Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

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.     

Recruitment and activation of tumor-specific immune T cells in situ. CD8+ cells predominate the secondary response in sponge matrices and exert both delayed-type hypersensitivity-like and cytotoxic T lymphocyte activity

This study analyzes the involvement of CD4+ and CD8+ T cells in a secondary cellular immune response to the highly metastatic murine lymphoma ESb in situ. This tumor line expresses tumor-associated transplantation Ag which can induce protective immunity in vivo and specific CTL in vitro. In tumor-immune mice the injection of a tumor vaccine (x-irradiated ESb tumor cells) into s.c. implanted vascularized sponges resulted in the generation of a specific secondary immune response characterized by massive leukocyte recruitment and generation of strong CTL activity at the restimulation site. During the antitumor immune response the CD4+:CD8+ T cell ratio decreased significantly and specifically in the restimulated sponges. Depletion of CD8+ but not CD4+ T cells from the tumor immune mice before restimulation significantly reduced the delayed-type hypersensitivity-like response and totally blocked the generation of tumor-specific CTL activity in situ. Only a minority of the CD8+ immune T cells which predominated the secondary response in situ expressed IL-2R and lymph node homing receptors as detected by the mAb MEL-14.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

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.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conditions for antitumor cytotoxic T-lymphocyte formation and inhibition of their activity by T-suppressors in mixed culture of human lymphocytes and tumor cells

Conditions for antitumour autolytic T lymphocytes (CTL) induction during human mixed lymphocyte tumour cell culturing (MLTC), as well as the patterns of CTL activation abolition in the experimental system by suppressor cells were investigated. The responders used in MLTC were peripheral blood lymphocytes of colorectal cancer patients fractionated by means of multilayer Percoll gradients centrifugation (the density of layers being 1.077, 1.067, 1.056 g/ml). The cells of the second fraction collected in the density interphase of 1.077-1.067 g/ml (more than 90% of population belonging to T lymphocytes), when used as responders in MLTC, developed an autolytic activity against autologous and allogeneic tumour target cells. The cell of the first fraction (collected in the interphase of 1.067-1.056 g/ml) added to the second fraction cells at the beginning of MLTC, prevented the following CTL induction. The first fraction contained T-suppressor cells capable of strongly interfering with antitumour CTL activity.     

Clonal analysis of expression of tumor-associated transplantation antigens and of metastatic capacity

Summary ESb, a spontaneous high metastatic variant of the chemically induced T lymphoma Eb, was found previously to express a tumor-associated transplantation antigen (TATA) that was different from that of the parental line. Syngeneic tumor-specific cytolytic T lymphocytes (CTL) were able to recognize the different TATAs of Eb and ESb in vitro and could therefore be used for routine typing. The object of this study was to investigate tumor antigen expression on a clonal level and to compare the in vitro data with the in vivo behavior of the same cell lines.Our CTL typing analysis of cloned tumor lines revealed that the two populations, Eb and ESb, are distinct and relatively homogeneous with regard to their TATA expression. Furthermore, all ESb clones formed rosettes with antibody-coated erythrocytes, while none of the parental type Eb clones showed this characteristic. The sensitivity to tumor-specific CTL lysis varied with time of tumor cell culture in vitro in a clone-dependent manner.Variability was also noted in vivo in tumor growth and metastatic spread. Of over 50 ESb clones tested, the majority were highly metastatic while a minority were significantly lower in metastatic capacity. High and low metastatic ESb clones could not be distinguished by their expression of TATAs and of Fc receptors. There was also a considerable individual variability in the hosts, although they were genetically identical. This variability was most probably due to differences in the immune status of the animals.     

Capacity of different cell types to stimulate cytotoxic T lymphocyte precursor cells in the presence of interleukin 2

Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis.     

Type I interferon as a stimulus for cross-priming

Type I interferon (IFN-alpha/beta) is induced rapidly by infection and is well recognised for its crucial role in innate defence. However, it is evident that IFN-alpha/beta also serves as a signal for the generation of adaptive immune responses. In this review, we focus on the involvement of IFN-alpha/beta in the induction of CD8+ T cell responses by cross-priming.     

Immune recognition of tumor cells in mice infected with Pichinde virus

Summary Pichinde virus (PV), a member of the Arenaviridae family, protects mice from a lethal inoculation with the sarcoma 180 (S180) tumor cell line. Virus replication, which is required for protection, occurs primarily in the spleen and tumor. During the first 4 days, elevated natural killer (NK) cell activity parallels an increase in serum interferon in PV-infected mice. On day 7 after infection virus-specific cytotoxic T cells (CTLs) are found in the mouse. This strong response peaks on day 13 and gradually declines over the next 17 days. The tumor-specific CTL response appears more slowly and is less intense than the virus-specific response, especially in the uninfected mouse. However, CTLs from either type of mouse recognize PV-infected tissue culture S180 target cells better than uninfected ones. Even though the primary tumor-specific immune response appears weak, mice that have cleared both virus and tumor are refractory to a subsequent challenge with S180 cells and rapidly produce tumor-specific CTLs. Thus, our data indicate a number of ways in which virus infection could lead to immune elimination of tumors: (1) Virus-induced interferon stimulates NK-cell activity, which in turn could control tumor load until a specific response is mounted against the S180 cells; (2) early onset of the tumor-specific T-cell response could be brought about by viral-enhanced tumor antigen presentation to the immune system; and (3) the tumor-specific T-cell response could be augmented through a bystander phenomenon involving factors associated with T cells responding specifically and vigorously to the virus itself.Deceased     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.     

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     

Priming of influenza virus-specific cytotoxic T lymphocytes vivo by short synthetic peptides.

Influenza virus-specific CTL were primed in vivo by immunization with short synthetic peptides representing major CTL epitopes from the nucleoprotein of type A influenza virus. The resultant CTL after in vitro boosting of primed spleen cells recognized both virus-infected and peptide-pulsed target cells. The requirement of CD4+ T cell activation was investigated in several ways. First the addition of helper epitopes to the CTL epitope did not enhance CTL generation, suggesting that helper activity was either not limiting or not required. However, in vivo depletion of CD4+ T cells completely inhibited the generation of CTL by peptide immunization. The inclusion of anti-CD4 in the in vitro restimulation with peptide also prevented the generation of CTL, whereas in vitro reactivation of virus immune spleen cells with peptide was not inhibited by anti-CD4. Thus there appears to be heterogeneity in the requirement of CD4+ T cell proliferation in CTL generation. One possibility is that virus infected cells can stimulate higher affinity T cells that are less helper T cell dependent.     

In vitro expression of secondary antitumor immunity by in vivo tumor-sensitized T cells: Inhibition by tumor-induced suppressor T cells

Summary In vitro cultivation of memory immune cells from P815- or P388-immune mice with corresponding irradiated tumor cells induced generation of cytolytic T cells (CTL). The induction of CTL generation, as well as the cytolytic activity itself, was tumor-specific. The in vitro generation of CTL from P815- or P388-immune cells was suppressed by spleen cells from mice bearing corresponding progressive tumors (tumor size 15 mm). The tumor-induced suppressor cells suppressed the in vitro generation of CTL, but did not affect their cytolytic function. The suppression was tumor-specific and was mediated by Ly1⁺2^–L3T4⁺ T cells. Treatment of suppressor cell donors with cyclophosphamide or sublethal -radiation completely abolished the ability of their spleen cells to inhibit the in vitro CTL generation.     

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.