Antigen recognition by cloned cytotoxic T lymphocytes follows rules predicted by the altered-self hypothesis

Radiation chimeras prepared by injecting H-2 heterozygous F1 stem cells into lethally irradiated parental hosts show a marked, but not absolute, preference for host-type H-2 antigens in the H-2-restricted cytotoxic T lymphocyte (CTL) response to minor histocompatibility (minor H) antigens. We have selected for the anti-minor HCTL that are restricted to the parental H-2 type absent from the chimeric host and found that in two out of eight cases, such CTL lysed target cells of either parental H-2 type. From one of these CTL populations that lysed H-2d and H-2k target cells expressing BALB minor H antigens, clones were derived and further analyzed. The results showed that: (a) lysis of both H-2d and H-2k target cells was H-2 restricted; (b) H-2d restriction mapped to Dd, and H-2k restriction mapped to Kk; (c) testing against various H-2d and H-2k strains of different and partially overlapping minor H backgrounds as well as against the appropriate F1 crosses revealed that in Dd- and Kk-restricted killing, different minor H antigens were recognized. In a second system, a CTL population was selected from normal (H-2d x H-2k)F1 mice that was specific for H-2d plus minor H antigens and for H-2k plus trinitrophenylated bovine serum albumin. We interpret these findings in terms of the altered-self hypothesis: The association of one H-2 antigen with one conventional antigen X may be recognized by the same T cell receptor specific for the complex formed by a different H-2 antigen in association with a second conventional antigen Y. The implications of these observations for the influence of self H-2 on the generation of the T cell receptor repertoire are discussed.     

Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Confirmation of genetic control of response to trinitrophenyl-modified H-2 antigens by the use of anti-H-2 and anti-Ia antibodies

Splenic lymphocytes from B10.A and B10.D2 mice were sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells. The effector cells generated were assayed in a 51Cr-release assay on TNP-modified syngeneic or congenic spleen target cells. Effector cells from B10.A donors lysed TNP-modified H-2Kk- but not H-2Dd-region products, whereas B10.D2 effectors reacted with modified products of both the H-2Kd and H-2Dd regions. As an independent confirmation that this selective K-end lysis by B10.A effector cells is due to an H-2-linked responder cell defect (4), anti-H-2Kk but not anti-H-2Dd sera were shown to inhibit the lysis of B10.A-TNP targets by B10.A effectors. In contrast, anti-H-2Dd sera inhibited the lysis of B10.A-TNP targets by B10.D2 effectors. Anti-Ia antibodies had no detectable effect on lysis. Anti-TNP-keyhole limpet hemocyanin sera blocked the lysis of TNP-modified targets, irrespective of whether the effector cells were directed against TNP-modified autologous H-2 products or H-2 alloantigens. These results independently verify that B10. A responding lymphocytes do not generate effector cells to TNP-modified H-2Dd products, whereas B10.D2 lymphocytes do (4), and suggest that some TNP groups are sterically close to (or part of) the serologically defined H-2K- and H-2D-region antigens.     

Quantitative differences in the expression of parentally-derived H-2 antigens in F1 hybrid mice affect T-cell responses

Quantitative absorption with specific anti-H-2 sera has shown that the H-2Kb and H-2Dd antigens coded by the B10.A(5R) haplotype are expressed in about fourfold lower amount on the spleen cells of [B10.A(5R) X B10.A(2R)]F1 hybrids than on parental B10.A(5R) cells. In contrast, the H-2Kk and H-2Db antigens of B10.A(2R) are expressed equally on parental and F1 cells. These quantitative differences are reflected in cytotoxic T-cell (Tc-cell) function. Macrophage target cells from F1 mice are killed less efficiently than B10.A(5R) targets by alloreactive or H-2 restricted Tc cells specific for H-2Kb or H-2Dd, and spleen cells of F1 mice are less efficient stimulators of alloreactive Tc cells specific for B10.A(5R) H-2 antigens, whereas F1 and B10.A(2R) cells are equal as targets and stimulators for Tc cells recognizing B10.A(2R) H-2 antigens.     

