Immune responses to complex protein antigens I. MHC control of immune responses to bovine albumin

Murine T cell proliferative and antibody responses to the multi-determinant protein bovine serum albumin (BSA) are controlled by Ir genes mapping within the H-2 gene complex. Strains possessing the H-2k, H-2a, and H-2d haplotypes are classified as high responders to BSA. In contrast, H-2b strains are low responders to BSA. Genetic mapping experiments employing strains with recombinant H-2 haplotypes indicate that both T cell proliferative and antibody responses are at least in part regulated by genes within the I-A subregion. Studies on the inhibition of T cell proliferation by monoclonal anti-Ia antibodies are consistent with the assignment of an Ir gene for BSA to the I-A subregion and strongly suggest a role for genes within the I-E/C subregions as well. The MHC-mediated control of antibody responses did not affect the affinity or the isotype of the antibody produced. The relative quantities of antibody specific for each of the three domains of BSA appears to be regulated by H-2-linked BSA Ir genes, and domain III antigenic determinants were found to be dominant in the responses of low-responder mice and in the early response of high-responder mice. This domain III epitope dominance essentially disappears by the tertiary response of high-responder mice.     

Role of macrophages in the augementation of mouse natural killer cell activity by poly I:C and interferon.

The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one "nonsense" peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the "nonsense" peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship between Ir genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu40ala60) (GA) and poly(glu51lys34tyr15) (GLT15). The response to GA was found to be controlled by an Ir gene in the I-A subregion, whereas the anti-GLT15 response was shown to be under dual control, one Ir gene mapping probably in the I-A subregion, and the other in the I-E subregion. We obtained two different lines of evidence suggesting identity of Ir and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derived H-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT15 response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation of Ir genes.     

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.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). V. T cell proliferative response and cellular interactions

We previously demonstrated that in vivo antibody production to HBsAg in the mouse is regulated by at least two immune response (Ir) genes mapping in the I-A (HBs-Ir-1) and I-C (HBs-Ir-2) subregions of the H-2 locus. To confirm that H-2-linked Ir genes regulate the immune response to HBsAg at the T cell level and to determine if the same Ir genes function in T cell activation as in B cell activation, the HBsAg-specific T cell responses of H-2 congenic and intra-H-2 recombinant strains were analyzed. HBsAg-specific T cell proliferation, IL 2 production, and the surface marker phenotype of the proliferating T cells were evaluated. Additionally, T cell-antigen-presenting cell (APC) interactions were examined with respect to genetic restriction and the role of Ia molecules in HBsAg presentation. The HBsAg-specific T cell proliferative responses of H-2 congenic and intra-H-2 recombinant strains generally paralleled in vivo anti-HBs production in terms of the Ir genes involved, the hierarchy of responses status among H-2 haplotypes, antigen specificity, and kinetics. However, the correlation was not absolute in that several strains capable of producing group-specific anti-HBs in vivo did not demonstrate a group-specific T cell proliferative response to HBsAg. The proliferative responses to subtype- and group-specific determinants of HBsAg were mediated by Thy-1+, Lyt-1+2- T cells, and a possible suppressive role for Lyt-1-2+ T cells was observed. In addition to T cell proliferation, HBsAg-specific T cell activation could be measured in terms of IL 2 production, because anti-HBs responder but not nonresponder HBs-Ag-primed T cells quantitatively produced Il 2 in vitro. Finally, the T cell proliferative response to HBsAg was APC dependent and genetically restricted in that responder but not nonresponder parental APC could reconstitute the T cell response of (responder X nonresponder)F1 mice, and Ia molecules encoded in both the I-A and I-E subregion are involved in HBsAg-presenting cell function.     

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10)

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.     

Molecular events in the processing of avidin by antigen-presenting cells (APC)

The immune response of T lymphocytes to avidin was measured by proliferative assays, antibody production and delayed-type hypersensitivity. Mice ofH-2 ^k haplotypes were found to be low responders, whereas mice of other haplotypes, and particularly ofH-2 ^s , were high responders.Ir genes controlling this response were mapped to theI subregion ofH-2. Helper T cells were found to be responsible for the Ir phenotype of antibody production. These results indicate the feasibility of using the avidin-biotin complex as a tool for studying molecular mechanisms by which antigens underIr gene control are processed and presented to T lymphocytes.Abbreviations used in this paper Ir genes, immune-response genes - H-2 murine major histocompatibility complex - APC antigen-presenting cell - OA ovalbumin - BSA bovine serum albumin - DNP dinitrophenyl - DNP-OA DNP-ovalbumin - DNP-Av DNP-avidin - DNP-BSA DNP-bovine serum albumin - CFA complete Freund's adjuvant - PPD purified protein derivative - PBS phosphatebuffered saline - IP intraperitoneal - LNC lymph-node cells - DTH delayed-type hypersensitivity     

