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.     

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 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.     

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.     

MHC class I-restricted recognition of a melanoma antigen by a human CD4+ tumor infiltrating lymphocyte

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.     

Human allogeneic melanoma-reactive T-helper lymphocyte clones: functional analysis of lymphocyte-melanoma interactions.

Lymphocyte clones were isolated from CD4+ peripheral-blood lymphocytes (PBL) of melanoma (Me) patient 9923 (HLA-DR7, DQw2, w6), co-cultured for 30 days with autologous accessory cells, allogeneic Me (Me 1811) (HLA-DR7, DQw1, w2), IL-1 beta (2 U/ml) and IL-2 (15 IU/ml). The 55 clones tested displayed a CD3+, CD4+, CD8-, T-cell receptor (TCR) alpha/beta+, gamma/delta- phenotype. Twenty clones were assayed for proliferation in the presence of Me 1811 and B-lymphoblastoid cell line (LCL) 1811, both expressing HLA-class-I and -II (DR7 and DQw2 shared with patient 9923), intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-function-associated antigen-3 (LFA-3) molecules. Eight clones were found to be reactive to Me 1811 but not to LCL 1811. Specificity analysis of these 8 clones revealed that each of them proliferated only to Me 1811, not to other 14 Me and 12 different LCL, suggesting recognition of melanoma-associated antigen (MAA) expressed on the stimulating Me. One clone (103) was analyzed in more detail. A wider specificity analysis showed that it reacted to Me 1811 but not to 10 other Me expressing or not HLA-DR7, 5 normal melanocyte cultures (2 of them typing HLA-DR7-positive when exposed to interferon-gamma--IFN-gamma), 4 tumors other than Me and 20 different LCL. Clones did not show proliferation in the presence of autologous Me cells. Clone proliferation in response to Me 1811 was significantly inhibited by monoclonal antibodies (MAbs) directed to CD3, TCR alpha/beta, TCR beta chain V12, CD4 and HLA-DR. Moreover, following stimulation with Me 1811, clone 103 showed increased surface expression of CD25 (IL-2 receptor) and CD71 (transferrin receptor) and produced significant amounts of IL-2 and IFN-gamma. The supernatant taken from co-culture of clone 103 with Me 1811 augmented the cytotoxicity of PBL 9923 and other allogeneic PBL against K562 and Me 1811. Thus, the lymphocyte clone 103 is a CD4+ Th clone which uses its CD3/TCR alpha/beta complex to recognize an MAA in conjunction with HLA-DR7. Availability of this type of reagent may prove useful to identify and characterize MAA recognized by T lymphocytes.     

Altered T cell repertoire usage in CD4 and CD8 subsets of multiple myeloma patients, a Study of the Eastern Cooperative Oncology Group (E9487)

Previous investigations have demonstrated that an expanding circulating T cell population is able to modulate the malignant clone in multiple myeloma. More recently, an expansion of T cell subsets exhibiting a restricted T cell repertoire has been detected in some MM patients. To further elucidate if a selected T cell expansion occurs in MM, we studied the T cell receptor (TCR) variable (V) region expression from a cohort of previously diagnosed and treated MM patients (N=37). The latter was done by assessing the reactivity of a panel of monoclonal antibodies specific for different V region families (alpha or beta) in combination with anti-CD4 or anti-CD8, for purified blood T cells from MM patients. TCR V region usage in MM patients was compared to blood T cells from age matched (N=13) control individuals. The multivariate analysis of variance did not uncover a difference for distribution of TCR V region usage between the normal controls and the MM cohort. However, there were individual MM patients who had expanded T cells with specific TCR V region expression when compared to the control group. Several MM patients had multiple, expanded CD4 and/or CD8 subsets based on TCR V region expression. The majority of MM patients had expanded T cell subsets that constituted less than 10% of the total blood T cell pool. However, a few MM patients (N=3) had larger percentages (range 34-84%) of these expanded T cell subsets within their blood T (CD3+) cells. The stage of disease and treatment status (currently on or off therapy) did not associate with the pattern of restricted T cell repertoire. Finally, a smaller cohort of newly diagnosed, untreated MM patients (N=13) also demonstrated an expanded T cell repertoire. However, these patients had more CD4 than CD8 cell subsets involved in the altered V region expression in several Vbeta families. Thus, these results add to the evidence that this malignant B cell disorder whether newly diagnosed or of longer duration, may be accompanied by an altered T cell repertoire characterized in part by expanded T cell clones.     