Neonatal tolerance of major histocompatibility complex antigens alters Ir gene control of the cytotoxic T cell response to vaccinia virus

The K region of H-2 controls the Tc cell response to vaccinia-Db. The Kb, Kd, and Kq alleles allow good Tc cell responses against vaccinia- Db. In contrast, the presence of Kk in H-2 recombinants 2R (Kk,Db) and 4R (Kk,Db) or in F1 hybrids greatly reduces the anti-vaccinia-Db response. The defect does not lie in antigen presentation, as infected 4R cells can stimulate anti-vaccinia-Db Tc cells in vitro. Furthermore, nonresponder animals possess Tc cell precursors for vaccinia-Db, as transfer of F1 nonresponder spleen cells into infected, lethally irradiated responder recipients allowed generation of anti-vaccinia-Db effector Tc cells. Secondary responses to vaccinia-Db can also be obtained in vitro from T cells of 4R animals. Feedback inhibition was excluded in experiments with mixed chimeras in which Kk and Db were expressed on separate cell populations. Neonatal tolerance of B10 animals to Kk suppressed the anti-vaccinia-Db response but did not affect anti-vaccinia-Kb, anti-lymphocytic choriomeningitis virus, or anti-H-2d responses. In cold target competition experiments, H-2k competitors inhibited vaccinia-Db-specific target cell lysis by Tc cells, which suggests that anti-vaccinia-Db and anti-H-2Kk Tc cells may cross-react. Therefore, we propose that the suppressive influence of Kk on anti-vaccinia-Db Tc cell responses is a consequence of self- tolerance and that suppression of anti-Kk Tc cells results in cross- reactive suppression of anti-vaccinia-Db Tc cells.     

H-2-linked genetic control of murine T-cell-mediated lympholysis to autologous cells modified with low concentrations of trinitrobenzene sulfonate

Spleen cells from B10.BR and C57BL/10 (B10) mice were compared for their ability to generate primary in vitro cytotoxic responses to syngeneic cells modified with different concentrations (from 10 to 0.031 mM) of trinitrobenzene sulfonate (TNBS) (TNP-self). Although both strains generated effector cells to TNP-self in the range of 10-0.25 mM TNBS modification, effector activity of B10 cells was weaker than that of B10.BR cells. B10 spleen cells did not respond to syngeneic stimulating cells modified at 0.1 mM or lower, whereas B10.BR cells generated effector activity even when stimulated by TNP-self modified with as low as 0.031 mM TNBS. Fluorescence analysis of the modified cells using the FACS II indicated that equivalent quantities of TNP were conjugated to the surfaces of B10.BR and B10 spleen cells for any given concentration of TNBS modification. Similar strain-dependent differences were observed when the TNP was diluted out in the cultures by reducing the number of stimulating cells modified with 10 mM TNBS. These response patterns were verified by stimulating cultures of B10.BR and B10 spleen cells either with TNP conjugated to bovine serum albumin or bovine gamma globulin (B10.BR but not B10 cells responded to TNP-conjugated proteins) or with TNBS-modified glass-adherent spleen cells. The strain-dependent differences could also be detected at the effector phase, because optimally stimulated B10.BR, but not B10 effector cells, could lyse 0.1 mM TNBS-modified syngeneic target cells. The genetic parameters associated with the response and nonresponse patterns of B10.BR and B10 mice were further investigated by comparing the cytotoxic responses to low doses of TNP-self of spleen cells from the following strains: (a) C3H/HeJ (H-2k) and C3H.SW (H-2b); (b) BALB.K (H-2k) and BALb.b (h-2b); and (c) B10.A (H-2a) and B10.D2 (H-2d). The H-2k and H-2a, but not the H-2b and H-2d, strains generated cytotoxic responses to TNP-self when the syngeneic stimulators were modified with 0.1 mM TNBS. Further studies using (B10 X B10.BR)F1 responding cells and parental or F1-modified stimulating cells, indicated that the F1 cells generated cytotoxic activity to low doses of TNP in association with H-2k but not in association with H-2b self products. The results of this study indicate that H-2-linked genetic factors, expressed in the target as well as in the responding and/or stimulating cell populations, control the ability of inbred mouse strains to generate cytotoxic effector cells to low doses of TNP-self. Such dose-dependent genetic effects may be important in the regulation of immune responses activated in vivo by chronic exposure to infectious agents.     

Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness

H-2 dependent and virus-specific Ir genes regulate the generation of primary virus-specific K or D restricted cytotoxic T-cell responses in vivo. The following examples have been analyzed in some detail: first, Dk restricted responses to vaccinia in Sendai viruses are at least 30 times lower than the corresponding K-restricted responses irrespective of the H-2 haplotypes (k, b, d, dxs, dxq) of K and I regions; in contrast, LCMV infection generates high responses to Dk. These findings are consistent with but do not prove that this Ir gene maps to D. Second, Db restricted responses to vaccinia and Sendai viruses are high in strains possessing the Kq or KbIb, KbaIb haplotype, are very low in strains with Kk, and relatively low in mouse strains of the KdI-Ad haplotype; LCMV generates high Db restricted response in the presence of Kk. This Ir gene for the response to vaccinia and Sendai viruses maps to K since B10.BYR (KqIkdDb) is a responder and B10.A (2R) is a nonresponder (KkIkdDb). Third, virus and K or D allele specific nonresponsiveness is dominant with variable penetrance; in heterozygous mice the nonresponder Kk allele over-rides responsiveness normally found in KbDb or KqDb combinations. Fourth, when (responder X nonresponder)F1 lymphocytes are stimulated in an environment expressing vaccinia virus plus only a high responder Kb or Kq allelle and Db, response to vaccinia Db is high; in contrast when the same F1 cells are stimulated in an environment expressing the low responder allele Kk, response to vaccinia Db is low. Thus absence of Kk during immunization allows generation of high responsive Db restricted vaccinia specific cytotoxic T cells. The Dk dependent low response to vaccinia Dk can be explained by a preclusion rule or by failure of vaccinia to complex with Db; however the analysis of Kk dependent low response to vaccinia Db does not support these explanations or that self-tolerance is responsible for this Ir effect but is compatible with the interpretation that Kk vaccinia is immunodominant over Db vaccinia. These results are discussed with respect to (a) possible mechanisms of regulation by Ir genes and (b) H-2 polymorphism and HLA-disease association.     

Self-recognition specificity expressed by T cells from nude mice. Absence of detectable Ia-restricted T cells in nude mice that do exhibit self-K/D-restricted T cell responses

The presence in athymic nude mice of precursor T cells with self-recognition specificity for either H-2 K/D or H-2 I region determinants was investigated. Chimeras were constructed of lethally irradiated parental mice receiving a mixture of F1 nude mouse (6-8 wk old) spleen and bone marrow cells. The donor inoculum was deliberately not subjected to any T cell depletion procedure, so that any potential major histocompatibility complex-committed precursor T cells were allowed to differentiate and expand in the normal parental recipients. 3 mo after reconstitution, the chimeras were immunized with several protein antigens in complete Freund's adjuvant in the footpads and their purified draining lymph node T cells tested 10 d later for ability to recognize antigen on antigen-presenting cells of either parental haplotype. Also, their spleen and lymph node cells were tested for ability to generate a cytotoxic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified stimulator cells of either parental haplotype. It was demonstrated that T cell proliferative responses of these F1(nude)----parent chimeras were restricted solely to recognizing parental host I region determinants as self and expressed the Ir gene phenotype of the host. In contrast, CTL responses could be generated (in the presence of interleukin 2) to TNP-modified stimulator cells of either parental haplotype. Thus these results indicate that nude mice which do have CTL with self-specificity for K/D region determinants lack proliferating T cells with self-specificity for I region determinants. These results provide evidence for the concepts that development of the I region-restricted T cell repertoire is strictly an intrathymically determined event and that young nude mice lack the unique thymic elements responsible for education of I region-restricted T cells.     

