Regulation of the in vitro presentation of minor lymphocyte stimulating determinants by major histocompatibility complex-encoded immune response genes

Activation of murine B lymphocytes in a splenocyte stimulator population with affinity-purified goat anti-mouse IgD (G alpha M delta) antibody was previously shown by this laboratory to enhance the presentation of strongly stimulatory major histocompatibility complex (MHC) and minor lymphocyte-stimulating (Mlsa,d) determinants in a primary mixed lymphocyte reaction. In the present study, the G alpha M delta treatment of murine splenocytes was employed to enhance the detection of the weakly stimulatory non-MHC Mlsc determinant in order to study the role the MHC might play as a restricting element for the recognition of these minor antigens in a primary mixed lymphocyte reaction. Indeed, enhanced T cell proliferation to Mlsc determinants presented on G alpha M delta-treated splenocytes was observed when the responder and activated H-2-compatible stimulator cell shared certain MHC haplotypes. High responsiveness was associated with the H-2a,k,j,p haplotypes, intermediate responsiveness was associated with the H-2f,g haplotypes and low responsiveness was associated with the H-2b,s haplotypes. (Low X high responder)F1 T cells preferentially responded to the Mlsc determinants presented on G alpha M delta-treated stimulator cells of the F1 or parental high responder H-2 haplotype. When mitomycin C instead of irradiation was used to inactivate normal (non-IgD-treated) splenocytes, a similar preferential response of T cells to Mlsc determinants presented on stimulator cells of a high responder H-2 haplotype was also observed. The inability of G alpha M delta-treated splenocytes of the low responder haplotype to elicit substantial levels of T cell proliferation across an Mlsc difference could not be attributed to the failure of these stimulator cells to become activated by the anti-Ig antibody. In addition, co-culture experiments could not identify the poor T cell response to Mlsc determinants presented on certain MHC haplotypes as being caused by the induction of nonspecific suppressor cells. Presentation of Mlsc determinants caused by transgene product complementation was detectable in F1 mice derived by crossing one parent that had the Mlsc non-MHC genes and a poorly permissive H-2 haplotype with a parent that expressed a permissive H-2 haplotype but lacked the Mlsc non-MHC genes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Major histocompatibility complex-conferred resistance to Theiler's virus-induced demyelinating disease is inherited as a dominant trait in B10 congenic mice.

Intracerebral inoculation of Theiler's murine encephalomyelitis virus into susceptible strains of mice produces chronic demyelinating disease in the central nervous system characterized by persistent viral infection. Immunogenetic data suggest that genes from both major histocompatibility complex (MHC) and non-MHC loci are important in determining susceptibility or resistance to demyelination. The role of the MHC in determining resistance or susceptibility to disease can be interpreted either as the presence of antigen-presenting molecules that confer resistance to viral infection or as the ability of MHC products to contribute to pathogenesis by acting as viral receptors or by mediating immune attack against virally infected cells. These alternatives can be distinguished by determining whether the contribution of the MHC to resistance is inherited as a recessive or dominant trait. Congenic mice with different MHC haplotypes on identical B10 backgrounds were crossed and quantitatively analyzed for demyelination, infectious virus, and local virus antigen production. F1 hybrid progeny derived from resistant B10 (H-2b), B10.D2 (H-2d), or B10.K (H-2k) and susceptible B10.R111 (H-2r), B10.M (H-2f), or B10.BR (H-2k) parental mice exhibited no or minimal demyelination, indicating that on a B10 background, resistance is inherited as a dominant trait. Although infectious virus, as measured by viral plaque assay, was cleared inefficiently from the central nervous systems of resistant F1 hybrid progeny mice, we found a direct correlation between local viral antigen production and demyelination. These data are consistent with our hypothesis that the immunological basis for resistance is determined by efficient presentation of the viral antigen to the immune system, resulting in local virus clearance and absence of subsequent demyelination.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

The role of H-2 and non-H-2 antigens and genes in the rejection of murine cardiac allografts