Expression of superantigen-like specificities on murine sjl/j-B lymphomas - antitumor v-Beta-17a+ T-lymphocytes use a diverse set of T-cell receptor v-alpha-chain gene-sequences

Previously we have demonstrated that the tumor infiltrating lymphocytes found in SJL/J mice with reticulum cell sarcomas (RCS) are CD4+ T cells which express T cell receptor (TCR) beta chain molecules comprised principally of Vbeta17a encoded variable gene segments. Herein we report both the cellular and molecular characterization of a panel of twenty RCS-specific T cell hybridomas derived from tumor infiltrating T cells of the mesenteric lymph nodes of an SJL/J mouse bearing the transplanted RCS LA-12 tumor. We determined by flow cytometry that all of the RCS reactive T cell hybridomas expressed TCR containing Vbeta17a encoded variable gene segments. Moreover, most of these T cell hybridomas (17 of 20) co-expressed the CD4 molecule, as did the tumor infiltrating T cells from which they were derived. Using Southern blot analysis of beta chain gene rearrangements. we determined that the predominant Vbeta17a gene usage seen in these hybridomas was not the result of the expansion, either in vivo or in vitro, of a single Vbeta17a+ RCS-reactive T cell clone. The hybridization patterns of hybridoma DNA, as revealed by a Jbeta2 probe, were dissimilar and indicated that multiple unique Vbeta17a+ T cells participate in the RCS response. Furthermore, using Northern blot analysis, we were able to determine that the T cell hybridomas used a varied set of TCR Valpha chain gene segments. The T cell hybridomas, for which the Valpha chain gene segments were determined, used Valpha chains comprised of either Valpha1, Valpha4 or Valpha8 family gene segments. Based on these findings we conclude that Valpha chain usage, if not random, is disparate in response to RCS tumors. Furthermore, these data suggest that the important reactivity for the RCS tumor antigen(s) lies with the beta chain (Vbeta17a) but not with the alpha chain used by the T cell. This pattern of alpha and beta chain utilization is consistent with reported TCR usage patterns exhibited by T cells in response to superantigens - where the beta chain and not the a chain dictates antigenic responsiveness. Therefore, our current findings suggest that the tumor antigen(s) presented by this RCS tumor is similar in nature to that of a superantigen.     

Differential regulation by interleukin-4 and interferon-gamma of an autologous melanoma-specific cytotoxic T-cell clone and the tumor-infiltrating lymphocytes from which it was established

To investigate the specificity of human tumor-infiltrating lymphocytes (TIL) against autologous tumors, TIL from five metastatic melanoma patients were expanded with rIL-2 and assessed for cytotoxicity in chromium release assays. TIL from a patient showing preferential cytotoxicity against autologous melanoma cells were further analysed. TIL were cloned by limiting dilution. Four out of 27 clones showed substantial cytotoxicity against autologous melanoma and one clone, designated as No. 8a-5 (CD3+, CD4-, CD8+, CD56-), selectively killed autologous melanoma but did not kill six different allogeneic melanoma, K562, or autologous or allogeneic Con A lymphoblast targets. Cytotoxicity of No. 8a-5 cells was inhibited by anti-HLA class I MAb (w6/32), by anti-beta 2-microglobulin MAb, and by anti-CD3 (OKT3) MAb, suggesting that the specific cytotoxicity was HLA class I-restricted and that the clone utilized the T-cell receptor complex for recognition of targets. Pretreatment with rIFN-gamma increased the sensitivity of autologous melanoma targets to lysis by No. 8a-5 cells. Exogenous rIL-4 enhanced [3H]TdR incorporation by these TIL. In contrast, rIFN-gamma reduced the sensitivity of the autologous melanoma to lysis by uncloned TIL, and rIL-4 suppressed the cytotoxicity and cell proliferation of uncloned TIL. These results indicate that both specific and non-specific cytotoxic cells can be developed from the same TIL and that these can be differentially regulated.     

Dominant rearrangements among human tumor-infiltrating lymphocytes. Analysis of T-cells derived from 32 patients with melanoma, lung, and renal cell carcinoma.

