The T-cell receptor V beta gene usage in tumor-infiltrating lymphocytes and blood of patients with hepatocellular carcinoma.

To determine a possibly restricted T-cell receptor (TCR) repertoire in tumor-infiltrating lymphocytes (TIL) in response to tumor-associated antigens in patients with hepatocellular carcinoma (HCC), freshly isolated TIL (n = 5) and peripheral blood lymphocytes (PBL; n = 6; 3 paired with TIL) were studied for expression of TCR variable (V) beta regions. RNA purified from TIL or PBL was reverse-transcribed into complementary DNA. This complementary DNA was amplified by quantitative polymerase chain reaction with 22 primers specific for 20 TCR V beta gene families and a 3' constant (C) beta primer. As a reference for later quantitation, a fragment of TCR C alpha was coamplified with each V beta region. Using 32P-labeled 3' primers, the percentage of total V beta expression was calculated by measuring the cpm of each of the amplified products. In contrast to PBL of 6 control, healthy individuals, whose range of expression of each TCR V beta gene varied from 0 to 13%, the expression of some V beta genes in HCC TIL was as high as 33%, indicating a restricted TCR V beta usage in HCC TIL. When polymerase chain reaction-amplified complementary DNAs of the V beta 1 or V beta 3 genes obtained from two TIL preparations were cloned and sequenced, the same rearrangements were found in the majority of DNA clones. The particular V beta genes that were over- or underrepresented in TIL varied among the patients. In 3 of 6 PBL and 3 of 5 TIL, the V beta 3 gene was expressed with a relatively high frequency. The V beta 4 gene expression was consistently low in patients' TIL or PBL. In 3 paired PBL and TIL, V beta expression was similar. In 5 of 6 cases, HCC PBL had different TCR V beta frequencies from those seen in normal PBL. This analysis of TCR V beta usage in freshly isolated TIL and in PBL indicated that T-lymphocytes in patients with HCC might have restricted immunological reactivity and that V beta 3-restricted TIL might represent antitumor effector cells.     

T-cell distribution and adhesion receptor expression in metastatic melanoma.

PURPOSE: Metastatic malignant melanoma is a devastating disease with a poor prognosis. Recent therapeutic trials have focused on immunotherapy to induce development of endogenous antitumor immune responses. To date, such protocols have shown success in activation of tumor-specific CTL but no overall improvement in survival. To kill tumor, antigen-specific CTL must efficiently target and enter tumor tissue. The purpose of this study was to examine the pathway of leukocyte migration to metastatic melanoma. EXPERIMENTAL DESIGN: Peripheral blood and metastatic melanoma tissues (n = 65) were evaluated for expression of adhesion molecules using immunohistochemistry of tumor sections and flow cytometry of tumor-associated and peripheral blood CTL and compared with healthy controls. CTL expressing T-cell receptors for the melanoma antigen MART-1 were identified in a subset of samples by reactivity with HLA-A2 tetramers loaded with MART-1 peptide. RESULTS: Results show that the majority of metastatic melanoma samples examined do not express the vascular adhesion receptors E-selectin (CD62E), P-selectin (CD62P), and intercellular adhesion molecule-1 (CD54) on vessels within the tumor boundaries. Strong adhesion receptor expression was noted on vessels within adjacent tissue. Tumor-associated T lymphocytes accumulate preferentially in these adjacent areas and are not enriched for skin- or lymph node-homing receptor phenotype. CONCLUSION: Expression of leukocyte homing receptors is dysregulated on the vasculature of metastatic melanoma. This results in a block to recruitment of activated tumor-specific CTL to melanoma metastases and is a likely factor limiting the effectiveness of current immunotherapy protocols.     

T-cell activation marker expression on tumor-infiltrating lymphocytes as prognostic factor in cutaneous malignant melanoma.