Genes of the major histocompatibility complex (MHC) in the mouse (H-2 complex) have been shown to be an important factor in determining the immune responsiveness of various strains of mice to isolated antigens (e. g., lysozyme). The role of MHC genes in controlling the responsiveness of mice to multiple alloantigens is less well-defined, and although non-MHC genes have been shown to be important in determining responsiveness in some systems (e. g., haptens), they have not been demonstrated as yet to influence the rejection of vascularized organ allografts. In this study, the responsiveness of mice to vascularized cardiac allografts transplanted across well-defined major (H-2) and minor (non-H-2) histocompatibility barriers was investigated using congenic mice in 32 different donor/recipient combinations. The results show that both H-2 and non-H-2 gene products can act as target alloantigens for rejection. At the responder level, they may interact to effect responsiveness of a recipient strain to multiple alloantigens. In no case in this study has any one gene or group of genes been found to confer universal high or low responder status.     

Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes.

Experimental autoimmune uveoretinitis (EAU) in animals is a T cell-mediated autoimmune response directed against cells of the neural retina, in particular the photoreceptors. EAU can be induced in susceptible strains of mice by immunization with purified retinal Ag, and serves as a model for human uveitis. Because strong HLA associations have been noted in a number of human uveitic diseases, we investigated the role of MHC vs non-MHC genes in the control of susceptibility to ocular autoimmunity using the mouse EAU model. Selected strains representing most of the known independent H-2 haplotypes, as well as several H-2-recombinant and congenic strains, were immunized with interphotoreceptor retinoid-binding protein. Ocular pathology was induced in strains of the H-2k haplotype and their I-A-matched congenics, as well as in strains of the H-2r, H-2b, and H-2d haplotypes. In a series of experiments utilizing intra-H-2 recombinant strains, MHC control of susceptibility was tentatively mapped to the I-A subregion of the H-2k. Expression of the I-Ek gene product was not required for susceptibility to EAU, and in fact appeared to have an ameliorating effect on disease. Incidence and severity of disease obtained in strains sharing the same H-2 on a different background, or sharing the same background in the context of a different H-2, indicated that non-MHC genes contribute significantly to the regulation of EAU. Disease expression of susceptible H-2 haplotypes was highest in strains with B10 background (permissive) and ranged from intermediate to absent in strains with other (nonpermissive) backgrounds. The data suggest that although the ability to develop ocular pathology is dependent on the I-A subregion of the H-2, the final expression of disease in susceptible haplotypes is largely determined by background, non-MHC genes.     

Intrinsic in vitro abnormalities in dendritic cell generation caused by non-MHC non-obese diabetic genes

Genes outside the MHC create a general susceptibility to autoimmunity in non-obese diabetic (NOD) mice. In this study, we describe marked differences in dendritic cell generation, in vitro, caused by non-MHC NOD genes. Bone marrow cells from NOD.H-2k mice cultured in vitro with GM-CSF and IL-4 generated a reduced yield of dendritic cells when compared to bone marrow cells from B10.H-2k mice. This was due to failure to pass through successive rounds of cell division and elevated levels of apoptosis in NOD.H-2k precursor cells. This aberrant response to GM-CSF and IL-4 was unique to the NOD.H-2k background when compared to bone marrow cells from other H-2k congenic strains, and coculture experiments showed that it was cell-autonomous. Overall, the results described in this study demonstrate a striking effect of non-MHC NOD genes on dendritic cell generation from myeloid precursors derived from the NOD.H-2k strain. These results identify a useful genetic model to explore the regulation of dendritic cell formation. Conceivably, the dysregulation of the dendritic cell system described here may contribute to the generalized defects in self-tolerance in the NOD strain.     

