Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. III. Single histocompatibility antigens dominate the male antigen

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions.     

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Immune recognition by cytotoxic T lymphocytes of minor histocompatibility antigens expressed on a murine colon carcinoma line

We have characterized in vivo and in vitro responses of mice to the BALB/c-derived carcinoma, C26. BALB/c mice were highly susceptible, in a dose-dependent fashion, to local tumor development following subcutaneous injection of C26. Other strains of mice, including allogeneic strains and major histocompatibility complex compatible strains of different minor histocompatibility (H) backgrounds, were resistant to C26-induced tumors. The basis for resistance of mice to C26 was studied using an in vitro-derived C26 line as target cells in microcytotoxicity assays, and as a source of antigen for in vivo priming. An H-2d-specific alloreactive cytotoxic T lymphocyte (CTL) line was isolated from C57BL/6 mice primed with C26, demonstrating the expression, and immune recognition, of MHC class I antigens on C26. C26 also expressed minor H antigens of BALB background as demonstrated by the ability of CTL specific for BALB minor H antigens to selectively lyse C26. Conversely, minor H antigens on C26 were immunogenic across a minor H barrier as demonstrated by the ability to raise anti-minor H CTL to C26 from minor H disparate strains. Collectively, those experiments indicate that C26 may be useful for immunologic and biochemical studies of murine minor H antigens, and for in vivo and in vitro studies of local immunity.     

H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.     

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.     

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.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.     

Genetics of histocompatibility in mice

The response to spleen cells incompatible at defined histocompatibility, H, loci was studied using the popliteal lymph-node enlargement test to establish its applicability as a method for detecting minor H antigens. This test was able to detect H-2 and H-Y antigens as well as most of the minor H antigens represented by 23 different strains congenic with C57BL/6By. Responses of both naive and immunized recipients were examined, and time-courses of the response were obtained for ten donor strains. These curves revealed that different antigens elicited responses that differed in timing and magnitude of peak enlargements, and that the two parameters were not closely correlated. Both the peak magnitude and the slope of the primary response were correlated with skin-graft rejection, however. The response to individual minor antigens did not appear to be dose-dependent above a threshold. Irradiation of donor cells had strain-dependent effects on the elicited response. Irradiation of recipients appeared to abrogate the primary response but not the responses of previously primed recipients in the combination tested. None of the responses studied had any demonstrable graft-versus-host component.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.     

Possible mechanisms by which the H-2Kbm3 mutation may decrease cytotoxic T-lymphocyte recognition of vesicular stomatitis virus nucleoprotein antigen.

Spleen cells from C57BL/6 (B6) mice generate a strong in vitro cytotoxic T-lymphocyte (CTL) response specific for vesicular stomatitis virus (VSV). Spleen cells from VSV-primed B6-H-2bm3 (bm3) mice, which have a mutation in H-2Kb, require approximately 10-fold more UV-inactivated VSV to generate in vitro secondary anti-VSV CTL, compared with spleen cells from primed B6 mice. Anti-VSV CTL elicited in both bm3 and B6 mice are primarily specific for the viral nucleocapsid protein (N protein), as demonstrated by using recombinant vaccinia viruses that express the VSV N protein. bm3 CTL were found to exhibit only a very low level of lytic activity when tested against autologous VSV-infected concanavalin A spleen cell blasts as well as several H-2b tumor cell lines. The weak anti-VSV response of bm3 CTL was found to be the result of a combination of inefficient recognition of VSV-infected target cells and decreased elicitation of secondary effector cells. VSV-infected bm3 target cells were not killed as well as B6 targets by either bm3 or B6 effectors. This is because of the inefficient recognition of targets, as demonstrated by the fact that VSV-infected bm3 cells were unable to competitively inhibit the lysis of VSV-infected B6 target cells by either bm3 or B6 effectors. By using cells from recombinant mice, it was shown that the CTL response restricted by H-2Kb was low in the bm3 mice, compared with that of the B6 mice. However, the H-2Db-restricted CTL activity was similarly low in both the B6 and bm3 mice. The possibility that the low response to VSV-infected bm3 cells is caused by differences between the bm3 and B6 cells in expression of either viral antigens or H-2K was investigated by radiolabeling and immunoprecipitation. VSV-infected B6 and bm3 cells were found to express equivalent levels of both viral antigens and H-2K. These results indicate that the bm3 mutation alters a functional site on the H-2Kb molecule that is involved in the recognition of VSV-infected cells. The observation that elicitation of bm3 CTL can occur at high antigen doses further suggests that the bm3 mutation results in a lower affinity of H-2K either for viral antigen or for receptor sites on the CTL.     

