiltrating Tregs, we obtained evidence of the presence of so-called public TCR sequences that are common to many individuals yet were tumor-specific in nature. Such comparisons also suggested that the Treg-Teff conversion process is not an active process at the tumor site or tumor-draining lymph nodes. Our findings strongly suggest that Treg infiltration of tumor tissue is followed by marked proliferation of a few dominant T-cell clones in the tumor.     

Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer.

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells. However, increased percentages of CD4(+)CD25(+) T cells were observed in the non-small cell lung cancer tumor-infiltrating lymphocytes and ovarian cancer tumor-associated lymphocytes. Furthermore, these CD4(+)CD25(+) T cells were found to secrete transforming growth factor-beta, consistent with the phenotype of regulatory T cells. Despite a generalized expression of lymphocyte activation markers in the tumor-associated T-cell populations, the CD8(+) T cells expressed low levels of CD25. To determine whether expression of CD25 could be restored on the CD8 cells, tumor-associated T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After stimulation, nearly all of the CD8 T cells expressed CD25. Furthermore, despite the low levels of interleukin 2, IFN-gamma, and tumor necrosis factor-alpha secretion by the tumor-associated and peripheral blood T cells at baseline, stimulation with anti-CD3 and anti-CD28 monoclonal antibodies significantly increased the fraction of cells producing these cytokines. Thus, tumor-associated T cells from patients with early and late-stage epithelial tumors contain increased proportions of CD4(+)CD25(+) T cells that secrete the immunosuppressive cytokine transforming growth factor-beta. Furthermore, our results are consistent with previous reports showing impaired expression of CD25 on CD8(+) T cells in cancer patients. Finally, increased lymphocyte costimulation provided by triggering the CD28 receptor is able to increase CD25 expression and cytokine secretion in tumor-associated T cells. These observations provide evidence for the contribution of regulatory T cells to immune dysfunction in cancer patients.     

Decreased absolute counts of T lymphocyte subsets and their relation to disease in squamous cell carcinoma of the head and neck.

PURPOSE: Apoptosis of circulating CD8+ T cells seen in patients with squamous cell carcinoma of the head and neck [SCCHN (Hoffmann T, et al. Clin Cancer Res 2002;8:2553-62)] suggested a possibility of lymphocyte imbalance. Therefore, absolute numbers and percentages of lymphocyte subsets were examined in the peripheral blood of SCCHN patients and controls. EXPERIMENTAL DESIGN: Venous blood was obtained from 146 patients with SCCHN and 54 normal volunteers. Absolute numbers of CD3+, CD4+, and CD8+ T lymphocytes were determined using fluorobeads in a flow cytometry-based technique. Percentages of T lymphocyte subsets were also evaluated by flow cytometry. The patients were grouped at the time of blood draw [active versus no evidence of disease (NED), type of therapy administered, and the length of follow-up]. RESULTS: Patients with SCCHN had significantly lower absolute numbers of CD3+ CD4+, and CD8+ T cells than normal controls. However, no differences in the percentages of T-cell subsets between patients and normal controls were observed. Patients with active disease had significantly lower CD3+ and CD4+ T-cell counts than those with NED. Patients who had NED after surgery and radiotherapy had the lowest T-cell counts among the NED cohort. Patients who had NED for >2 years did not recover their T-cell counts, and the T-cell imbalance was evident many years after curative surgery. The tumor-node-metastasis (TNM) stage or site of the disease was not related to the absolute T-cell count. Patients with recurrent disease at the time of blood draw tended to have the lowest CD4+ T-cell counts. CONCLUSIONS: Patients with SCCHN have altered lymphocyte homeostasis, which persists for months or years after curative therapies.     

Analysis of the T-cell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors

A significant enrichment of CD4(+)Foxp3(+) T cells (regulatory T cells, Treg) is frequently observed in murine and human carcinomas. As Tregs can limit effective antitumor immune responses, thereby promoting tumor progression, it is important that the mechanisms underpinning intratumoral accumulation of Tregs are identified. Because of evidence gathered mostly in vitro, the conversion of conventional T cells (Tconv) into Tregs has been proposed as one such mechanism. We assessed the contribution of conversion in vivo by analyzing the TCR (T-cell receptor) repertoires of Tconvs and Tregs in carcinogen-induced tumors in mice. Our results indicate that the TCR repertoires of Tregs and Tconvs within tumor-infiltrating lymphocytes (TIL) are largely distinct. Indeed, the cell population with the greatest degree of repertoire similarity with tumor-infiltrating Tregs was the Treg population from the tumor-draining lymph node. These findings demonstrate that conversion of Tconvs does not contribute significantly to the accumulation of tumor-infiltrating Tregs; rather, Tconvs and Tregs arise from different populations with unique TCR repertoires. Enrichment of Tregs within TILs most likely, therefore, reflects differences in the way that Tregs and Tconvs are influenced by the tumor microenvironment. Elucidating the nature of these influences may indicate how the balance between tumor-infiltrating Tregs and Tconvs can be manipulated for therapeutic purposes.     

Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers.

PURPOSE: CD4(+) T cells play a central role in initiating and maintaining anticancer immune responses. However, regulatory CD4(+)CD25(+) T cells which express Foxp3 have also been shown to inhibit antitumor effector T cells. In view of these heterogeneous CD4(+) T-cell populations, this study was designed to determine the prognostic value of various tumor-infiltrating CD4(+) T-cell populations in head and neck squamous cell carcinoma.EXPERIMENTAL DESIGN: Eighty-four newly diagnosed untreated patients with histologically proven primary head and neck squamous cell carcinoma were included in this study. Double or triple immunofluorescence staining was done to assess and quantify the activated CD4(+)CD69(+) T cells, regulatory CD4(+)Foxp3(+) T cells, and mixed CD4(+)CD25(+) T cells comprising both activated and regulatory T cells.RESULTS: On univariate analysis, high levels of tumor-infiltrating CD4(+)CD69(+) T cells were correlated with both better locoregional control (P = 0.01) and longer survival (P = 0.01). Infiltration by regulatory Foxp3(+)CD4(+) T cells was positively associated with a better locoregional control of the tumor. Multivariate analysis showed that the only significant prognostic factors related to locoregional control were T stage (P = 0.02) and CD4(+)Foxp3(+) T-cell infiltration of the tumor (P = 0.02). In the Cox multivariate analysis, only two variables influenced overall survival probability: T stage (P = 0.036) and CD4(+)CD69(+) T-cell infiltration (P = 0.017).CONCLUSION: This study shows that tumor-infiltrating activated CD4(+)CD69(+) T cells are associated with a good prognosis in head and neck squamous cell carcinoma. In addition, regulatory Foxp3(+)CD4(+) T cells are positively correlated with locoregional control may be through down-regulation of harmful inflammatory reaction, which could favor tumor progression.     

Tumor antigen-specific T-cell expansion is greatly facilitated by in vivo priming.

PURPOSE: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu-specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from cytomegalovirus (CMV)-seronegative and CMV-seropositive donors as well as HER-2/neu-positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide-based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4(+) T cells. RESULTS: The frequencies of CMV-specific T cells generated from CMV(+) donors were >11-fold higher than the frequencies from CMV(-) donors (P = 0.001), with 22-fold increase of total number of CD3(+) T cells. The frequencies of HER-2/neu-specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3(+) T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3(+)CD4(+) regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4(+) and CD8(+) HER-2/neu-specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). CONCLUSION: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.     

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.     

Chemokine C receptor 7 expression and protection of circulating CD8+ T lymphocytes from apoptosis.

Chemokine C receptor 7 (CCR7) expression is important for lymphocyte homing to tissues. We hypothesized that CCR7 also plays a role in CD8(+) T-cell protection from apoptosis. Its expression was determined on circulating T cells in patients with cancer and related to that of molecules responsible for lymphocyte susceptibility/resistance to apoptosis. Peripheral blood mononuclear cells were obtained from 36 patients with squamous cell carcinoma of the head and neck and 16 normal controls. Multicolor flow cytometry was used to evaluate CCR7, Fas, Bax, and Bcl-2 expression in CD8(+) T cells. Annexin V binding to CD8(+)CCR7(+) and CD8(+)CCR7(-) T-cell subsets was compared. Fewer CD8(+)CCR7(+) T cells bound Annexin V than CD8(+)CCR7(-) T cells in normal control and patients (P < 0.0001). CCR7 expression correlated with higher Bcl-2 but lower Bax and Fas expression levels in CD8(+) T cells in both normal control and patients (P < 0.0001). In patients, the CD8(+)CCR7(+) subset was reduced relative to normal control (P = 0.008) and replaced with an excess of apoptosis-sensitive CD8(+)CCR7(-) T cells. To study CCR7 signaling, CD8(+) T cells were stimulated with CCR7 ligands, chemokine C ligands 19 or 21. Ligand binding to CCR7 resulted in phosphorylation of Akt and increased Bcl-2 expression in CD8(+)CCR7(+) T cells, suggesting that CCR7 protects effector T cells from apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. The absence of CCR7 expression on the majority of CD8(+) T cells in the peripheral circulation of patients with squamous cell carcinoma of the head and neck contributes to apoptosis and a rapid turnover of these effector cells.     

