The rationale and practice of CTL therapy against cancer in the mouse and human

It is essential to investigate and elucidate the immune response especially T cell response to either syngeneic or autologous tumor for establishing a rational immunotherapy of cancer. We reported that major immune effector cells capable of inducing tumor regression are cytotoxic T lymphocytes (CTL). We found that there are at least two distinct CTL subsets directed to syngeneic tumor. One CTL subset which is selectively induced by syngeneic solid tumor is independent from CD4 positive helper T cells but requires a soluble factor (s) released from macrophage-like accessory cells designated killer T cell activating factor (KAF) in its induction and generation directed to the homologous tumor. The other CTL subset which is usually induced by syngeneic tumor of hematocytic origin is dependent on CD4 positive helper T cells in its induction. On the basis of our findings regarding the induction and activation mechanism of CTL to syngeneic tumors in the mouse, we have investigated the mechanisms of human CTL generation to autochthonous tumor in peripheral blood mononuclear cells of cancer patients. It was found that the nature of human CTL and its generation to autochthonous tumor are similar to those of murine CTL to syngeneic solid tumor. We are now establishing a rational cancer specific immunotherapy utilizing intravenous passive cell transfer of in vitro activated CTL to autochthonous tumor into an original cancer patient.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1/3000. Among these, the frequency of TATA-specific CTL was 1/16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.     

Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL

The roles of Class II-restricted L3T4+ T cells and of accessory cells (AC) during the in vitro generation of Class I-restricted Lyt-2+ cytotoxic T cells (CTL) specific for a Class II-negative syngeneic tumor cell line, FBL, was examined. Treatment of responder FBL-immune spleen cells with alpha L3T4 plus complement before culture, as well as the direct addition of alpha L3T4 to cultures, diminished the generation of FBL-specific CTL. The contribution of L3T4+ cells could be completely replaced by the addition of exogenous cytokines. The data demonstrate that the optimal generation of FBL-specific Lyt-2+ CTL requires the presence of L3T4+ cells, presumably to provide necessary lymphokines. FBL-specific CTL could not be generated from purified FBL-immune T cells in the absence of AC. Syngeneic Ia+ macrophages (M phi), added at the initiation of culture, restored the response of purified T cells. Pretreatment of M phi with ammonium chloride or chloroquine, or the addition of monoclonal alpha I-Ab antibody at the initiation of culture, inhibited the ability of M phi to reconstitute the CTL response. Finally, the addition of exogenous helper factors could replace M phi and reconstitute the FBL-specific response of AC-depleted immune T cells. These results suggest that during the generation of Lyt-2+ CTL to a syngeneic tumor expressing only Class I MHC antigens, Ia+ AC are required to biochemically process antigen released from the tumor cells and present this modified antigen to Class II-restricted T helper cells.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Enhanced TNP-reactive helper T cell activity and its utilization in the induction of amplified tumor immunity that results in tumor regression

The present study was designed to investigate the generation of trinitrophenyl (TNP)-reactive helper T cell activity potent enough to induce the regression of a syngeneic tumor; this occurs by augmenting antitumor-specific immunity through T-T cell interaction. Mice whose skin was painted with trinitrochlorobenzene (TNCB) exhibited a variety of anti-TNP T cell responses, including delayed-type hypersensitivity (DTH) and cytotoxic T cell responses, as well as helper T cell activity. Pretreatment of C3H/He mice with TNP-conjugated copolymer of D-glutamic acid and lysine (TNP-D-GL) or cyclophosphamide, which have been shown, respectively, to inactivate TNP-specific suppressor T cells or suppressor T cells in general, exhibited a slight or marginal augmentation of DTH and cytotoxic potentials when tested 5 wk after TNCB painting. In contrast, the same pretreatment regimens induced an appreciably amplified generation of anti-TNP helper T cell activity. This amplified TNP-helper T cell activity was demonstrated to enhance cytotoxic responses to antigens other than TNP in an antigen-nonspecific way. In fact, such helper T cells enhanced antitumor CTL responses when co-cultured with spleen cells from syngeneic X5563 plasmacytoma-bearing mice in the presence of TNBS-modified X5563 tumor cells. This amplified TNP-helper cell system was utilized for its immunotherapeutic potential. When TNCB was injected into X5563 tumor mass of syngeneic C3H/He mice in which the amplified TNP-helper T cell activity had been generated, an appreciable number of growing tumors was observed to regress. This contrasted with the low incidence of tumor regression observed in mice in which TNP-helper activity had been induced by TNCB painting without inactivation of suppressors. Thus, the present model provides an effective immunotherapeutic manipulation for eliciting enhanced in vivo tumor regression, and emphasizes a role of helper T cells in augmentation of syngeneic tumor immunity.     