The central role of T cells in antitumor immunity is well established. However, tumor progression, often seen in the presence of substantial lymphocytic infiltration, suggests that these T cells are not capable of mounting an effective immune response to control tumor growth. Evidence has accumulated that T lymphocytes infiltrating human neoplasms are functionally defective, incompletely activated, or anergic. Therefore, when characterizing the immune competent cells within lymphoid infiltrates of tumors, it is important to assess their activation state. We investigated the expression of two T-cell activation markers, interleukin 2 receptor alpha (CD25) and OX40 (CD134), by immunohistochemistry in primary cutaneous melanoma samples of 76 patients and analyzed it in relation to tumor stage and tumor progression (>5 years follow-up), as well as to patients' survival. We found that the degree of infiltration by CD25(+) and intratumoral OX40(+) lymphocytes showed a tendency to decrease in thicker melanomas. The frequency of samples with high numbers of peritumoral CD25(+) and OX40(+) cells was significantly lower (P = 0.0009 and P = 0.0087, respectively) in melanomas developing distant visceral metastases, compared with nonmetastatic or lymph node metastatic tumors. For both activation markers studied, high peritumoral densities were associated with longer survival by univariate analysis (P = 0.0028 and P = 0.0255 for CD25 and OX40, respectively), whereas peritumoral OX40(+) lymphocyte infiltration had an impact on survival also in multivariate analysis (P = 0.035). The results suggest that the presence of lymphocytes expressing the T-cell activation markers CD25 or OX40 shows correlation with tumor progression as well as with patients' survival in cutaneous malignant melanoma.     

Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook

Immunotherapy using autologous T cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs), T cells transduced with high-affinity T cell receptors against major tumor antigens, and T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin light chains with endodomains of T-cell signaling molecules. Among these and other options for T-cell therapy, TILs together with high-dose interleukin 2 have had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined and undefined tumors antigens against all possible major histocompatibility complex, rather than the single specificity and limited major histocompatibility complex coverage of the newer T cell receptors and chimeric antigen receptor transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL-mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trial is needed in an attempt to gain regulatory approval of TILs as standard of care. Second, improvements in how we expand TILs for therapy are needed that minimize the time the T cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers to better select suitable patients for TIL therapy to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TILs will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TILs with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation should further increase therapeutic efficacy and durable complete response rates.     

T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer

Tumor-infiltrating lymphocytes (TIL) are often found in malignant breast tumors, and have been claimed to be of prognostic value. It has been proposed that TIL may represent an enriched population of tumor-specific cytotoxic lymphocytes, reacting with antigenic determinants on the tumor cell surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells. The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between phenotypes and expression of any particular V segments were found. Neither was there any correlation between the expression of specific V alpha/V beta segments and cytotoxicity against allogeneic tumor cells.     

T-cell receptor gene expression in tumour-infiltrating lymphocytes and peripheral blood lymphocytes of patients with nasopharyngeal carcinoma.

The T-cell receptor (TCR) repertoire expression of tumour-infiltrating lymphocytes (TILs) from 19 nasopharyngeal carcinoma (NPC) biopsies was compared with those of lymphocytes from 18 control nasopharyngeal biopsies. mRNA was extracted from these lymphocytes and the cDNA transcribed. A panel of 18 V alpha- and 21 V beta-specific primers was used to detect the TCR gene use from cDNA. The use of V alpha and V beta genes was restricted in TILs compared with lymphocytes from biopsies. The frequencies of V alpha 2, V alpha 3, V alpha 9, V alpha 10, V alpha 11, V alpha 13, V alpha 14, V alpha 15, V beta 11, V beta 15 and V beta 20 were decreased and the frequencies of V alpha 10 [Pc = 0.04; relative risk (RR) = 0.05], V alpha 11 (Pc = 0.02; RR = 0.07), V alpha 13 (Pc = 0.002; RR = 0), V alpha 14 (Pc = 0.04; RR = 0.05), V beta 14 (Pc = 0.001; RR = 0.03) and V beta 20 (Pc = 0.001; RR = 0.03) remained significantly reduced after correction for the number of families typed. The frequency of V alpha 17 was higher in NPC biopsies than in NPC PBLs (P = 0.05), and the frequency of V beta 15 was lower in NPC biopsies than in NPC PBLs (P = 0.02). The frequencies of V alpha 17 and V alpha 18 in HLA-B46+ patients were significantly lower (P = 0.009; P = 0.044) than in B46+ controls. The results suggest that the restriction of TCR gene use in NPC patients may be important in NPC pathogenesis.     

Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

Usage of T-cell receptor Vβ chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable β regions (Vβ). To perform the TCR-Vβ analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5′ primers specifically recognizing the sequences of 20 Vβ gene families and a 3′ primer annealing to the constant region of the β chain. The TCR-α constant region (Cα) gene was co-amplified as a standard for the calculation of the percentage of each TCR-Vβ gene expressed. The frequency of individual Vβ regions expressed on TIL was computed from the ratio of cpm Vβ to cpm Ca for each Vβ region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vβ genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of Vβ-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the Vβ-genes usually expressed in normal PBL were not expressed in fresh TIL. In melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of Vβ genes occurred. Although in 4/5 TIL cultures this selection involved the Vβ7 gene, no relationship could be established between Vβ gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3⁺ CD8⁺ T-cell lines with Vβ-gene expression restricted to I or 2 Vβ families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.     

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes

1. Download : Download high-res image (269KB)
2. Download : Download full-size image

     

NKG2D-mediated antitumor activity by tumor-infiltrating lymphocytes and antigen-specific T-cell clones isolated from melanoma patients.

PURPOSE: The role of NKG2D receptor in antitumor immunosurveillance has not been completely clarified. We addressed this issue by investigating the involvement of this receptor in tumor-specific immunologic response in melanoma patients. EXPERIMENTAL DESIGN: We determined the presence of NKG2D+ T cells among tumor-infiltrating lymphocytes (TIL) of 10 (one primary and 9 metastatic) melanoma samples and the expression of NKG2D ligands (NKG2DL) by these tumor cells. Moreover, the expression of NKG2D was assessed in a panel of antigen-specific T lymphocytes isolated from melanoma patients and the engagement of NKG2D in antitumor activity mediated by these T cells was determined. RESULTS: TILs located either in the periphery or within the tumor mass of melanoma samples expressed NKG2D and the expression of this receptor by T cells was retained after in vitro culture. However, NKG2DLs were weakly expressed, or not expressed, by most metastatic lesions with only the primary tumor being positive for all these molecules. In contrast, these ligands were expressed, although heterogeneously, by all in vitro established melanoma lines. Moreover, the engagement of NKG2D occurred in antitumor activity by both freshly isolated and in vitro cultured TILs. However, this receptor was involved to a different extent in the antitumor activity of antigen-specific T-cell clones. CONCLUSIONS: These findings indicate that NKG2D+ T cells have a role in the immunologic response against tumor. Thus, new immunotherapeutic treatments for melanoma patients should be designed aimed at augmenting the NKG2D+ T lymphocyte-mediated immune response.     

Proliferative and cytolytic potentials of purified human tumor-infiltrating T lymphocytes. Impaired response to mitogen-driven stimulation despite T-cell receptor expression

