The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

恶性肿瘤患者外周血中CTL、Ts细胞绝对数值的变化及临床意义分析

观察肿瘤患者外周血中CD8 CD28 (CTL)、CD8 CD28-(Ts)T细胞绝对数值的变化,分析其与肿瘤的相关性。方法:采用流式细胞术测定118例肿瘤患者外周血CTL、Ts细胞数,按能否手术和复发转移分成3组,即A组:手术后组;B组:肿瘤无法切除组;C组:复发转移组。并选择15例健康者作为对照,采用SPSS10．0软件进行各组间比较分析。结果:恶性肿瘤患者外周血中CTL细胞绝对数明显下降,Ts细胞显著升高,与对照组有显著差异(p＜0．05)。A组CTL细胞计数与B、C组有显著性差别,三组的CTL、Ts细胞与对照组有显著差别(p＜0．05),B、C两组之间CTL、Ts细胞均没有明显差别。结论:肿瘤患者外周血CTL细胞低表达、Ts细胞高表达,而且在肿瘤的不同发展阶段,其绝对值也存在显著差异。观察肿瘤患者外周血中CTL、Ts细胞绝对数值的变化,对了解肿瘤患者细胞免疫功能状态有一定的价值,从而为肿瘤的免疫治疗提供参考。     

Retinoic acid elicits cytostatic,cytotoxic and immunomodulatory effects on uveal melanoma cells

The current therapy of uveal melanoma (UM) metastases remains inefficient, which warrants the development of new treatment modalities. For the first time we investigated the effects of retinoic acid (RA) on a panel of UM cell lines and found that RA induces morphological changes compatible with differentiation, suppresses proliferation and causes apoptosis in these cells. RA treatment resulted in an increase of p21, p27 and p53 protein levels and G1 arrest in UM cells, which correlated with significant down-modulation of surface Her2/neu proto-oncogene expression. In addition, RA-treated UM cells exhibited increased sensitivity to both MHC class I-restricted killing by cytotoxic T lymphocytes and NK cell-mediated lysis that were accompanied by more efficient conjugate formation between UM cells and killer lymphocytes. Taken together, our results implicate UM as a new target for treatment with retinoids and suggest that retinoids and T- or NK-cell based immunotherapy can have mutually enhancing effects in UM patients.     

Identification of CTL epitopes in hepatitis C virus by a genome-wide computational scanning and a rational design of peptide vaccine

Developing a peptide-based vaccine for the highly variable hepatitis C virus (HCV) remains a challenging task. Variant viruses not only escape antigen presentation but also persist in a patient as quasi-species. Such variants are often antagonistic to the responding T cell repertoire. To overcome these problems, we herein propose a cocktail vaccine consisting of a few epitope peptides, which make it possible to outpace the emergence of variant viruses. To design such a vaccine, we developed a way to identify HLA-A*2402-binding peptides efficiently by means of the computational scanning of the whole genome of the pathogen. Most of the predicted peptides exhibited strong binding to the HLA-A*2402 molecule, while also inducing CD8 T cell responses from the patients’ peripheral blood mononuclear cells (PBMCs). Peptide-induced T cells were capable of lysing HCV-expressing HepG2 cells which process antigens endogenously. The amount of HCV core antigen in the patients’ livers suggested that the lytic activity of the peptide-induced T cells was clearly in a range suitable for therapeutic use. If T cells were activated under optimal conditions by high density peptides, then they tended to be relatively tolerant of single amino acid variations for cytolysis. Finally, an analysis of the viral population isolated in Japan suggested no obvious changes due to immune evasion in the viral genome even in a host population highly biased toward HLA-A*2402. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

CD8<Superscript>+</Superscript> T lymphocytes isolated from renal cancer patients recognize tumour cells through an HLA- and TCR/CD3-independent pathway

PURPOSE: The aim of this study was to characterize the immune response of patients affected by renal cell carcinoma (RCC). METHODS: Long-term RCC lines were established by retroviral-mediated transfer of the large T-antigen of SV40 into fresh carcinoma cells. Reactive T cell effectors were generated by iterative stimulations of patients' PBMC with autologous tumour cells. RESULTS: This protocol led to the induction of CD8(+) T cell clones reactive against the autologous tumour, but not against NK-sensitive cell lines. However, some of these effectors recognize normal renal cells, allogeneic renal carcinoma cell lines and colon and non-small cell lung carcinomas but not melanomas and lymphoblastoid lines, without evidence of shared classical HLA class I (HLA-I) molecules. Further characterization performed on the CD8(+) TCR alpha/beta(+) clone, CTL30, demonstrated that neither expression of CD1, HLA-Ia nor HLA-Ib, correlated with the T cells' recognition. Moreover, beta2m expression by target cells was not required to achieve interaction of tumour-effector cells. In agreement with this observation, the lytic activity of CTL30 was not inhibited by anti-HLA-I Ab, and antigen expression was not affected by inhibitors of antigen processing. Lytic activity of CTL30, while partially inhibited by anti-NKG2D, could not be abolished by anti-CD3 Abs. Moreover, growth and expansion of CTL30 was sustained only by T cell interaction with antigen-expressing tumour cells; unspecific mitogenic stimuli, such as anti-CD3 and PHA, did not allow T cell expansion. These results demonstrated the existence of an alpha/beta T cell population, recognizing epithelial tumour cells through an HLA-unrestricted, CD3-independent mechanism.     