Dominant rearrangements of T-cell receptor (TCR) beta-chain genes are reported among tumor-infiltrating lymphocytes (TIL). After interleukin-2 expansion of TIL from renal and lung carcinoma and melanoma biopsy tissues, rearrangements of TCR beta-chain genes were analyzed by Southern blotting. Nongermline restriction fragments, indicating dominant rearrangements, were detected among the TIL from all 6 patients with renal cell carcinoma, 17 of 20 patients with melanoma, and 3 of 6 patients with lung tumors. The restriction-fragment sizes of these dominant rearrangements were heterogeneous among the various patients. Rearrangements into C beta 1 were more common than C beta 2 rearrangements. Phenotypic analyses indicated that dominant rearrangements occurred in both CD4 and CD8 predominant TIL populations. The TIL populations that were extracted were expanded to derive large cell numbers suitable for in vivo transfer in an interleukin-2 and TIL immunotherapy program. The data indicated that the cells delivered to these patients usually were characterized by dominant populations of T-cells with selective TCR gene rearrangements. The significance of selective TCR use requires evaluation of the function and specificity of the TIL comprising these dominant populations both in their native in vivo setting and in the context of therapeutic transfer.     

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.     

Human autologous tumor-specific T cells in malignant melanoma

T cell lines and clones with autologous tumor-specific activity have been developed in malignant melanoma by stimulating peripheral blood lymphocytes (PBL), lymph node lymphocytes or tumor-infiltrating lymphocytes (TIL) with autologous melanoma cells in the presence of recombinant interleukin 2 (rIL2). T-cell lines and clones have been developed with specific cytotoxicity and/or proliferative responses for autologous melanoma targets but not for allogeneic melanoma tumor cells, autologous normal cells or natural killer (NK)-sensitive targets. The concentration of rIL2 is critical for the generation of autologous tumor-specific T-cell lines, with low rIL2 concentrations (up to 800 IU/ml) facilitating the growth of T-cell lines with tumor-specific activity. The T-cell receptor (TCR) and the CD3 antigen are involved in specific cytotoxicity and/or proliferative responses of these T-cell lines and clones. An oligoclonal pattern of -chain TCR gene rearrangements was observed on T-cell lines and clones with autologous tumor-specific cytotoxicity, suggesting that they are comprised of T cells that have undergone a clonal expansion in response to particular antigen. Autologous tumor-specific cytotoxic T cells are HLA-restricted and recognize on the melanoma tumor cells HLA Class I or possibly Class II antigens plus a tumor-specific determinant. TIL from patients with metastatic melanoma have unique characteristics in comparison with PBL and lymph node lymphocytes and they appear to contain substantial proportions of T cells that have been locally sensitized to autologous tumor cells. Single stimulation of TIL with autologous tumor cells in the presence of rIL2 is sufficient for the generation of T cell lines with autologous tumor-specific activity, whereas, multiple stimulation of PBL and lymph node lymphocytes was required to achieve the same purpose. TIL-derived T cell lines have been expanded in rIL2 in vitro by at least 1,500-fold without losing their activity. Approximately, 40% of the patients exhibited complete or partial responses to adoptive immunotherapy with melanoma TIL and rIL2.     

Common tcr-Beta chain rearrangements in short-term cultured lymphocytes infiltrating ovarian-tumors

We have recently reported that tumor reactive T cell lines and clones can be developed from lymphocytes infiltrating ovarian malignant tumors and ascites (TIL/TAL). These cells are currently used for immunotherapy of ovarian cancer. In this study we investigated the presence of common non-germline rearrangements at TCR Cbeta1 among freshly isolated and cultured ovarian TIL/TAL. The cultured ovarian TIL/TAL show a characteristic T cell phenotype (>95% CD3+) and mediate tumor target lysis. Our results show that non-germline rearrangements can be detected in cultured ovarian TAL isolated from ascites but not in freshly isolated TAL. Certain of rearranged bands were common among cultured TAL. In contrast, in samples of freshly isolated TIL from solid ovarian tumors localized at different organ sites, common rearrangements were found among fresh and cultured TIL. The results suggest that the observed polyclonality of T cells isolated from freshly collected ascitic tumors may be due, at least in part, to either dilution by passenger lymphocytes, or the lack of an in vivo oligoclonal response to autologous tumor, and indicate the usefulness of molecular analysis of TCR V gene segments in T cells used for immunotherapy.     