Analysis of Ir gene control of cytotoxic response to hapten-modified self: helper T cells specific for a sulfhydryl hapten can substitute for an anti-TNP-H-2b self helper cell defect

Helper T cells specific for N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine (I-AED) were generated in (C56BL/6 X C3H/He)F1 mice by immunization with I-AED-modified syngeneic cells (AED-self). The requirements for activation of hapten-induced helper cells were investigated. The results demonstrated that activation of AED and trinitrophenyl- (TNP) helper cells was strictly hapten specific. In addition, F1 AEd-helpers could be activated efficiently by either I-AED-modified H-2b or H-2k self components to enhance the anti-AED self-CTL responses. This contrasts with the previous findings demonstrating the failure of TNP-H-2b self to activate F1 TNP-helper cells. After AED-helpers were activated, they were capable of augmenting sensitization of cytotoxic T cells (CTL) against TNP-self. These results indicate that although the activation of hapten-reactive helper cells is antigen (hapten)-specific, the subsequent helper activity, as determined by augmentation of CTL responses against another hapten, is antigen nonspecific. Since helper function was antigen nonspecific, F1 AED-helper cells activated by AED-H-2b or AED-H-2k self were tested for their ability to augment the F1 and anti-TNP-H-2b CTL response. The results indicate that the Ir gene defect in the ability of F1 spleen cells to respond to TNP-H-2b self could not be corrected by these helper cells. These results are discussed in the light of Ir gene controlled differences in the activation of AED and TNP-helper cells and possible models for augmenting CTL responses against various antigens in strains that generate marginal helper activity to TNP-self.     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

Enhancement of tumor growth correlates with suppression of the tumor-specific cytolytic T lymphocyte response in mice chronically infected by Trypanosoma cruzi

The immunity of BALB.B mice to syngeneic Gross murine leukemia virus (MuLV)-induced B.GV cells was studied at various times after infection by Trypanosoma cruzi. BALB.B mice chronically infected by the parasite do not develop an effective immune response against B.GV tumor cells, and B.GV tumor growth in vivo is consequently facilitated. The tumor-specific cytolytic T lymphocyte (CTL) compartment in these mice was studied in vitro because CTL are known to participate actively in syngeneic tumor rejection. These analyses showed that: a) CTL differentiation is suppressed in mice with chronic T. cruzi infections; b) suppression is at the level of CTL precursor cell activation; c) suppression is not antigen-specific; and d) suppression is mediated by macrophages and Lyt-2+ T lymphocytes.     

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.     

Evaluation of in vivo and in vitro effectivity of immune defense against a spontaneously arising,nonlymphoid rat tumor