Immune response genes control T killer cell response against Moloney tumor antigen cytolysis regulating reactions against the best available H-2 + viral antigen association

Cytolytic T lymphocytes (CTL) specific for the virus-induced and leukemia-associated Friend, Moloney, Rauscher (FMR) antigen are easily detected in the spleens of primary and secondary stimulated H-2b or H- 2d mice. They react, respectively, with H-2Db + FMR and H-2Kd + FMR; Dd and Kb never being involved. On the other hand, recombinant (KbDd) mice are relatively low responders that produce CTL only after secondary stimulation. Competition and blocking experiments with monospecific anti-H-2 antibodies have demonstrated that on the same H-2b tumor cells, C57BL/6 (H-2b) lymphocytes recognize Db + FMR, whereas B10.A(5R) lymphocytes recognize Kb + FMR, the restriction cannot, therefore be explained by a specific association of viral molecules with certain H-2 products. The CTL response of (B10 X 5R)F1 hybrids is (a) easily detected in primary reaction, the high responder anti-FMR phenotype being dominant and (b) directed against Db + FMR, F1 mice being low responder against Kb + FMR like the B10 parent. These results suggest that a D region-associated immune response gene controls the cell- mediated anti-FMR reaction, the best available H-2 + FMR antigenic association being chosen by CTL precursors.     

Genetic control of cytolytic T-lymphocyte responses. I. Ir gene control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells

The ability of cytotoxic T lymphocytes (CTL) induced in vitro to trinitrophenyl (TNP)-modified syngeneic cells to cross-reactively lyse a TNP allogeneic spleen target varies among inbred mouse strains. The cross-reactive CTL phenotype was found to be histocompatibility 2 (H-2) linked and to be dominant in F1 hybrid mice. All strains investigated demonstrated cross-reactivity except for some strains bearing portions of the H-2k haplotype. The gene(s) controlling this response maps to the K and/or I-A region of the H-2 complex. We have termed the immune response (Ir) gene responsible for controlling the specificity of CTL induced to TNP-modified syngeneic cells Ir-X-TNP.     

Nonrandom inclusion of H-2K and H-2D antigens in Friend virus particles from mice of various strains

Friend murine leukemia virus (FV), isolated from infectious serum of several mouse strains, has been examined for the presence of H-2 antigens. Following banding of the virus on a discontinuous sucrose gradient, pelleting, and disruption with Nonidet P-40 detergent, virus preparations were tested for their capacity to inhibit the lysis of target cells mediated by various anti-H-2K or anti-H-2D antisera. Virus from mice homozygous for the H-2b, H-2d, H-2g H-2k, and H-2ol haplotypes or heterozygous for the H-2g/H-2k, H-2b/H-2d, and H-2b/H-2k haplotypes was used. Of the six H-2D or H-2K alleles examined, the products of only two, H-2Db and H-2Kk were detected. Virus preparations contained no, one, or both antigens, depending on the genotype of the host animal. Control preparations from normal mouse serum and preparations in which the virus had not been disrupted demonstrated no H-2 activity. Furthermore, attempts to neutralize FV spleen focus forming activity with anti-H-2Db or anti-H-2Kk antisera yielded negative results.     

Growth of SJL/J-derived transplantable reticulum cell sarcoma as related to its ability to induce T-cell proliferation in the host. I. Dominant negative genetic influences of other parent haplotype in F1 hybrids of SJL/J mice

