Membrane structures involved in auto-tumor recognition

Tumor patients' blood lymphocytes have the capacity to recognize autologous tumor cells in vitro. A consequence of this recognition is the proliferation of small-size, high-density, resting T cells. Both helper (CD4+) and cytotoxic/suppressor (CD8+) T lymphocytes proliferate in the mixed lymphocyte-tumor cell cultures. In contrast to the autologous mixed lymphocyte cultures, both the auto-erythrocyte rosetting and non-rosetting (AE+ and AE-) T cells participate in the auto-tumor response. In contrast to stimulation by virus-infected or hapten-modified cells, DR antigen expression is not essential for stimulation by autologous tumor cells. In a proportion of cancer patients, blood lymphocytes have the capacity to lyse the patients' own tumor cells in vitro. There are two populations of lymphocytes with auto-tumor cytotoxic function. The first is characterized by low buoyant density and by non-adaptive cytotoxicity. In contrast to the recognition of hapten-modified or virus-infected target cells by the CTL, recognition of autologous tumor cells by the cytotoxic LD cells occurs even when the MHC class I antigens are blocked by mAb. The CD3 complex is also not involved in LD-mediated lysis. The other population with auto-tumor cytotoxic function comprises high-density, resting T cells. Recognition of autologous tumor cells by cytotoxic HD lymphocytes shares the characteristics of CTLs, i.e., their function is abrogated by pretreatment of the effectors with mAbs directed to the T3 receptor complex and by preincubation of the targets with mAb to the MHC class I antigens. Cytotoxicity of HD cells is restricted to the autologous tumor cells. This selectivity and the characteristics shared with CTL suggest that the auto-tumor reactivity of HD lymphocytes reflects an immune response against the autologous tumor.     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Auto-tumor recognition following in vitro induction of MHC antigen expression on solid human tumors: stimulation of lymphocytes and generation of cytotoxicity against the original MHC-antigen-negative tumor cells

Summary Expression of major histocompatibility complex (MHC) class I antigens was induced in eight out of nine freshly prepared tumor cell suspensions by exposure to interferon (IFN) and tumor necrosis factor (TNF) in vitro. The untreated, class-I-antigen-negative, and the treated, antigen-positive, cells of three tumors (one breast carcinoma, one plasmocytoma and one ovarian carcinoma) were compared for the capacity to stimulate autologous and allogeneic blood lymphocytes, to generate auto-tumor cytotoxicity and for sensitivity to the lytic effect induced in autologous mixed lymphocyte tumor cell culture (MLTC). The MHC class I-negative cells did not stimulate, while the cells induced for expression of antigens did. On the other hand, when the autologous cytotoxic cells were generated in the MLTC by the class I antigen-positive tumor cells the class I-negative tumor cells were also damaged. Lysis of the class-I-positive tumor cells was abrogated by the W6/32 monoclonal antibody directed against the monomorphic part of the class I molecules.     

Analysis of cellular immune response to EBV by using cloned T cell lines

Eight cloned T cell lines specific for Epstein Barr virus-transformed B lymphocytes were derived. In the presence of the autologous virus-infected B cells, the T cell lines show HLA-restricted cytotoxic activity and also secrete alpha-interferon in sufficient amounts to inhibit infection and transformation. Four of these clones showed restriction to a single HLA locus (two for A3, and two for B7) and three showed exquisite self-restriction lysing only autologous targets. These seven clones expressed the classical cell surface phenotype of cytotoxic T cells being T3, 8, 11, and la-positive and T4-negative. An eighth clone that lacked the T8 surface marker appeared to recognize both B7 and BW51. HLA restriction was confirmed: 1) by the ability of a monoclonal antibody against an HLA-A,B,C framework antigen (W6-32) to block the cytotoxicity; 2) the failure of the clones to lyse Daudi, an EBV-positive, HLA-A,B, C-negative cell line; and 3) successful competition of the cytotoxicity by autologous but not allogeneic cold targets. The cloned T cells do not kill EBV-negative targets such as autologous pokeweed mitogen blasts and cell lines including CEM and the natural killer cell target K562. The results suggest T cell clones may be generated against an EBV-associated membrane antigen on transformed B cells, perhaps equivalent to the lymphocyte-determined membrane antigen, and that the recognition is restricted by a single HLA determinant. We propose that single T cells can play multiple roles in controlling EBV infection in vitro and in vivo including the elimination of transformed cells by cytotoxicity and the prevention by secreted interferon of further re-infection and transformation.     