H-2-associated and background genes influence the development of a murine retrovirus-induced immunodeficiency syndrome

Host genetic determinants of resistance or susceptibility to a retrovirus-induced immunodeficiency syndrome, termed MAIDS, were evaluated in Fv-1b mice infected with the mixture of ecotropic, MCF, and defective murine leukemia viruses designated LP-BM5 murine leukemia viruses. Genes of the MHC were shown to exert a major influence on the development of disease and the extent of virus spread in mice infected as adults. Strains bearing the b, f, k, q, r, and s haplotypes were moderately to highly susceptible to MAIDS whereas mice of the d haplotype were the most resistant. Resistance to disease was strongly associated with inhibition of mink cell focus-inducing virus spread and, to a lesser extent, with inhibition of ecotropic virus expression. Mapping studies localizing resistance associated with the d haplotype to H-2Dd were confirmed by the demonstration that B6 mice carrying this gene as a transgene or by recombination were resistant to disease. Penetrance of resistance to disease associated with expression of H-2Dd was markedly influenced by MHC genes mapping to the left of H-2D and by non-MHC loci such that some strains bearing this gene were highly susceptible to MAIDS. The combined effects of MHC and background genes among 40 strains examined yielded a remarkably wide spectrum of disease phenotypes with the onset of advanced disease ranging from 10 wk to 72 wk postinfection. Resistance to disease in moderately to highly resistant strains was shown to develop with age. Unexpectedly, the disease resistant phenotype was found to be a recessive trait.     

Immune response gene control of determinant selection. II. Genetic control of the murine T lymphocyte proliferative response to insulin.

The genetic control of the murine T cell proliferative response to insulin was examined. It was found for two responder strains of mice that each recognizes a different determinant on the insulin molecule. H-2b mice recognize a determinant in the A chain loop of insulin whereas H-2d mice recognize a determinant that resides in the B chain, possibly in the last eight amino acids. Using H-2 recombinant strains of mice, the location of Ir gene control of the response to both determinants was mapped to the K region and/or I-A subregion of H-2. The possibility of non-MHC regulation of MHC-controlled immune responses is suggested by studies of recombinant inbred strains of mice.     

Limited T cell receptor beta-chain usage in the sperm whale myoglobin 110-121/E alpha dA beta d response by H-2d congenic mouse strains.

The specificity and TCR gene usage of a panel of sperm whale myoglobin (SpWMb)-reactive T cell clones from DBA/2 mice have previously been characterized, to study structure-function relationships between components of the ternary complex consisting of Ag, TCR, and MHC class II molecules, whose interaction leads to Th cell activation. These DBA/2 clones were specific for epitopes within the residue 110 to 121 region of SpWMb, in the context of the mixed isotype molecule E alpha dA beta d, and expressed the TCR V beta 8.2 gene element. SpWMb-specific T cell hybridomas from the H-2d-congenic B10.D2 mouse strain, which differs from the DBA/2 strain only in the non-MHC background, were generated and compared with the T cell hybridomas from DBA/2 mice, in order to investigate the influence of non-MHC genes on the specificity of the T cell response to the 110-121 epitope. V beta usage by these hybridomas was very homogeneous; three of three DBA/2 and eight of nine B10.D2 hybridomas specific for the 110-121 epitope, in the context of the mixed isotype molecule E alpha dA beta d, expressed the V beta 8.2 gene product. Nucleotide and amino acid sequences of D beta, J beta, and N regions were also similar. One 110-121/E alpha dA beta d-specific B10.D2 hybridoma used V beta 7, a V beta that is clonally deleted in DBA/2 mice. These experiments suggest that a similar set of TCR beta genes are used to respond to a given epitope, regardless of non-MHC background, and they support the hypothesis that, despite great variability between individuals in their non-MHC background genes, human HLA-associated diseases might result from the formation of a particular ternary complex consisting of a shared MHC molecule, a common "disease-associated" epitope, and a shared TCR.     

