Generation Of Cytolytic T Lymphocytes Research Articles

IL-2 and IL-6 cooperate to enhance the generation of influenza-specific CD8 T cells responding to live influenza virus in aged mice and humans

An age-related decline in cytolytic activity has been described in CD8+ T cells and we have previously shown that the poor CD8+ effector T cell responses to influenza A/H3N2 challenge result from a decline in the proportion and function of these cytolytic T lymphocytes (CTL). Here, we describe that addition of exogenous cytokines to influenza-stimulated PBMC from both aged mice and humans, enhances the generation of influenza specific CD8 CTL by increasing their proliferation and survival. Our data show that the addition of IL-2 and IL-6 to splenocytes from mice previously infected with influenza virus restores the aged CD8+ T cell response to that observed in young mice. In humans, IL-2 plus IL-6 also reduces the proportion of apoptotic effector CD8+ T cells to levels resembling those of younger adults. In HLA-A2+ donors, MHC Class I tetramer staining showed that adding both exogenous IL-2 and IL-6 resulted in greater differentiation into influenza-specific effector CD8+ T cells. Since this effect of IL-2/IL-6 supplementation can be reproduced with the addition of Toll-like receptor agonists, it may be possible to exploit this mechanism and design new vaccines to improve the CD8 T cell response to influenza vaccination in older adults.

Differential requirements for CTL generation by novel immunostimulants: APC tropism, use of the TAP-independent processing pathway, and dependency on CD80/CD86 costimulation

A major drawback of subunit vaccines is their inability to generate cytolytic T lymphocytes (CTL), a deficit attributed to segregation of the class I and class II antigen-processing pathways. We sought to understand processes involved in CTL induction by three proprietary adjuvants: Tomatine, PROVAX™, and a synthesized glycolipid (Glc-N-(8/16), Glycolipid). We used in vivo models to investigate antigen uptake, macrophage involvement, TAP-independent processing, and costimulatory molecule dependencies. Glycolipid required splenic and lymph node macrophages, whereas Tomatine generated CTL independently of either macrophage population. In contrast, PROVAX™ showed partial macrophage requirements. Immunized TAP knockout mice revealed that ovalbumin (OVA)–Tomatine and OVA–PROVAX™, but not OVA–Glycolipid, generate class I–peptide complexes. All three immunostimulants also elicited CD86-dependent TH1 cytokine responses.

Cytolytic T Lymphocytes Recognize an Antigen Encoded by MAGE-A10 on a Human Melanoma

Abstract From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.

Construction and characterization of a recombinant adenovirus directing expression of the MAGE-1 tumor-specific antigen.

The finding that many human melanomas express distinct antigens that can be recognised by specific cytolytic T lymphocytes (CTL) implies that immunotherapeutic strategies against this cancer might prove effective. The ex vivo delivery of a tumour-associated antigen to autologous cells and the subsequent re-administration of these cells to the patient might prove effective in boosting the T cell immune response. Recombinant human adenoviral vectors provide an efficient delivery system and have many advantages over other viral and non-viral delivery vehicles. Infection of a panel of human melanoma cell lines by AdCMVMAGE-1, a novel recombinant adenovirus which incorporates the full-length MAGE-1 cDNA, was shown to induce production of high levels of MAGE-1 protein. Incubation of transduced HLA-A1 expressing melanoma cell lines with 2 anti-MAGE-1.A1 CTL clones resulted in specific recognition and lysis of target cells, indicating that the exogenous MAGE-1 protein was processed and presented in a normal manner. Furthermore, quantitative analyses demonstrated a correlation between the efficiency of transduction and the proportion of cells lysed. Importantly for future clinical trials, stimulation of peripheral blood lymphocytes (PBLs) from a melanoma patient by AdCMVMAGE-1-transduced autologous cells resulted in the generation of specific CTLs against the MAGE-1 antigen. Together, our data emphasize the utility of adenoviruses as vaccination vehicles and highlight the potential efficacy of this approach for the treatment of melanoma.