V gamma 9V delta 2 T cell response to colon carcinoma cells

During analysis of CD8 T cells derived from ascites of a colon cancer patient, we isolated a Vgamma9Vdelta2 T cell clone showing strong reactivity against autologous tumor cell lines. This clone killed a large fraction of allogeneic colon carcinoma and melanoma cell lines, but did not affect a normal colon cell line, colon fibroblasts, or melanocytes. Tumor cell recognition was TCR and NKG2D dependent and induced TNF-alpha and IFN-gamma secretion by the clone; accordingly, tumor targets expressed several NKG2D ligands, such as MHC class I chain-related gene A and UL16-binding protein molecules. Colon tumor recognition by Vgamma9Vdelta2 T cells was highly dependent on isopentenyl pyrophosphate production and ICAM-1 expression by target cells. Finally, similar reactivity patterns against colon carcinoma cell lines were observed using polyclonal Vgamma9Vdelta2 T cells of various origins, and Vgamma9Vdelta2 lymphocytes were present in the majority of colon tumor samples studied. Together, these results suggest that Vgamma9Vdelta2 T cells contribute to the natural immune surveillance against colon cancers. Therefore, this study provides a strong rationale for the use of Vgamma9Vdelta2 T cell agonists in immunotherapies targeting colon tumors.     

Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.     

Staphylococcal enterotoxins direct and trigger CTL killing of autologous HLA-DR+ mononuclear leukocytes and freshly prepared leukemia cells

Attempts have been made to induce cytolytic T cells to kill target cells that do not express the appropriate target molecules by crosslinking the T cells and the target cells in various ways. One successful strategy has been to use heteroconjugates or bispecific monoclonal antibodies reacting with T cell molecules with activating properties (e.g., mab directed to CD3/TCR) and target cell surface antigens. In this report we show that Staphylococcal enterotoxins (SE) direct human T lymphocytes to execute cytotoxicity toward MHC class II-expressing Raji cells, but not against MHC class II-deficient Raji mutant RJ 2.2.5. Both HLA-DR+ and HLA-DR- effector T lymphocytes are effective in the killing of Raji cells coated with SE. The Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity (SDCC) is a rapid T lymphocyte-mediated cytolytic mechanism killing the targets within an hour of incubation. HLA-DR+ target cells are sensitized to be killed within minutes of incubation with picomolar concentrations of SE. SE-sensitized Raji cells remain targets for SDCC after overnight culture at 37 degrees C, demonstrating that the sensitive state is relatively stable. SEA- and SEB-selective cytolytic T cell lines were established to illustrate the clonal variability of SDCC effectors with respect to SE specificity. We also demonstrate that autologous monocytes and activated T lymphocytes as well as B lymphocytes and freshly prepared HLA-DR+ leukemic cells are excellent targets in SDCC.     

Antitumor activity in vitro in chronic myelogenous leukaemia revealed after treating peripheral cells with cytosine arabinoside

Summary Cytosine arabinoside (Ara-C) treatment of peripheral blood mononuclear cells from 12/12 chronicphase chronic myelogenous leukaemia (CML) patients revealed a proliferative response stimulated by their untreated leukaemic cells. Specific recognition of tumour cells by patients' normal lymphocytes was suggested by the finding that cells of siblings genotypically identical for human leukocyte antigen caused no stimulation. Lymphocytes thus stimulated by tumour cells from one of these patients were cloned by limiting dilution and tested for antileukaemic effects in cytotoxicity and proliferation assays. Cytotoxic lines were isolated that killed autologous CML targets but only a limited number of allogeneic fresh leukaemias or cell lines. These results show that anti-leukaemia effectors can be isolated from chronic-phase CML patients and suggest their potential application in adoptive immunotherapy.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 120) Abbreviations used: ANLL, acute non-lymphocytic leukaemia; Ara-C, cytosine arabinoside; CML, chronic myelogenous leukaemia; IL, interleukin; LAK, lymphokine-activated killer; NK, natural killer; PBMC, peripheral blood mononuclear cells; HLA, human leukocyte antigen     

Expression of the adhesion molecule ICAM-1 and major histocompatibility complex class I antigens on human tumor cells is required for their interaction with autologous lymphocytes in vitro