Differential expression of the repertoire in in vitro and in vivo responses to minor alloantigens

The response to multiple minor alloantigens in the secondary mixed lymphocyte culture (MLC) between mouse strains that are identical at the major histocompatibility complex (MHC) generally yields effector cytotoxic T lymphocytes (CTL) which show cross-reactive killing of most or all third-party mouse strains which also share MHC haplotypes. We have investigated the clonal diversity of CTL responses in vivo versus in vitro by examination of such cross-reactions, using CTL effector cells derived from a primary response, an in vivo secondary response, and an in vitro secondary MLC. CTL from these three responses were assayed on a panel of H-2' targets. Restimulation of antigen-primed spleen cells in vitro yielded CTL which were strongly cross-reactive on all targets, whereas the in vivo responses were much less so. We conclude that the set of clones which become cytotoxic effectors in vivo is much less diverse than the set which is primed on a first encounter with antigen and that powerful constraints must therefore operate on the specificity of in vivo responses to non-MHC antigens.     

Detection of shared H-2Kk and H-2Dk antigens that function as self determinants for cell-mediated lympholysis to trinitrophenyl-self

Spleen cells from C3H/He mice immunized in vivo to trinitrophenyl (TNP)-self were sensitized in vitro to TNP-self. These spleen cells displayed strong lysis on TNP-modified H-2D end-matched (Kd-Dk) targets as well as enhanced cytotoxicity against H-2 matched (Kk-Dk) or H-2K end-matched (Kk-Dd) target cells. Cold target-blocking studies showed that the lysis of TNP-Kd-Dk targets could be blocked by the addition of TNP-modified Kk-Dk, Kk-Dd Kk-Db, or Kd-Dk, but not by TNP-modified Kd-Dd, Kb-Db and Kq-Kq spleen cells. These results demonstrate that the lysis of TNP-Kd-Dk targets is not due to cross-reactive clones against TNP-Kd-Dd, Kb-Db or Kq-Dq antigens. Inhibition of the TNP-Kd-Dk target lysis by TNP-Kk-matched (Kk-Dd or Kk-Db) as well as TNP-Dk-matched (Kd-Dk) blockers also reveals that this target is lysed by clones directed against shared antigens between Kk-TNP and Dk-TNP, indicating that no cytotoxic response restricted for Dk-TNP only could be detected even after in vivo priming.     

T helper cell lines that augment in vivo cytotoxic T-cell responses to minor alloantigens

We have investigated the ability of long-term cultured T helper (Th) cell lines to help an in vivo cytotoxic T lymphocyte (CTL) response to non-H-2 alloantigens (minor antigens). Th cell lines specific for various single or undefined minor antigens were selected by regular restimulation with antigen in vitro. They were antigen specific and H-2 restricted in proliferation assays and were found to be able to help primary CTL responses to multiple minor antigens and secondary CTL responses to single minor antigens. Although the Th were antigen specific they did not determine the specificity of the CTL. Th cells were both necessary and limiting for an effective CTL response indicating that "helper-independent" CTL are not in themselves sufficient to generate a strong in vivo response. Under conditions where a CTL response was clearly H-2 restricted, Th cells were not. Thus, the Th cells appeared to be activated by reprocessed antigen rather than antigen on the surface of the injected antigenic cells even though the CTL themselves reacted directly to the injected antigen.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.