Growth of three transplantable reticulum cell sarcomas (RCS) was studied in a variety of F1 hybrids of SJL/J mice by determination of lymph node (LN) and spleen: body weights ratios 7 and 14 d after intravenous injection of RCS cells. Comparison of BIO.S x SJL and A.SW x SJL with SJL/J showed a negative effect of both the A and the BIO non- H-2 genes, particularly on growth in LN. F1 hybrid resistance was noted with F1 hybrids that carried H-2Dd and was much more evident with F1 hybrids from BIO- than from A-background mice. This resistance was less marked at 14 than at 7 d and was partially overcome by injection of higher tumor doses. Changing the I region in the F1 parent from H-2d to H-2b or H-2f had no effect on growth, but changing to H-2k or H-2d virtually abolished the ability to support tumor growth. This effect appeared partially as a result of the I-E/C and partially of the I-A(B) region and was not overcome by higher tumor dose or longer intervals after injection. There also appeared to be a negative influence on growth of H-2Kk, but this was difficult to differentiate from the I-Ak effect with the available strains. The known proliferative responsiveness that SJL/J Lyt-1 T cells exhibit to Ia determinants on gamma-irradiated RCS cells in vitro was also compared with that of cells from various F1 hybrids. Responsiveness of F1 LN cells was expressed as a percentage of the response in SJL/J LN cells to the same RCS cells, measured as [3H]thymidine incorporation. There was a striking degree of correlation between proliferative responsiveness of F1 LN cells to RCS and the ability of the F1 mice to support tumor growth. This correlation was especially clear with respect to the negative influences of non-H-2 genes, and of H-2 loci in the I region, particularly of I-Ak or -d and of I-E/Ck or -d, but there also appeared to be a (smaller) negative effect of I-Ab or -f. Negative influence of H-2Dd on growth, however, was not reflected in a similarly large effect on the proliferative response. Additional findings showed that LN cells from all F1 hybrids exhibited equivalent syngeneic mixed lymphocyte responses in the presence of polyethylene glycol to mitomycin-treated spleen cells from both the SJL/J and the other parent. The extra high response of F1 cells to RCS cells, as compared with SJL spleen cells, however, was always absent when Ik or -d was contributed by one of the F1 parents. The results suggest a promoting effect of the proliferative response on RCS growth in vivo and, furthermore, an interesting effect of I-A and I-E/C genes, possibly via an interaction product, on the ability of LN cells to be stimulated by Ia determinants on RCS cells.     

Bifunctional major histocompatibility-linked genetic regulation of cell- mediated lympholysis to trinitrophenyl-modified autologous lymphocytes

Murine thymus-derived lymphocytes can be sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells (1, 2). Cytotoxic effector cells were generated which were specific for TNP-modified target cells expressing the same H-2K and H-2D serological regions as the modified stimulator cells (3, 7). Spleen cells from two C57BL/10 congenic strains of mice sharing common I-C, S, and D regions, but differing at K, I-A, and I-B regions, generated different levels of lytic responses to the shared modified H-2Dd products upon sensitization with auto logous TNP-modified cells. Lymphocytes from an F1 between responder and nonresponder strain generated a level of cytolysis toward the H-2Dd modified specificity which was of the same order of magnitude as that obtained with the high responder, irrespective of whether F 1 or either parental strain of modified stimulator cell was used. These results suggest that the modification of H-2Dd products resulted in formation of new antigenic determinants in both parental strains. However, the difference observed in responsiveness appeared to be due to a gene or genes mapping in the K, I-A, or I-B region which influenced the ability of the responding lymphocytes to react to these modified H-2Dd products. Responsiveness was expressed as a dominant trait in the F1.     

Sendai virus-specific, H-2-restricted cytotoxic T lymphocyte responses of nude mice grafted with allogeneic or semi-allogeneic thymus glands

The in vitro secondary cytotoxic T lymphocyte (CTL) response to Sendai virus-treated stimulator cells by primed spleen cells from thymus gland-grafted nude mice was examined. BALB/c (H-2d) nude mice grafted with allogeneic C57BL/10 (H-2b) thymus glands developed CTL responses directed exclusively to Sendai virus-infected H-2d target cells. (C57BL/6 X BALB/c)F1 nude mice grafted with thymus glands of either parent developed CTL responses preferentially against infected target cells expressing the MHC antigens present in the parental thymus graft, but also had detectable activity for infected target cells of the parental haplotype not expressed in the thymus. These results provide evidence against the concept that self recognition by MHC-restricted CTL is directed exclusively by the MCH type of the thymus.     

Mutual recognition of parental and F1 lymphocytes. Selective abrogation of cytotoxic potential of F1 lymphocytes by parental lymphocytes

Four different combinations of F1 hybrid mice [(C57BL/10 X B10.A)F1, (C57BL/10 X B10.BR)F1, B6D2F1, and AKD2F1] were injected intravenously with spleen cells from parental strains. The T-cell-mediated cytotoxic potential of spleen cells from the injected F1 mice was assessed from 4 to 21 d later by in vitro sensitization with trinitrophenyl-modified parental or syngeneic F1 spleen cells (TNP-self) or with allogeneic spleen cells. The cytotoxic potential of the F1 mice to TNP-self as well as to alloantigens was abolished or severely depressed throughout this period when the respective H-2k,a,d parental spleen cells were injected. In contrast, the cytotoxic potential was unaffected or only marginally reduced when H-2b parental cells were injected. The induction of depressed cytotoxic activity was shown to be a result of a population of parental radiosensitive T lymphocytes. The results should be discussed with respect to (a) the genetic and mechanistic parameters associated with the differential depressive effects of parental cells expressing H-2b vs. H-2k,a,d antigens, and (b) the use of this system for investigating allogeneic receptors on T-lymphocyte populations.     