Construction and characterization of a recombinant adenovirus directing expression of the MAGE‐1 tumor‐specific antigen

The finding that many human melanomas express distinct antigens that can be recognised by specific cytolytic T lymphocytes (CTL) implies that immunotherapeutic strategies against this cancer might prove effective. The ex vivo delivery of a tumour-associated antigen to autologous cells and the subsequent re-administration of these cells to the patient might prove effective in boosting the T cell immune response. Recombinant human adenoviral vectors provide an efficient delivery system and have many advantages over other viral and non-viral delivery vehicles. Infection of a panel of human melanoma cell lines by AdCMVMAGE-1, a novel recombinant adenovirus which incorporates the full-length MAGE-1 cDNA, was shown to induce production of high levels of MAGE-1 protein. Incubation of transduced HLA-A1 expressing melanoma cell lines with 2 anti-MAGE-1.A1 CTL clones resulted in specific recognition and lysis of target cells, indicating that the exogenous MAGE-1 protein was processed and presented in a normal manner. Furthermore, quantitative analyses demonstrated a correlation between the efficiency of transduction and the proportion of cells lysed. Importantly for future clinical trials, stimulation of peripheral blood lymphocytes (PBLs) from a melanoma patient by AdCMVMAGE-1-transduced autologous cells resulted in the generation of specific CTLs against the MAGE-1 antigen. Together, our data emphasize the utility of adenoviruses as vaccination vehicles and highlight the potential efficacy of this approach for the treatment of melanoma. Int. J. Cancer 72:1045–1055, 1997. © 1997 Wiley-Liss, Inc.

IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro.

CD8+ cytolytic T lymphocytes (CTLs) are important mediators for resistance to infections and malignant diseases. IL-12 enhances proliferative and cytolytic responses by killer cells, but its function in the generation of human antiviral CD8+ T cell responses has not been defined. We therefore evaluated the role of IL-12 in the generation of CTLs to influenza-infected dendritic cells. IL-12 was not detectable in supernatants of infected-dendritic cells, or during CTL generation. Furthermore, anti-IL-12 antibody did not block CTL generation. However, exogenous IL-12 (30-300 pg/ml) enhanced CD8+ T cell proliferative and cytolytic responses. The effect was greatest in individuals with weak reactivity to influenza virus or at antigen-presenting cell (APC):T cell ratios of 1:100 or less. IL-12 augmented interferon-gamma production during CTL generation. The CTL enhancing effects of the cytokine, however, could not be blocked by neutralizing anti-interferon-gamma antibody. Together with IL-12, antigen-pulsed dendritic cells may be a useful approach for boosting CTL responses against infectious agents and malignancies.

Impaired generation of anti-AKR/Gross murine leukemia virus cytotoxic T lymphocytes in mice experimentally infected with MuLV.

C57BL/6 (B6) and C57BL/6.Fv-1n (B6.Fv-1n) mice mount AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses following primary and secondary stimulation with AKR/Gross MuLV-induced tumor cells. In contrast, mice exposed to infectious virus rather than virus-infected cells generate little, if any, antiviral CTL activity. In this report, we show that inoculation of B6 or B6.Fv-1n mice with MuLV prior to priming with H-2-matched AKR/Gross virus antigen-positive tumor cells resulted in a profound inhibition of the virus-specific CTL response. Antiallogeneic major and minor histocompatibility antigen-specific CTL responses were not significantly diminished in MuLV-infected mice. The AKR/Gross MuLV-specific CTL response in B6 mice was inhibited by NB-tropic (SL3-3NB, Friend and Moloney), but not N-tropic (AKR623) MuLV, suggesting that productive infection of host cells was required. We were unable to inhibit the in vitro generation of virus-specific CTL by adding modulator cells from virus-infected mice to mixed lymphocyte-tumor cell cultures (MLTC) of spleen cells from uninfected animals. We also failed to augment CTL generation in MLTC from virus-infected animals by adding exogenous IL-2 or CD4+ lymphocytes from uninfected, tumor-primed mice. Taken together, the data suggested that the inhibition resulted from either a direct or an indirect effect on the in vivo priming of virus-specific CD8+ cells. It is therefore interesting that MuLV such as Friend and Moloney, which do not encode the immunodominant epitope recognized by anti-AKR/Gross MuLV CTL, are nonetheless able to specifically inhibit this response. These results demonstrate a potentially important mechanism by which retroviruses may escape CTL-mediated immunity.