Using limiting dilution analysis (LDA) we have previously shown that in most instances, the frequency (F) of proliferative T lymphocyte precursors (PTL-P) was strikingly reduced in tumor-infiltrating lymphocytes (TIL). In this study involving 19 cases, we show that the impaired clonogenic potential of CD2+ TILs is primarily caused by an intrinsic defect rather than to suppressor T cells or to a direct effect of the tumor cells usually present in the culture system. This was demonstrated by experiments in which the F of PTL-Ps was quantitated both in highly purified CD2+ TILs (using a cell-sorter) and in non-purified TIL suspensions (still containing tumor cells), which originated from the same biopsy specimen. The F of PTL-Ps was virtually identical in either sorted or nonsorted suspensions and the data from LDA were always consistent with the single-hit Poisson model, indicating that no suppressor cells interfered with growth of CD2+ TIL. Stimulation of sorted CD2+ TIL in low-density cultures by either phytohemagglutinin or anti-CD3-monoclonal antibody (MAb) indicated that the antigen-dependent activation pathway was impaired, although structurally intact T-cell receptor (TCR) complexes were apparently expressed, as assessed by immunofluorescence. The depressed proliferative response of CD2+ TIL could not be reversed in vitro when phorbol-esters were used in combination with ionomycin, which bypass the TCR. Nevertheless, 180 clones obtained from 8 cases were analyzed for their cytolytic activity. The majority mediated specific lytic activity (against unknown antigens), as assessed by lectin-dependent cell cytotoxicity, whereas only 6% of them manifested lymphokine-activated killing on appropriate targets.     

Characterization of T-cell memory phenotype after in vitro expansion of tumor-infiltrating lymphocytes from melanoma patients

Memory T-cell populations in human antitumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not been fully characterized. Our studies demonstrated that CD62L, CD27 and CD28 positive effector memory T-cells were present in the TIL samples from the tumor tissues of melanoma patients and T-cell expansion led to the significant loss of memory T-cells. CD27- and CD28-positive T-cells had high levels of CD44 expression. T-Cell expansion resulted in significant down-regulation of CD44 expression. Interleukin-2 (IL-2) and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8(+) T-cells during expansion. Furthermore, CD44 down-regulation using small interfering RNA (siRNA) on TILs dramatically reduced interferon-gamma and IL-2 release upon tumor stimulation. These results suggest that the regulation of CD44 expression in TILs may play an important role in memory T-cell maintenance and antitumor immune response.     

T-cell receptor v-Beta usage of tumor-infiltrating lymphocyte lines cloned from human breast-tumor and melanoma

A total of forty-one tumor infiltrating T cell lines (TIL) were cloned, in the presence of interleukin-2, from nine breast tumor and five melanoma specimens with limiting dilution in a microculture system. Nineteen (46%) of the lines/clones reacted to autologous tumor targets. The T cell receptor (TcR) V beta gene usage was analyzed using polymerase chain reaction (PCR) technique and a set of oligonucleotide primers specific for 20 V beta families. T cell lines generated from paired peripheral blood lymphocytes (PBL) under similar condition were used as control. Our data revealed a limited heterogeneity in TcR V beta gene usage with a biased expression of V beta 6 in both breast tumor- and melanoma-derived TIL lines/clones. In contrast, a random pattern of TcR V beta usage was observed in 27 control T cell lines derived from PBL of patients with breast cancer and melanoma. The results lend support to oligoclonal expansion of TIL at tumor sites but fail to directly correlate the preferential expression of V beta 6 with the functional property of the TIL in recognition of tumor antigens.     

Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma

Human autologous tumor-specific T-helper 2 (Th2) cells were investigated in melanoma tumor-infiltrating lymphocytes (TILs). Both a CD4⁺ T-cell line and its 5 potential T-cell clones established from TILs of a patient with metastatic melanoma produced significant levels of IL-4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to autologous, but not any of 12 allogeneic, melanoma cell lines. They also produced IL-3 and IL-8 but not IL-2, IFN-γ, TNF-α or TNF-β in response to autologous tumor cells. Furthermore, they showed autologous melanoma-specific cytotoxicity only in an 18-hr ⁵¹Cr-release assay. Specific IL-4, IL-6 or IL-10 production by the CD4⁺ M73 T-cell line and its clone was inhibited by anti-class 11 DR (but not anti-class 1) MAb, whereas their specific cytotoxicity was inhibited by anti-class 1 (but not anti-class 11) MAb. Anti-CD3 and -CD4 MAb (but not anti-CD8) abrogated both IL-4, IL6 and IL-10 production and cytotoxicity, while anti-IL-4 antibody did not inhibit cytotoxicity. CD4⁺ potential T-cell clones, but not CD8⁺ clones, that were established from freshly isolated TILs without in vitro sensitization by autologous tumor cells also produced IL-4, IL-6 and IL-10 but not IFN-γ or tumor necrosis factor (TNF)α in an autologous tumor-specific fashion. These Th2 cells were neither reactive to EBV-B cells nor suppressive against CD8⁺ T-cell clones. PMA and PHA stimulated these potential T-cell clones, regardless of their specific lymphokine production, to produce IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFα and IFN-γ. Our results demonstrate the presence of autologous tumor-specific Th2 cells at the melanoma sites.     