Contribution of CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis and its animal models

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by multi-focal demyelination, axonal loss, and immune cell infiltration. Numerous immune mediators are detected within MS lesions, including CD4⁺ and CD8⁺ T lymphocytes suggesting that they participate in the related pathogenesis. Although CD4⁺ T lymphocytes are traditionally considered the main actors in MS immunopathology, multiple lines of evidence suggest that CD8⁺ T lymphocytes are also implicated in the pathogenesis. In this review, we outline the recent literature pertaining to the potential roles of CD8⁺ T lymphocytes both in MS and its animal models. The CD8⁺ T lymphocytes detected in MS lesions demonstrate characteristics of activated and clonally expanded cells supporting the notion that these cells actively contribute to the observed injury. Moreover, several experimental in vivo models mediated by CD8⁺ T lymphocytes recapitulate important features of the human disease. Whether the CD8⁺ T cells can induce or aggravate tissue destruction in the CNS needs to be fully explored. Strengthening our understanding of the pathogenic potential of CD8⁺ T cells in MS should provide promising new avenues for the treatment of this disabling inflammatory disease.     

脉络膜黑色素瘤中整合素αvβ3表达的研究

目的：整合素αvβ3整合素家族成员之一,是一类跨膜粘附分子,其在多种肿瘤细胞及新生内皮血管细胞中高表达而在成熟血管内皮、上皮细胞及正常细胞中表达较低或无表达。基于这些特征,整合素αvβ3年来成为分子影像研究的热点之一。鉴于整合素αvβ3人眼脉络膜黑色素瘤中是否存在表达尚不可知,本课题拟研究整合素αvβ3人脉络膜黑色素瘤中表达情况,为以整合素αvβ3基础的分子显像提供理论基础。方法：培养人源脉络膜黑色素瘤细胞株OCM-1,使用蛋白免疫印迹方法检测整合素αvβ3OCM-1中表达水平,并使用免疫组化方法检测人脉络膜黑色素瘤病理切片中整合素αvβ3达分布。结果：蛋白免疫印迹实验表明在OCM-1细胞株中存在整合素αvβ3达,其表达水平介于已知高表达整合素αvβ3头颈鳞癌细胞株HEP-2和较低表达整合素αvβ3头颈鳞癌细胞株CNE-1之间,免疫组化方法检测人脉络膜黑色素瘤病理切片中也存在整合素αvβ3达。结论：人脉络膜黑色素瘤中具有整合素αvβ3达,可以为后期研究基于整合素αvβ3分子显像技术提供依据。     

Therapeutic use of a long-term cytotoxic T cell line recognizing a common tumour-associated antigen: The pattern of in vitro reactivity predicts the in vivo effect on different tumours

Summary A long-term-cultured cytotoxic T lymphocyte (CTL) line (E/88) was obtained from splenic lymphocytes of BALB/c (H-2 ^d) mice bearing the weakly immunogenic colonic carcinoma C26. This line was shown to be /TCR⁺V6⁺CD3⁺CD8⁺CD4^– and to recognize a common tumour-associated antigen on syngeneic carcinomas and sarcomas in a major-histocompatibility—complex-restricted and T-cell-receptor(TCR)-mediated fashion. The assessment of cytotoxic activity on a panel of 30 normal and neoplastic target cells of differing etiology and histotype showed that E/88 CTL lysed syngeneic colon carcinomas and some fibrosarcomas but not leukemias, lymphomas or mammary carcinomas. Clones derived from the E/88 line exhibited the same lytic pattern. Moreover, anti-T3, anti-Lyt2.2, anti-/TCR and anti-V6 mAbs as well as anti-H-2^d antisera abolished cytotoxicity when used in blocking experiments. The therapeutic activity of E/88 CTL upon in vivo transfer was assessed in mice bearing either experimental or spontaneous metastases of C26. In both models therapy with E/88 lymphocytes in combination or not with interleukin-2 was highly effective. Adoptive immunotherapy carried out with two clones obtained from line E/88 showed comparable therapeutic effects. In addition, treatment of syngeneic mice bearing experimental metastases of in vitro E/88-lysable or E/88-resistant tumours, showed that E/88 CTL can eradicate metastases of the former but not of the latter neoplasms. These data indicate that long-term CTL lines recognizing common tumour-associated antigens can be derived from tumourbearing animals and used in adoptive immunotherapy of tumours previously shown to be lysed in vitro by these effectors.     