Nonresponsiveness of AKR.H-2 b congenic mice for anti-AKR/Gross MuLV CTL responses: involvement of inhibitory cells as defined by adoptive transfer experiments

AKR.H-2 b mice are unable to elicit AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses. The participation of inhibitory cells was addressed through adoptive transfer experiments utilizing young AKR.H-2 b:Fv-1 b congenic responder mice as the recipients of AKR.H-2 b donor cells. The adoptive transfer of unfractionated viable splenocytes led to inhibition of virus-specific CTL responsiveness without affecting minor histocompatibility or allogeneic (H-2 d)-specific CTL responses. Negative cell selection studies indicated that of the donor AKR.H-2 b spleen cells that mediate specific inhibition, B lymphocytes, CD4 −CD8 + and CD4 +CD8 − T lymphocytes, but not macrophages, even though they are viral antigen positive (as are B and T lymphocytes), were the cells responsible for the diminution of the generation of AKR/Gross virus-specific CTL by AKR.H-2 b:Fv-1 b mice. To evoke maximal inhibition, the adoptive transfer of AKR.H-2 b cells had to be performed prior to in vivo priming with viral antigen. Anti-AKR/Gross MuLV nonresponsiveness of AKR.H-2 b mice could not be overcome through utilization of exogenous IL-2 at either the priming or in vitro restimulation phases of CTL generation. These results illustrate the complex interaction between retroviruses and lymphocytes and are relevant to understanding how retrovirus-infected cells may not only escape immune surveillance themselves, but also may inhibit the cytolytic T cell response directed at other infected cells, such as tumor cells.

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells.

Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells.

Effect of Interleukin-4 on Cytolytic T Cells

Recent studies indicate that interleukin-4 (IL-4) is a potent T-cell growth factor which may have a significant stimulatory effect on alloreactive cytolytic T lymphocytes (CTL). In this study, it was found that exposure of a recombinant retrovirus (RVpC)-specific, MHC-restricted, polyclonal CTL line to IL-4 resulted in the generation of CTL which efficiently lysed RVpC-infected as well as uninfected autologous target cells. A number of CTL cloned lines was generated from the parental line grown for 2 weeks with autologous stimulator cells in IL-4-containing media which again lysed RVpC-expressing as well as uninfected autologous target cells but not their allogeneic HLA-mismatched counterparts. These results extend previous observations in allogeneic systems and suggest that, in a syngeneic system, IL-4 may stimulate the recruitment of autoreactive CTL in vitro. This is a previously unrecognised property of IL-4, the in vivo significance of which remains to be established.

Interleukin 7 enhances cytolytic T lymphocyte generation and induces lymphokine-activated killer cells from human peripheral blood.

The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.

Forskolin and prostaglandin E2 regulate the generation of human cytolytic T lymphocytes

In this paper we examine the characteristics of human cytolytic T lymphocytes (CTL) generated in the presence of forskolin and PGE2. Forskolin and PGE2 suppressed the generation of class-I-specific CTL. The CTL generated in the presence of forskolin and PGE2 had different characteristics which included their ability to proliferate in response to the alloantigen and their lectin-mediated cytolytic activity. The CTL generated in the presence of forskolin had normal proliferative response to the alloantigen, whereas the CTL generated in the presence of PGE2 showed a suppressed proliferative ability to the alloantigen. The two groups of CTL were then tested for their activity in the process of lectin-dependent cell-mediated cytotoxicity. After the addition of PHA into the chromium release assay the CTL generated in the presence of forskolin normally lysed the nonspecific targets, whereas the CTL generated in the presence of PGE2 did not show the normal response in lysing the nonspecific targets. The results suggest that the cytolytic machinery was intact when the CTL were generated in the presence of forskolin but CTL were not able to either recognize or lyse the target cell. However, the CTL generated in the presence of PGE2 did not share the same characteristics as the CTL generated in the presence of forskolin because the CTL generated in the presence of PGE2 were unable to kill even in the presence of lectin. It appears that the inhibitory effects of forskolin were mediated by cAMP and not by its effects on the potassium channels because the 1,9-dideoxy derivative of forskolin which did not activate adenylate cyclase also did not suppress the generation of CTL. However, it was not established whether the diverse effects of PGE2 on the generation of CTL were mediated by cAMP-dependent, -independent or by both mechanisms.

Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10 −8–10 −4 M) suppressed the generation of class I specific CTL but, at 10 −4 M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H 2 antagonist ranitidine but not with the H 1 antagonist pyrilamine. These data suggest that suppression was mediated by the H 2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T Cells.

Amplification of altered self-reactive cytolytic T lymphocyte responses by cloned, allospecific human Th cells.

The effect of a cloned allospecific human Th cell, termed 86, on the in vitro generation of altered self-reactive cytolytic T lymphocytes (CTL) was investigated. Utilizing the induction of hapten altered self-reactive CTL as a model for virus or tumor-specific cell-mediated immunity, we determined that the presence of small numbers of clone 86 cells markedly amplified the generation of hapten altered self-reactive CTL. The killer cells induced belong to the CD4-, CD8+ subset, are specific for the hapten-modified autologous stimulator cells present in culture, and are MHC class I restricted. The CTL induced under these culture conditions are readily expanded in the presence of IL-2 with maintenance of efficient and specific altered self-killing. Of interest, clone 86 cells preferentially enhance the growth of CD8+ T cells and selectively amplify altered self-cytolysis but not NK cell activity. Although in vitro clone 86 cells mediate help for CTL generation via the production of lymphokines (IL-4 but little IL-2), one can envision immunotherapeutic strategies for human disease that involve the adoptive transfer of Th cells functionally analogous to clone 86.

Inhibition of alloantigen-induced cytolytic T lymphocytes in vitro with (2R,5R)-6-heptyne-2,5-diamine, an irreversible inhibitor of ornithine decarboxylase

The objective of the present investigation was to examine the effect of a new potent irreversible inhibitor of ornithine decarboxylase, (2R,5R)-6-heptyne-2,5-diamine (MAP) (MDL 72,175), on the induction of functionally reactive T-cell populations in vitro. We examined alloantigen-activated cytolytic T lymphocytes (CTL) and T-helper (T H) lymphocytes generated during a one-way mixed-leukocyte culture (MLC). The addition of MAP (1 m M) at the initiation of cell culture reduced intracellular putrescine, spermidine, and spermine levels by 81.9, 82.4, and 55.8% respectively. MAP reduced CTL induction 93.8, 78.4, and 37.5% when added at 0, 24, or 48 hr of culture, respectively. A dose-dependent inhibition of CTL induction and polyamine levels was observed following MAP treatment. In direct comparison with another ODC inhibitor, α-difluoromethylornithine (DFMO), MAP was five- to sixfold more potent in reducing CTL induction. CTL generation is dependent upon the endogenous production of the T H-cell product interleukin 2 (IL-2). MAP treatment reduced detectable IL-2 activity in a MLC by 54.8%. These results indicate that MAP is a potent inhibitor of alloantigen-activated CTL in vitro and deserves further investigation as a potential immunosuppressive agent.

Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine.

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.

Regulation of cytolytic T-lymphocyte generation by B-cell stimulatory factor.

The growth and differentiation of cytolytic T lymphocytes (CTL) is regulated by soluble growth hormones, of which interleukin-2 (IL-2) is considered to be of prime importance. Here we report that the lymphokine B-cell stimulatory factor (BSF-1 or interleukin-4) also has profound effects on the generation of these functionally active T cells. In particular, BSF-1 acts as a potent helper factor for the generation of CTL in primary mixed leukocyte culture (MLC) and induces cytolytic activity in in vitro primed, MLC memory populations. Direct comparison of purified recombinant BSF-1 and IL-2 reveals BSF-1 to be the more potent CTL helper factor in primary MLC. Interestingly, the two lymphokines differed in that IL-2, but not BSF-1, induced a lytic population in cultures of unprimed cells without an overt antigenic stimulus. Collectively, our data provide a direct demonstration of a heretofore undefined mechanism by which CTL activation and amplification can occur.