Summary In a group of 30 human tumors, comprising 12 lung, 14 ovarian, 2 breast carcinomas, 1 hypernephroma and 1 mid-gut carcinoid, the expression of major histocompatibility complex (MHC) class I molecules and the intercellular adhesion molecule 1 (ICAM-1, CD54) was found to vary independently. Some tumors expressed both or neither of these molecules. Among 9/13 ICAM-1⁺ tumors, in which >50% cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) (LB-2), the class I antigen was also detected on >50% of the cells. Only 2 ICAM-1⁺ tumors were class-I^–. In 5/17 cases the tumors were MHC-class-I⁺ and ICAM-1^–. Lymphocytes collected from the blood or from the tumor site were assayed for recognition on the tumor cells in the auto-tumor cytotoxicity test and in mixed lymphocyte tumor cell culture (MLTC). Positive results were obtained only with the MHC-class-I⁺/ICAM-1⁺ tumors. In vitro treatment of the tumor cell suspensions with interferon and tumor necrosis factor (TNF) induced or enhanced the ICAM-1 and/or class I antigen expression in 8/12 cases. Of the tumor samples treated, 8/9 aquired stimulatory capacity and 3/10 became susceptible to lysis by the lymphocytes. In 6/6 MLTC performed with the cytokine-treated tumor cells, cytotoxicity against the autologous tumor was generated. Three of these MLTC lymphocytes also lysed the untreated targets. mAb directed to class I antigens or to ICAM-1 inhibited both the stimulation by and the lysis of tumor cells when confronted with fresh lymphocytes. The cytotoxicity generated in the MLTC was also inhibited. If, however, the cytotoxic function was induced in MLTC containing interleukin-2 (5 U/ml), inhibition was obtained only by pretreatment of the targets with mAb against ICAM-1. The results show thus (a) that the lymphocytes react in vitro with tumor cells only if these express both MHC class I molecules and ICAM-1; (b) that expression of these molecules can be induced by interferon and TNF; (c) that cytotoxic effectors generated in the MLTC with cytokine-treated tumors can also act on the untreated tumor cells. The requirement of the two surface moieties for the interaction with lymphocytes was also substantiated by blockade with relevant mAb.     

Human cytotoxic T lymphocytes (CTL) against Epstein-Barr virus (EBV) infected cells: EBV specificity and involvement of major histocompatibility complex determinants in the lysis exerted by anti-EBV CTL toward HLA-compatible and allogeneic target cells

Human peripheral blood lymphocytes (PBL), from anti-Epstein-Barr virus (EBV)-seropositive donors, were stimulated by EBV and were shown to be cytotoxic toward autologous, HLA-compatible, and fully allogeneic EBV-transformed target cells. The lysis was not due to natural killer (NK) cells since the target cells used were resistant to lysis by fresh PBL and by virus-stimulated PBL-depleted of AET-SRBC-rosetting T cells (the latter being still fully cytotoxic on K562 NK-susceptible target cells). Conversely only E-rosette-purified (T) lymphocytes killed EBV-transformed HLA-compatible and allogeneic target cells. Moreover, anti-MHC antibodies inhibited the cytotoxicity exerted by EBV-induced cytotoxic T lymphocytes (CTL) on both autologous and allogeneic target cells. Finally the lysis was EBV specific since PHA blasts were not killed and since only EBV-transformed cells could compete for lysis with the EBV-positive target cells. Efficient competition was achieved by EBV-transformed cells autologous or allogeneic to the targets, even when effector and target cells were fully allogeneic. All together, the data suggest that human anti-EBV CTL may recognize nonpolymorphic HLA determinants on the target cells in association with the virus-induced antigens.     

Stimulation of human lymphocytes with irradiated cells of the autologous Epstein-Barr virus-transformed cell line. I. Virus-specific and nonspecific components of the cytotoxic response

Peripheral blood lymphocytes were cocultivated with irradiated cells of the autologous EB virus-transformed cell line at different responder:stimulator (R:S) ratios and the cytotoxic response was assayed up to 12 days later. In cocultures set up at a R:S ratio of 4:1, the response from both EB virus antibody-positive (seropositive) and negative donors was dominated by a broad-ranging NK-like cytotoxicity which did not segregate within the E-rosette-forming subpopulation of effector cells. In contrast, cocultures set up at a R:S ratio of 40:1 and harvested after 10 to 12 days gave rise, in the case of seropositive donors only, to effector T-cell preparations which appeared to be both EB virus specific and HLA-A and B antigen restricted. Strong lysis of the autologous virus-transformed cell line and demonstrable activity against certain allogeneic HLA-A and/or B antigen-related virus-transformed lines occurred in the absence of any significant killing either of the corresponding lines from HLA-unrelated donors or of a variety of EB virus genome-negative target cells (K562, HSB2, BJAB) particularly sensitive to NK-like cytotoxicity; furthermore, lysis of the autologous cell line by these effector T cells was specifically inhibited by monoclonal antibodies binding to HLA-A, B, and C antigens on the target cell surface. This work demonstrates that an HLA-restricted EB virus-specific cytotoxic T-cell response can indeed be induced in vitro by stimulation of fresh lymphocytes with autologous EB virus-transformed cells providing cocultures are set up at the correct R:S ratio.     