Prognostic value of beta1 integrin expression in metastatic melanoma

The expression of integrin-type cell adhesion receptors is frequently changed in malignant transformation. Despite their important role in cancer cell behaviour, the value of integrins as prognostic markers is mostly unknown. We have examined the expression of beta1 integrins in 38 metastatic melanomas obtained from 27 patients treated with combined chemoimmunotherapy. On the basis of beta1 integrin expression, the melanoma samples were divided into two groups: beta1-negative tumours (<10% beta1 integrin immunostained cells) and beta1-positive tumours (with > or = 10% positive cells). Patients with beta1-positive tumours (n = 15) had significantly longer disease-free survival (median 38 versus 7 months, P < 0.0001) and overall survival (median 70 versus 23 months, P = 0.0001) evaluated after the diagnosis of primary disease compared with patients with beta1-negative metastases (n = 11). Moreover, the survival of the patients with beta1-positive tumours after the initiation of chemoimmunotherapy was significantly prolonged (median 18 versus 9 months, P = 0.017). The independent nature of beta1 integrin expression as a significant prognostic factor for survival after therapy was confirmed using Cox's multivariate analysis (P = 0.014). Our results indicate that the expression of beta1 integrins might have some major tumour growth regulatory role and can be used as a predictor for prognosis in patients with metastatic melanoma.     

Selective cytokine gene expression in renal cell carcinoma tumor cells and tumor-infiltrating lymphocytes

The progression of tumors such as renal cell carcinoma (RCC), despite the presence of substantial lymphocytic infiltrates (TIL), suggests that the ability of the local immune response to control tumor growth is impaired. Cytokine gene expression was examined to further investigate the nature of this response. Initial studies were performed with frozen tumors using PCR-assisted mRNA amplification with cytokine-specific primers for interleukin 10 (IL-10), interleukin 2 (IL-2) and interferon-gamma (IFN-γ). IL-2 mRNA was not detected, despite the presence of T cells as defined by the expression of CD3γ mRNA. In contrast, mRNA for IFN-γ was expressed in 4/9 and for IL-10 in 5/9 tumors. To confirm this, 5 fresh tumor specimens were examined, and PCR demonstrated that IL-10 mRNA was detectable in 4/5 tumors from which RNA was isolated at the time of nephrectomy. In these experiments multiple cycles and dilutions were employed to semi-quantitate the expression of IL-10. To identify potential sources of this cytokine in the tumor bed, IL-10 mRNA expression in freshly isolated lymphocytes and tumor cells, TIL lines, cultured RCC and established RCC lines was examined. Our studies demonstrate that within the tumor TIL may be one source of IL-10. Lymphocyte-enriched populations from 4/5 tumors expressed IL-10 mRNA as did 4/6 freshly isolated tumor cell preparations. IL-10 gene expression was not detected, however, in tumor cells after one passage in vitro in short-term cultured RCC tumor cells (passages 2–5) or in established RCC tumor cell lines. Finally, 4/9 CD4⁺ and 2/5 CD8⁺ TIL lines expressed IL-10 mRNA either constitutively or after stimulation with anti-CD3 antibody. This finding was associated with IL-10 production in vitro. Our studies demonstrate that IL-10 mRNA is frequently present in RCC tumors and may originate from the tumor-infiltrating mononuclear cell population. © 1995 Wiley-Liss, Inc.     

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.