Clonal deletion of postthymic T cells: veto cells kill precursor cytotoxic T lymphocytes

Veto cell-mediated suppression of cytotoxic T lymphocyte (CTL) responses has been proposed as one mechanism by which self-tolerance is maintained in mature T cell populations. We have previously reported that murine bone marrow cells cultured in the presence of high-dose interleukin 2 (IL-2) (activated bone marrow cells [ABM]) mediate strong veto suppressor function. To examine mechanisms by which ABM may suppress precursor CTL (p-CTL) responses, we used p-CTL generated from spleen cells of transgenic mice expressing a T cell receptor specific for H-2 Ld. It was demonstrated that the cytotoxic response by these p-CTL after stimulation with irradiated H-2d/k spleen cells was suppressed by DBA/2 (H-2d) ABM, but not by B10.BR (H-2k) ABM or dm1 (Dd, Ld mutant) ABM. Flow cytometry analysis with propidium iodide staining revealed that these p-CTL were specifically deleted by incubation with H-2d ABM, but not with H-2k ABM. These data indicate that ABM veto cells kill p-CTL with specificity for antigens expressed on the surface of the ABM, and that the mechanism for veto cell activity of ABM is clonal deletion of p-CTL.     

Early development of the T cell repertoire. In vivo treatment of neonatal mice with anti-Ia antibodies interferes with differentiation of I-restricted T cells but not K/D-restricted T cells

Monoclonal antibodies to I-Ak were injected into neonatal H-2k mice for a period of 3 wk. The spleens of such mice are devoid of Ia-positive cells. Allo- and trinitrophenyl (TNP)-self-specific cytotoxic T lymphocyte (CTL) responses in such anti-I-A-treated mice were almost completely abrogated at the end of the 2-3 wk in vivo treatment period. Development of suppressor cells, carry-over of blocking antibodies, lack of responder accessory cells, or defective CTL function were not responsible for the observed defect. As concanavalin A supernatant could restore the defect, it is more likely that the defect is due to the absence of competent Ia-specific T helper cells. In addition, anti-I-A-treated mice exhibit reduced I-A antigen expression in the thymus and defective Ia-bearing accessory cell function in the spleen. It is postulated that, for development of Ia-specific T cells to occur, precursor T cells need to interact with Ia-encoded products in the thymus, and anti-Ia treatment interferes with this process. Finally, the mechanism of this interference was shown to be due to actual removal or functional inactivation of those I-A-positive elements responsible for the education of I-A-recognizing T cells, since in (H-2b X H-2k)F1 mice, treatment with anti-I-Ak antibodies results in abrogation of CTL responses to TNP in association with both parental haplotypes, while in the thymus of these mice expression of both I-Ak and I-Ab was reduced.     

Anti-tumor vaccination in heterozygous congenic F1 mice: presentation of tumor-associated antigen by the two parental class I alleles

Peptide vaccination of homozygous mice against syngeneic tumors using single major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) epitopes elicits effective immune responses against metastatic growth. So far, single-peptide vaccination of patients against their autologous tumors seems to elicit less satisfactory results. In this study, the authors tried to determine whether effective anti-metastatic immunity requires the presentation of peptides restricted by the two parental class I major histocompatibility complex alleles in heterozygous hosts. The immune response against the H-2b-derived 3LL Lewis lung carcinoma was evaluated in heterozygous recombinant congenic F1 mice (Kk x K(b)) and (Kd x K(b)). Vaccination of such heterozygous animals with dendritic cells expressing the two parental H-2K alleles, pulsed with total tumor extract, elicited a potent anti-metastatic response. A comparable response was obtained after vaccination with tumor cells genetically modified to express the two class I alleles. In contrast, vaccination of the heterozygous mice with dendritic cells expressing only one of the parental F1 H-2K alleles or with tumors expressing only one H-2K allele failed to elicit effective immunity against tumor metastasis in recombinant congenic F1 mice. It appears, therefore, that to achieve effective anti-metastatic immunotherapy in heterozygous organisms, presentation of cytotoxic T lymphocyte epitopes restricted by the two parental class I alleles is required.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. I. Differences in activation of helper T cells in C57BL/10 and BALB/c congenic strains