Spontaneous apoptosis of CD8+ T lymphocytes in peripheral blood of patients with advanced melanoma.

Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion (P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas+ and tumor necrosis factor receptor 1-positive (TNFR1+) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apoptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-alpha. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-alpha. Three-color flow cytometry revealed that CD3+ Fas+ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher (P = 0.01) in the CD3+ CD8+ T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells, resulting in a decreased immunocompetence.     

Characterization of bone marrow T cells in monoclonal gammopathy of undetermined significance, multiple myeloma, and plasma cell leukemia demonstrates increased infiltration by cytotoxic/Th1 T cells demonstrating a squed TCR-Vbeta repertoire

BACKGROUND: The majority of studies published to date regarding the role of the bone marrow (BM) microenvironment in the pathogenesis of monoclonal gammopathies (MG) have focused on the interaction between stroma cells and plasma cells, whereas information concerning the lymphocytes infiltrating the tumor microenvironment is scanty. METHODS: The authors measured the distribution, TCR-Vbeta repertoire, immunophenotype, and functional characteristics of different subsets of BM T lymphocytes from 61 nontreated patients with MG (30 patients with MG of undetermined significance [MGUS], 27 patients with multiple myeloma [MM], and 4 patients with plasma cell leukemia [PCL]). RESULTS: The authors found a significantly increased rate of BM infiltration by T cells in all patient groups, at the expense of CD4+CD8- and CD4-CD8- T lymphocytes and both CD4+CD28- and CD8+CD28- cytotoxic/effector T cell subsets, and associated with TCR-Vbeta expansions in both CD4+ and CD8+ BM T cells in the majority of patients with MGUS, MM, and PCL. Moreover, the percentage of T cells secreting interferon (IFN)-gamma was found to be increased (P < or = 0.05) both in CD4+ and CD8+ T cells in MGUS and MM patients, and a higher plasma concentration of IFN-gamma was found in patients with MM. It is interesting to note that a positive correlation was noted between the proportion of CD28- and both the percentage of IFN-gamma-secreting cells and the proportion of expanded TCR-Vbeta lymphocytes within the total BM CD4+ T cells. CONCLUSIONS: The results of the current study demonstrated an increased infiltration of BM by T cells associated with frequent TCR-Vbeta expansions and a more prominent cytotoxic/Th1 phenotype in all the patient groups studied.     

Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.     

Spontaneous apoptosis of circulating T lymphocytes in patients with head and neck cancer and its clinical importance.

Spontaneous apoptosis was observed in a proportion of peripheral blood mononuclear cells obtained from patients with head and neck cancer (HNC) but not from normal healthy donors (T. Saito et al., Clin. Cancer Res., 5: 1263-1273, 1999). To further investigate this phenomenon, peripheral blood mononuclear cells were obtained from patients with HNC or normal controls (NCs) and evaluated for expression of apoptosis markers (annexin V binding and caspase-3 activation), T-cell receptor-associated zeta chain, and the death receptor Fas (APO-1, CD95) in CD3(+) T cells by multicolor flow cytometry. Soluble Fas ligand (sFasL) in the sera of these individuals was quantitated by ELISA. In patients with HNC, 74 +/- 15% (mean +/- SD) of CD3(+) T cells were Fas(+) compared with 52 +/- 13% in NCs (P < 0.0001). Furthermore, 29 +/- 16% of the Fas(+) CD3(+) T cells bound annexin V in patients and only 14% +/- 7% of the Fas(+) CD3(+) T cells bound annexin V in NCs (P < 0.0001). In patients, Fas(+) CD3(+) cells preferentially underwent apoptosis and showed a loss of zeta chain expression. Significantly greater proportions of CD8(+) T cells than CD4(+) T cells were apoptotic (P < 0.0002), which indicates that CD8(+) T cells were especially sensitive to apoptosis. Serum levels of sFasL were lower in HNC patients with active disease than in NCs or in patients with no evident disease (P < 0.0183). This suggested utilization of sFasL produced in vivo and activation of the Fas/Fas ligand (FasL) pathway in Fas(+) T cells. Proportions of apoptotic T cells were higher in HNC patients than in NCs (P < 0.0001), and a subset of HNC patients with active disease had the highest proportions of circulating Fas(+) annexin V(+) T lymphocytes. The data indicate that the Fas/FasL pathway is involved in spontaneous apoptosis of circulating Fas(+) T lymphocytes in cancer patients. Fas/FasL interactions might lead to excessive turnover of T cells in the circulation and, consequently, to reduced immune competence in patients with HNC.