Melanoma Antigen and Transforming Gene

We summarize our recent studies on the analysis of melanoma antigen and the melanoma gene recently cloned. The melanoma antigen analyzed by syngeneic monoclonal antibodies is composed of a complex of GM3 ganglioside and protein molecules. The epitope recognized by antimelanoma cytotoxic T lymphocytes seemed to be the combinatorial determinants of GM3 and proteins, whereas the epitope for the suppressor T cells was found to be solely GM3 (NeuAc). The genomic DNA controlling the melanoma antigen was recently isolated and was found to possess transforming activity. The structure of this transforming gene is discussed based on the sequence data of the corresponding cDNA clone.     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

Dietary restriction reduces the incidence of 3-methylcholanthrene-induced tumors in mice: Close correlation with its potentiating effect on host T cell functions

Summary All mice treated with 3-methylcholanthrene (MC) suffered with tumor 114 days after treatment. However, 40% dietary restriction caused a great inhibition of tumor incidence. In order to understand the mechanisms by which dietary restriction decreased the occurrence of tumor in mice, we investigated the correlation between tumor incidence and host T cell immune responses. At 114 days after MC administration, the mice were sacrificed and their T cell immune responses were assessed. Flow cytometry studies demonstrated that dietary restriction caused a marked increase of the proportion of Thy1.2⁺, L3T4⁺ T cells in MC-treated diet-restricted mice. Consistent with this result, T cell responses against concanavalin A and interleukin-2 were also potentiated in spleen cells obtained from MC-treated diet-restricted mice, while spleen cells obtained from MC-treated unrestricted mice showed decreased T cell responses because of their tumor burden. Such potentiation of T cell functions by dietary restriction was also observed at earlier stages of MC-induced tumorigenesis. During the course of carcinogenesis, spleen cells obtained from diet-restricted mice showed decreased natural killer activity in vivo. However, in vitro induction of cytotoxic T cells was markedly augmented in MC-treated diet-restricted mice compared with unrestricted mice. These results strongly suggest that the increase of host T cell immune responses might be one of the major causes for the reduction of tumor occurrence by dietary restriction.     

Cytotoxic T cell lines recognize autologous and allogeneic melanomas with shared or cross-reactive HLA-A

Summary Cytotoxic T lymphocytes (CTL), CD3⁺, / T-cell-receptor-positive, are important effector cells with specific immunity in melanoma patients. The establishment and expansion in vitro of CTL of a specific phenotype to tumor cells strongly depends on the method of activation and sensitization with tumor cells. We generated CD3⁺ CTL lines to melanoma by co-culturing peripheral blood lymphocytes with autologous irradiated melanoma cells and repetitive stimulation with high-dose interleukin-4 in a cocktail culture medium. CTL lines were investigated for their specificity to kill autologous and allogeneic melanoma. Histocompatibility locus antigen (HLA) class I (A, B) molecules are important restrictive recognition antigens for CTL. Although these antigens are highly polymorphic, they can share a similar immunogenic molecular epitope(s) and can be immunologically cross-reactive. The CTL lines generated were found to kill not only autologous melanoma, but also allogeneic melanomas having class I HLA-A antigens shared or cross-reactive with autologous HLA-A. These CTL lines were poor killers of melanomas bearing non-shared or non-cross-reactive HLA-A. Cold-target inhibition assays demonstrated this CTL cross-reactivity to allogeneic melanoma specificity. Epstein-Barr-virus-transformed autologous and allogeneic B lymphoblastoid cell lines failed to block autologous melanoma killing, indicating that CTL were not recognizing major histocompatibility complex antigens, serum proteins or culture medium products as the primary target antigen. HLA-A2 was the major shared HLA-A antigen recognized by CTL lines on the melanoma lines studied. CTL lines also recognized shared HLA-A11 and A24 on allogeneic melanoma. There were no CTL lines showing restriction to HLA-B. These results suggest that common tumor-associated antigens are present on melanomas and are recognized in association with distinct HLA-A epitopes by CTL.This study was supported by grant CA12 582 awarded by the National Cancer Institute, USA     

Restriction fragment analysis of the HLA Class II region: Application to tissue transplantation

Southern blotting and hybridization with locus specific probes is a reliable method for the determination of HLA Class II specificities and in conjunction with several other techniques is providing valuable information on the nature of Class II polymorphisms in man. In terms of the identification of Class II specificities in transplantation however, the advanced nature of DR serology means that DR restriction fragment analysis is probably limited only to those situations where serological methods have been unsuccessful.Whether DQ and DP incompatibilities can evoke rejection responsesin vivo remains to be fully ascertained although our preliminary data suggest that DQ mismatches probably have little effect upon graft outcome. Restriction fragment analysis is particularly attractive in that in enables reliable retrospective studies to be performed in conjunction with prolonged clinical follow up, and the technique will clearly continue to contribute significantly to our understanding of the importance of these molecules in transplantation.