Dominant TCRB-V-J chain usage and clonal expansion of sarcoma-reactive CD4+ HLA-DR-restricted T cells suggest a limited set of immunodominant sarcoma antigens

Tumor-reactive CD4⁺ T cells have been isolated from tumor patients, and their specifity but not T-cell receptor (TCR) repertoire has been analyzed. Since we have described CD4⁺ sacoma-reactive T-cell clones, we now sought to determine whether the TCR repertoire of these clones provides information on the spectrum of recognized sarcoma antigens. We analyzed the TCR beta (TCRB) chain repertoire of 19 CD4⁺ HLA-DR-restricted T-cell clones reactive with the autologous sarcoma cell line MZ-MES-1, with HLA-DR-matched tumor cell lines of different tissue origins and B-cell blasts. We identified 7 different clonotypes, which used a limited set of TCRBV and TCRBJ segments. Although the CDR3 of the different clonotypes was diverse, repeated restimulation with sarcoma cells led to a monoclonal expansion of T cells with particular TCR clonotypes in 5/6 mixed lymphocyte tumor cell cultures (MLTC). One clonotype was found in 2 independent MLTC experiments. Sarcoma-reactive T cells were demonstrated in patient tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) by clonotypic PCR. Our results indicate a limited number of immunodominant antigens expressed by the sarcoma cells. The junctional diversity of the TCR clonotypes shows that these antigens did not lead to extensive negative thymic selection as classical autoantigens would have. Therefore, the recognized antigens might represent cryptic autoantigens related to cellular transformation or proliferation. Int. J. Cancer 72:403–407, 1997. © 1997 Wiley-Liss, Inc.     

Radiation leukemia virus-induced T-cell lymphomas with common T-cell receptor variable region structure and similar binding specificity for retrovirus

The 5C2 cell line was derived following culture of mouse spleen cells exposed in vivo and in vitro to radiation leukemia virus (RadLV) containing supernatants from the C6VL/1 T cell lymphoma. This cell line has been found to express an alpha beta T-cell receptor (TCR) identifiable with the Mab124-40 anti-clonotypic antibody which is specific for C6VL/1. It has been shown to be genetically and phenotypically distinct from C6VL/1 with a unique phenotype, i.e. CD4-, CD8-, CD3+, TCR-alpha beta. 5C2 has been shown to express high levels of alpha and beta chain mRNA and to utilize the same or similar V alpha and V beta region genes as C6VL/1. Whereas C6VL/1 binds cross-reactively to both RadLV/C6VL and an unrelated isolate RadLV/VL3, 5C2 has binding specificity for only RadLV/C6VL, which induced its proliferation. The anti-clonotypic antibody Mab124-40 specifically and completely inhibits binding of 5C2 to RadLV/C6VL at concentrations as low as 300 ng/ml. The 5C2 cell line can also be stimulated to increased proliferation by RadLV/C6VL. All of these data are consistent with the role of a TCR alpha beta heterodimer in binding and stimulation by RadLV and satisfy one prediction of the receptor-mediated leukemogenesis hypothesis that T-cell clones identifiable by their T-cell receptor clonotype may be targets for transformation by a particular retrovirus.     

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.     

结直肠癌T细胞克隆TCRβ基因CDR3区氨基酸序列的初步分析

 目的　通过建立结直肠癌肿瘤浸润性淋巴细胞 (tumorinfiltratinglymphocytes ,TIL)和术前外周血 (peripheralbloodlymphocytes ,PBL)中抗肿瘤T细胞克隆的TCRβ基因谱型 ,确定抗肿瘤T细胞克隆的分子特征。方法　采用RT PCR方法扩增结直肠癌TIL和PBL的TCRβ基因的 2 4个不同V家族基因片段 ,经过测序凝胶分离和特殊银染色 ,结合测序结果确定TCRβ基因谱型。 结果　结直肠癌患者具有TIL和 /或PBL中T淋巴细胞的克隆性增殖。有些T细胞克隆表达相同的CDR3区氨基酸基序。CDR3区不同位点具有特征性的疏水性和极性氨基酸以及甘氨酸的分布。结论　通过分析结直肠癌抗肿瘤T细胞克隆特征性的CDR3区氨基酸序列 ,将有益于发现新的抗肿瘤免疫治疗方法。