Genetic control of the immune response in mice to Leishmania mexicana surface protease

Congenic mouse strains were tested in the lymphocyte proliferation assay for their response to the purified surface protease of Leishmania mexicana (gp63). The data obtained allow us to distinguish three different patterns of response, influenced both by H-2 (class II) and non-H-2 genes. Mice of the C57BL/10 (B10) background carrying H-2 haplotypes b,q, and r were found to be high responders; those carrying H-2 haplotypes d, j, v, and z were low responders; and those with H-2a, H-2f, H-2k, H-2p, and H-2u haplotypes were intermediate responders. Studies with H-2 recombinant strains indicated that the high responsiveness on the B10 background was determined by the Ab allele and the low responsiveness influenced by the Ad allele. Other genes besides H-2 appear to have a role in the immune response as shown by the fact that some strains with BALB, DBA, or C3H background differed in their pattern of responsiveness from B10 background strains carrying the corresponding H-2 haplotypes. By using recombinant protein, the influence of the leishmanial surface lipophosphoglycan that might co-purify with gp63, on the MHC restriction of the response to gp63 was excluded. The immune response to gp63 did not correlate with susceptibility of mouse strains to cutaneous infection with L. mexicana promastigotes.     

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

Alloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2^pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2^b), BALB/c (H2^d), CBA (H2^k), and DBA/1 (H2^q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Genetic control of sensitivity to Moloney leukemia virus in mice V. Role of H-2-linked genes in the control of anti-FMR cytolytic T lymphocytes

Three H-2-linked genes, Rmv1, Rmv2, and Rmv3 control the resistance of mice against Moloney virus (MLV)-induced leukemias. It has been shown previously that they function as immune response (Ir) genes regulating the level of antivirus antibodies. In the present experiments, the cell-mediated anti-tumor response has been studied in a series of inbred strains selected for their resistance or sensitivity to the MLV-induced disease. We failed to detect any relationship between resistance and sensitivity and the ability to produce cytolytic T lymphocytes (CTL) directed against the virus-induced FMR cell surface antigen. Furthermore, the role of each Rmv gene has been studied separately using congenic pairs of mice differing at only one of these loci: we failed to evidence any influence of these genes in the cell-mediated antitumor reactions as measured by the ability to lyse syngeneic FMR(+) target cells. Nevertheless a gene mapping in the D region of the MHC but probably different from Rmv3 controls the response of a subset of anti-FMR CTL restricted by the H-2K^k antigens, with higher response in H-2D^d than in H-2D^b animals. This observation confirms the existence of H-2D region associated Ir genes regulating the CTL-mediated antitumor immune responses by choosing the subset of responder CTL, and suggests that a fourth H-2-linked gene plays some role in the genetic control of the anti-FMR immune response.     

NK gene complex and chromosome 19 loci enhance MHC resistance to murine cytomegalovirus infection

An H-2^k MHC locus is critical for murine cytomegalovirus (MCMV) resistance in MA/My mice and virus control is abolished if H-2^k is replaced with H-2^b MHC genes from MCMV-susceptible C57L mice. Yet, H-2^k resistance varies with genetic background; thus, modifiers of virus resistance must exist. To identify non-MHC resistance loci, spleen and liver MCMV levels and genome-wide genotypes were assessed in (C57L × MA/My) and (MA/My × C57L) F₂ offspring (representing 550 meioses). Significantly, a non-Mendelian frequency of MHC genotypes was observed for offspring of the latter cross. Quantitative trait loci (QTL) and their interaction potential in MCMV resistance were assessed in R/qtl; QTL on chromosomes 17, 6, and 19 affected MCMV levels in infected animals. A chromosome 6 QTL was linked with the NK gene complex and acted in an additive fashion with an H-2^k MHC QTL to mitigate spleen MCMV levels. We provide biological confirmation that this chromosome 6 QTL provided MCMV control independent of H-2^k via NK cells. Importantly, both chromosome 6 and 19 QTLs contribute to virus control independent of H-2^k. Altogether, MHC and non-MHC MCMV-resistance QTL contribute in early resistance to MCMV infection in this genetic system.     