Summary It was evaluated in vitro whether it is possible to induce immunogenicity by haptenization of a nonlymphoid rat tumor, BSp6AS, which is known to be antigenic, but non-immunogenic, in the syngeneic host. The effectivity of induced immunogenicity was tested in vivo.BSp6AS, an NK- and macrophage-resistant variant of a spontaneously arising fibrosarcoma in the BDX rat strain, does not induce a primary or a secondary T cell response after in vivo or in vitro priming. This deficiency in cytotoxic response is due solely to failure of activation of helper T cells (T_H), since (a) cytotoxic T cells (CTL) can be detected after in vitro stimulation in the presence of interleukin 2 (IL-2)-containing medium; and (b) there are no indications for down-regulation of a potential specific immune response by suppressor T cells (T_S). The lack of activation of tumor-specific T_H can be bypassed by activation of hapten-specific T_H. Upon coculture with haptenized tumor cells as a stimulator population, both hapten-specific and tumor-specific CTL are activated by hapten-specific T_H.In line with the findings in vitro, no transplant rejection of naive tumor cells was seen after a variety of immunization schedules. But immunized F₁ hybrids did reject tumor grafts, supporting the hypothesis of lacking help in the syngeneic situation. This could be confirmed in the syngeneic system by adoptive transfer experiments. Tumor-specific CTL, educated in vitro in the presence of IL-2, were ineffective. But complete protection against haptenized, and partial protection against native tumor cells was achieved in the additional presence of hapten-specific T_H.To our knowledge these experiments prove for the first time that hapten-specific T_H are efficient in inducing an immune response even against a nonlymphoid, nonimmunogenic tumor, i.e., it is possible (a) to activate tumor-specific CTL and (b) to initiate tumor graft rejection via hapten-specific T_H.Abbreviations BSS Hank's balanced salt solution - CFA complete Freund's adjuvant - ConA Concanavalin A - CTL cytotoxic T cell - iFA incomplete FA - IL-2 interleukin 2-containing supernatant of rat spleen cells cultured in ConA - IP intraperitoneal - IV intravenous - LNC lymph node cells - 2-ME 2-mercaptoethanol - MHC major histocompatibility complex - MLTC mixed lymphocyte tumor cell culture - RPMI 1640 supplemented with antibiotics, L-glutamine and fetal calf serum - SC spleen cells - SC subcutaneous - TAA tumor-associated antigen(s) - TB tumor bearer - TD₁₀₀ tumor cell dose resulting in 100% takes - T_H helper T cells - T_S suppressor T cells - TNBS trinitrobenzenesulfonic acid - TNP trinitrophenyl - WAB Wistar Nottingham rats     

Secondary in vitro generation of CTL specific for MM antigen by stimulation with somatic tumor hybrid, but not with parental tumor cells

Somatic tumor hybrid cells (L-FM3A#2), obtained by hybridization of MM tumor cells (FM3A/R) with HGPRT-less L cells, could induce CTL directed against MM antigen, a tumor-associated transplantation antigen that is expressed on some ascitic mammary tumor cell lines of C3H/He mice; parental tumor cells (FM3A/R) could not produce such CTL in syngeneic mice. In this study, the mechanisms of the generation of CTL by stimulation with L-FM3A#2 hybrid cells were investigated. In the secondary in vitro stimulation system, L cell component(s) that were introduced into hybrid cells by cell fusion play a role in the induction of CTL, as shown by the fact that stimulation with a mixture of L and FM3A/R cells could induce MM antigen-specific CTL, and that the killer helper effect of L cells could be replaced by cell free culture supernatants obtained from co-cultures of unprimed C3H spleen and L cells. IFN activity, but no IL 2 activity, was detected in the culture supernatants. Both IFN and killer helper activity were lost with pH 2 treatment; furthermore, CTL were generated by stimulation of primed spleen cells with FM3A/R cells in the presence of mouse beta-IFN, but not in its absence. These results suggest that IFN liberated by the helper cells that recognize L cell component(s) on the surfaces of tumor hybrid cells plays an essential role in the generation of CTL specific for MM antigen.     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Induction of anomalous killing activity from antigen-specific CTL clones by adding high doses of human recombinant interleukin 2

Four out of six long-term murine cytotoxic T lymphocyte (CTL) clones specific for trinitrophenyl (TNP)-modified spleen cells could develop an anomalous cytotoxicity against syngeneic and allogeneic tumor cells upon stimulation with TNP-modified spleen cells and high doses of human recombinant interleukin 2 (rIL-2). On FACS analysis, hyperactivated CTLs were positive for Thy-1, Ly 2 and LFA-1, but negative for L3T4 and asialo GM1. The staining profile of the cells with each antibody indicated that the CTL clones consisted of just one cell type. Monoclonal anti-Ly 2.2 and anti-LAA (lymphokine-activated cell-associated antigen) antibodies inhibited cytolysis of CTL and hyperactivated CTL clones against TNP-modified spleen cells, but failed to inhibit the anomalous killing of the hyperactivated CTL. The cold target competition test suggested the degeneracy of antigen specificity. The present study demonstrated that the CTL clone acquired a new specificity for tumor target cells upon stimulation with a high dose of rIL-2.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.