Induction of anti-AKR/gross virus cytolytic T lymphocytes in AKR.H-2b:Fv-1b congenic mice: age-dependent conversion to a nonresponder phenotype.

Previously, we reported that the generation of cytolytic T lymphocytes (CTL) specific for syngeneic tumors induced by AKR/Gross leukemia viruses was under multi-gene control. Thus, although carrying the required immune response gene(s) encoded by the H-2b haplotype and characteristic of responder strains such as C57BL/6, AKR.H-2b congenic mice failed to mount antiviral CTL responses. Young adult AKR.H-2b:Fv-1b "doubly congenic" mice, however, were able to generate specific anti-AKR/Gross virus CTL activity. These results demonstrated that the positive effect of MHC-encoded immune response gene control could be overcome by the action of the Fv-1n allele. The responder status of the B6.Fv-1n congenic, however, indicated that this Fv-1n-mediated inhibition was dependent on the interaction of Fv-1n with another gene(s) encoded by the AKR background. The results of experiments performed with AKXL recombinant inbred mice further suggested that a single additional genetic locus, encoding the Akv-1 provirus, was necessary along with Fv-1n to cause inhibition of antiviral CTL generation. Here we show that the responsiveness of AKR.H-2b:Fv-1b mice is dependent on their age. Thus, with moderate aging these doubly congenic mice converted to a nonresponder status with respect to anti-AKR/Gross virus CTL production: 85% of mice less than or equal to 9 wk of age responded compared with 0% of mice greater than 9 wk old. As with nonresponder AKR.H-2b mice, an inverse correlation was observed between CTL responsiveness and the expression of CTL-defined viral antigens by normal cells. Namely, spleen cells from young AKR.H-2b:Fv-1b mice showed little or no expression of such viral antigens, whereas with moderate aging there was a steady increase in their display. These results are discussed with reference to possible mechanisms of unresponsiveness of AKR.H-2b vs moderately aged AKR.H-2b:Fv-1b mice, and with respect to the utility of this system as a model for naturally occurring retrovirus infections and the interactions of retroviruses with the immune system.

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL.

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.

Immunodominance in the T-Cell response to multiple non-H-2 histocompatibility antigens

Immunization of C57BL/6 mice with BALB.B spleen cells in vivo and subsequent boosting in mixed lymphocyte culture result in the generation of cytolytic T lymphocytes (CTLs) which are specific for a limited number of immunodominant antigens. Experiments are described which suggest the existence of a hierarchy of immunodominance in this donor: host combination. Two antigens, CTT-1.3 and CTT-2.3, are dominant in the C57BL/6 anti-BALB.B CTL response. The distribution of these antigens among CXB recombinant inbred (RI) strains suggests that they segregate as single gene traits. Elimination of the CTT-1.3 and CTT-2.3 antigens by complementation in the responder, or elimination from the priming and boosting stages by the selection of CXB RI strain mice as responders or stimulators, reveals a second level of immunodominant antigens which include CTT-3.3 and CTT-4.3. CXB mice which express one of the CTT-1.3 or CTT-2.3 antigens will produce CTLs specific for the other antigen upon priming and boosting with BALB.B cells. Expression of both antigens in responders results in the generation of CTLs specific for the second level, dominant antigens. Immunodominance is not confined to the C57BL/6 anti-BALB.B system but can also be observed in the BALB.B anti-C57BL/6 and B10.D2 anti-DBA/2 systems. Finally, generation of CTLs following priming and boosting with dominant and dominated antigens presented on different cells confirmed that immunodominance can only be observed when the dominant and dominated antigens are presented on the same cells. These observations suggest that immunodominance is revealed at the level of antigen-presenting cells primarily involved in vivo priming.