Identification and characterization of T helper epitopes in the nucleoprotein of influenza A virus

By using a series of overlapping synthetic peptides that cover more than 95% of the amino acid sequence of nucleoprotein (NP) of influenza A/NT/60/68 virus, five Th cell epitopes in B10.S (H-2s), BALB/c (H-2d), CBA (H-2k), and B6 (H-2b) mice have been identified. The specificity of Th cell recognition of epitopes is largely dependent on the H-2 haplotype of the responding mouse strain. However, two out of the five Th epitopes defined could be recognized by mice of more than one haplotype, implying that the primary sequence of protein antigens could also influence the selection of dominant T cell epitopes by the immune system. Immunization of B10.S mice with peptide 260-283 generated strong Th cell response against type A influenza viruses. In the other three strains of mice tested, priming with helper peptides induced a stronger antipeptide than antiviral T cell response. However, the low responsiveness to virus in these mice could be partially overcome by immunization with a mixture of several helper peptides. The Th epitopes are defined by the ability of the peptides to stimulate class II MHC restricted CD4+ T cells to proliferate and to produce IL-2 in vitro. When compared with the known epitopes on NP recognised by class I restricted CD8+ cytotoxic T cells, it appears that Th and cytotoxic T cell epitopes are nonoverlapping. The AMPHI and Motifs methods were employed to analyze the sequence of NP and predict the potential dominant sites in the molecule. The predictions are compared with the experimental data obtained and the implications discussed.     

H-2 effects on cell-cell interactions in the response to single non-H2 alloantigens. IV. Variations in the proliferative response to H-Y and H-3

Individual mice were tested for their proliferation T-cell response to H-Y- and H-3-incompatible stimulator cells in secondary mixed lymphocyte culture. Responders expressing the H-2b haplotype were restricted in their response to stimulators presenting H-Y and H-3 in the context of H-2b. Lymphocytes from individual B10 females proliferated in response to H-Y presented with I-Ab and Db. The ratio of I-Ab/Db-restricted responses varied between individual responders, indicating significant qualitative variation between genetically identical responders. The majority of the proliferative response in all tested mice was restricted to the entire H-2b haplotype suggesting complementation of I-Ab- and Db-region genes in presenting the H-Y antigen. Similar observations were made in the response of individual B10.LP mice to the H-3 antigen. H-3-specific, proliferating T cells were restricted to H-3 antigen presented with KbAb and Db with significant variation between individuals in proliferative response to H-3 plus KbAb and Db. In contrast to the response to H-Y, the proliferative response to H-3 plus H-2b could be accounted for by the summation of the proliferative responses to H-3 plus KbAb and Db. These observations demonstrate that the proliferative response to non-H-2H antigens in the context of I-region determinants is not a sine qua non for the T-cell response to these antigens. Further, the individual qualitative and quantitative variation observed with individual genetically identical mice has strong implications for our knowledge of intrastrain variation in immune responsiveness and the characterization of inbred strains for immune responsiveness.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Studies of the mixed lymphocyte reaction by the virus plaque assay.

The virus plaque assay has been developed as a tool for enumerating activated lymphocytes. In previous studies using mitogens it was found that the assay detects activated T- but not activated B-lymphocytes. In the present studies, the mixed lymphocyte reaction was studied by the virus plaque assay as well as by incorporation of thymidine and development of cytotoxic lymphocytes. In combinations differing at the entire major histocompatibility complex, approximately 1% of the cells were activated and treatment with anti-thy.1 serum totally abrogated the virus plaque forming cell (V-PFC) response. In studies on the A.TH-A.TL recombinants and the AQR-B10/ 6R recombinant strains, incompatibility at the H-2-K or H-2-D loci were found not to be capable of activating T-cells to produce virus plaques. In contrast, differences in I or I plus S regions caused a marked T-cell activation, 3–6% of the cells being V-PFC. There was a general parallelism between thymidine incorporation and the V-PFC, and it was not possible to dissociate the cell types carrying out each of these functions by kinetic studies. However, preinfection of responding cells with the virus used in these studies, vesicular stomatitis virus, caused a complete abrogation of thymidine incorporation, indicating that the activation of virus is an earler stage than DNA synthesis, and that viral activation will block further cell replicaton and is probably a lytic event.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.