Disease-related lymphocyte cytotoxicity in vitro in patients with transitional cell carcinoma of the urinary bladder. Comparisons with other malignancies,acute cystitis,and healthy donors

Summary Blood lymphocytes from 100 patients with transitional cell carcinoma of the urinary bladder (TCC-bladder) were studied for their cytotoxicity in vitro against a panel of allogeneic tissue culture cell lines. Of the TCC-bladder patients, 45 were untreated for their disease, while 55 had been treated with local radiotherapy up to 12 years before testing. Control lymphocytes were obtained from (1) 45 untreated, age- and sex-matched patients with other neoplastic diseases, mainly urogenital cancers; (2) 19 patients with acute cystitis; and (3) 45 healthy donors. Lymphocytes from individual donors within all five groups were frequently cytotoxic to any one of the target cells. However, the lymphocytes from each of the two TCC-bladder groups were markedly more cytotoxic to two different bladder tumor targets than to control targets derived from normal bladder epithelium, from colon carcinoma, or from malignant melanoma. Similar comparisons made within each of the three control donor groups did not show this. The results indicate that the two bladder tumor targets were not more susceptible to lymphocyte-mediated lysis than the control targets. The mean cytotoxicity displayed by the lymphocytes from both TCC-bladder groups to the bladder tumor targets was significantly higher than that of the cancer control group and that of the healthy donors. No such elevation was seen when the cancer control group or the cystitis patients were compared with healthy donors. Although untreated TCC-patients with a larger tumor burden (stages T3–T4) appeared to be slightly less cytotoxic to all target cells than those with a smaller tumor burden (T1–T2), these differences were not statistically significant. On the other hand, among the treated TCC-patients, in the main those tested more than 1 year and up to 5 years after therapy exhibited a significantly elevated mean cytotoxicity to the bladder tumor targets. Within all five donor groups, the overall cytotoxicity to the bladder tumor targets and the normal bladder targets showed a statistically highly significant correlation. However, while there was no correlation for the untreated TCC-bladder patients and the clinical controls between cytotoxicity to the bladder tumor targets on one hand and non-bladder targets on the other, the cytotoxicity to the bladder tumor targets of the treated TCC-bladder patients was also correlated with that to the colon carcinoma and the melanoma targets. The results indicate that cytotoxicity in both TCC patients and controls reflects recognition by the lymphocytes of a variety of antigens, shared to different degrees by different groups of target cells. Furthermore, in TCC-bladder patients there is a superimposed cytotoxicity, which is related to their disease and which probably reflects reactions against one or several tumor-associated antigens.     

DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

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     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Characteristics of human NK clones: target specificity and phenotype

Clones derived from purified human large granular lymphocytes (LGL) of three different donors were expanded in culture medium supplemented with interleukin 2 (IL 2). Their cytotoxic activity was tested in a 51Cr-release cytotoxicity assay against a panel of three to five NK-susceptible tumor cell lines. Of 196 LGL clones tested, only 44 (22.4%) displayed significant cytotoxic activity. A heterogeneous pattern of reactivity was seen; 26 clones (59%) killed all the targets within the panel tested, whereas 18 clones (41%) had a more restricted specificity. Among these 18 clones, 12 lysed only one target (K562, six clones; ADCC, three clones; Daudi, two clones; MOLT-4, one clone), whereas the other six killed two different targets (ADCC and A1ab, one clone; K562 and MOLT-4, five clones). Clones derived from LGL preselected for positive reactivity with the monoclonal antibodies (MoAb) alpha OKM1, alpha OKT10 and alpha B73.1 also demonstrated either broad or restricted patterns of cytotoxicity. The LGL reactive with MoAb alpha B73.1 gave a high percentage of cytotoxic clones. Phenotype analysis showed that clones could express both antigens associated with T cells (i.e., OKT3, OKT4, and OKT8) and antigens shared by LGL (i.e., OKM1, OKT10, and B73.1). The pattern of surface markers varied considerably among the clones; however, no clear correlation between the pattern of antigenic phenotype and cytotoxic activity was seen. These data show that clones derives from purified preparations of LGL present different functional and antigenic characteristics, and support the hypothesis that the heterogeneity of the entire NK population is attributable, at least in part, to a mixture of clones that vary substantially in their target specificities and phenotypes.     

Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.     

Hodgkin’s lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.