B10.A(5R) mice immunized with C57BL/10 spleen cells demonstrate a normal T-cell-mediated cytotoxicity to H-2Db tumor cells but they do not mount any IgG antibody response to H-2Db alloantigens. B10.A(5R) mice do show a high titered IgG response when immunized with A.BY cells, which differ at H-2Db plus non-H-2 cell surface antigens, or with B10.A(2R) cells, which differ at H-2Db, H-2Kk, and H-2Ik cell surface antigens. These findings indicate a failure of the T-helper cells to induce the switch from IgM to IgG when the H-2Db alloantigens are the only difference on the immunizing cell. In immunizing H-2d mice with congenic H-g2 cells which differ only in the H-2Db region, mice of the C57BL/10 background made only IgM antibodies whereas mice of the BALB/c background made IgG antibodies. This comparison confirms that genes separate from H-2 regulate the T-cell helper function. The genes that influence the T-cell helper function do not regulate the T-cell- mediated cytotoxicity.     

Specificity of cytotoxic T cells from athymic mice

In normal mice, self-H-2 antigens in the thymus have a profound influence on T cell specificity. We have therefore investigated the properties of cytotoxic T lymphocyte (CTL) precursors from athymic nude mice (5) with the notion that they may provide a model system for the study of T cells whose receptro specificity is closer to the germ-line- encoded repertoire. It was found that the precursors of nude CTL are, themselves, THy-1+ cells. The possibility that these nude t cells were derived from the phenotypically normal mother by placental transfer was ruled out. In the presence of T cell growth factor, nude CTL can be induced by polyclonal activation with concanavalin A or by stimulation with allogeneic or trinitrophenyl (TNP)-modified syngeneic stimulator cells, but not by stimulation with minor H antigens in the context of self-H-2. Alloreactive, nude CTL--like those from normal mice-- recognize H-2K- and H-2D-region-encoded antigens in killer-target cell interactions, but, unlike normal CTL, did not cross-react on third- party target cells. Whereas the anti-TNP response of nude mice is H-2 restricted, it does not seem to be influenced by self-H-2 antigens in the same manner as in normal mice. This is suggested by the finding that the immunodominance of H-2k over H-2d in the anti-TNP-self response of normal (H-2d X H-2b)F1 mice is absent in (H-2d X H-2k)F1 nude mice. These observations are discussed in relation to the role of the thymus in the generation of the normal mature T cell receptor repertoire.     

Role of self and foreign antigenic determinants in allogeneic and self- restricted cytotoxic T cell recognition

Murine spleen cells were sensitized in vitro to H-2 disparate allogeneic spleen cells and assayed on syngeneic target cells conjugated with the trinitrophenyl (TNP)-self or the fluorescein isothiocyanate (FITC)-self haptens, or on syngeneic target cells expressing the male H-Y antigen (H-Y self). The results indicated that allo-induced cytotoxic T lymphocytes (CTL) contained effectors that lysed both hapten-self but not H-Y self targets. Furthermore, it was demonstrated that separate populations of those allogeneic CTL were responsible for the lysis of TNP-self and FITC-self targets. This study also showed that cytotoxic effectors generated against the H-Y antigen with lytic activity equal to or greater than that of an allogeneically induced CTL response were unable to lyse hapten-self targets. These findings provide the first evidence that H-2 alloantigens may be unique in their ability to induce effectors that lyse hapten-conjugated autologous targets. These observations are discussed with respect to the self and foreign antigenic determinants involved in allogeneic and self-restricted CTL models.