Genetic control of the murine immune response to cholera toxin

This study was undertaken to determine whether previously noted differences in the immune response of inbred strains of mice to cholera toxin (CT) might be under immune response gene control. A series of inbred, congenic, and intra-H-2I region recombinant mouse strains were tested for responsiveness to CT after i.p. immunization with 0.1 micrograms CT in alum. Samples of plasma were collected at intervals before and after priming and boosting. IgG and IgA anti-CT were measured by ELISA. In three different sets of congenic strains, the level of IgG anti-CT clearly depended on the H-2 haplotype of the strain rather than on any background or Igh genes. Strains with the H-2b and H-2q haplotypes were high responders, and strains with the H-2k, H-2s and H-2d haplotypes were low responders. Within the H-2 complex, the IgG anti-CT response was mapped to the I-A subregion with the use of congenic intra-H-2I region recombinant strains. In contrast to these results with IgG anti-CT, plasma IgA anti-CT levels were uniformly low and indeterminate. We conclude that the murine IgG anti-CT response is controlled by a locus within the I-A subregion of H-2--a remarkable finding, considering the known abilities of this toxin to bind to and to directly stimulate lymphocytes.     

Genetic control of immune response to sperm whale myoglobin in mice. I. T lymphocyte proliferative response under H-2-linked Ir gene control.

Studies on the genetic control of immune response to sperm whale myoglobin were initiated. As demonstrated in this paper, the T lymphocyte proliferative response to whale myoglobin is under H-2-linked Ir gene control. Mice of H-2d, H-2f, and H-2s haplotypes were high responders to the myoglobin, whereas haplotypes H-2b, H-2k, H-2p, H-2q, and H-2r were low responders. The Ir gene(s) was localized between H-2K and H2D regions, since the recombinant strain A.TL (KsIkSkDd) was a low responder and A.TH (KsIsSsDd) was a high responder. Further studies with recombinant strains revealed that the expression of the high-responder I-Ad or Ias alleles was sufficient to give a good response, since strains D2.GD (d d b b b b b b) and B10.HTT (s s s s k k k d) were high responders. The expression of the I-Cd allele in strains B10.A (k k k k k d d d) and B10.A(5R) (b b b k k d d d) also gave high response, and thus suggested a second Ir gene, derived from the H-2d haplotype. The finding that expression of the I-Cs allele in B10.S(8R) (k k ? ? s s s s) did not result in high response suggests the lack of the second Ir gene in the high-responder H-2s haplotype.     

T lymphocyte-enriched murine peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera.

The recent development of a reliable murine T lymphocyte proliferation assay has facilitated the study of T lymphocyte function in vitro. In this paper, the effect of anti-histocompatibility antisera on the proliferative response was investigated. The continuous presence of anti-Ia antisera in the cultures was found to inhibit the responses to the antigens poly (Glu58 Lys38 Tyr4) [GLT], poly (Tyr, Glu) ploy D,L Ala-poly Lys [(T,G)-A--L], poly (Phe, Glu)-poly D,L Ala-poly Lys [(phi, G)-A--L], lactate dehydrogenase H4, staphylococcal nuclease, and the IgA myeloma protein, TEPC 15. The T lymphocyte proliferative responses to all of these antigens have previously been shown to be under the genetic control of major histocompatibility-linked immune response genes. The anti-Ia antisera were also capable of inhibiting proliferative responses to antigens such as PPD, to which all strains respond. In contrast, antisera directed solely against H-2K or H-2D antigens did not give significant inhibition. Anti-Ia antisera capable of reacting with antigens coded for by genetically defined subregions of the I locus were capable of completely inhibiting the proliferative response. In the two cases studied, GLT and (T,G)-A--L, an Ir gene controlling the T lymphocyte proliferative response to the antigen had been previously mapped to the same subregion as that which coded for the Ia antigens recognized by the blocking antisera. Finally, in F1 hybrids between responder and nonresponder strains, the anti-Ia antisera showed haplotype-specific inhibition. That is, anti-Ia antisera directed against the responder haplotype could completely block the antigen response controlled by Ir genes of that haplotype; anti-Ia antisera directed against Ia antigens of the nonresponder haplotype gave only partial or no inhibition. Since this selective inhibition was reciprocal depending on which antigen was used, it suggested that the mechanism of anti-Ia antisera inhibition was not cell killing or a nonspecific turning off of the cell but rather a blockade of antigen stimulation at the cell surface. Furthermore, the selective inhibition demonstrates a phenotypic linkage between Ir gene products and Ia antigens at the cell surface. These results, coupled with the known genetic linkage of Ir genes and the genes coding for Ia antigens, suggest that Ia antigens are determinants on Ir gene products.     

Complementation between a gene of theI-E subregion and a non-H-2 gene in the class-specific suppression of IgG2a antibody to sheep erythrocytes

A low level of IgG2a antibodies is observed in B10 mice after primary immunization with SRBC. Analysis of the response in different H-2^b mice and among B10 animals with differentH-2 haplotypes reveals that this selective isotype deficiency is under the control of at least two genes: a background gene and anH-2-linked gene. Responses ofH-2 recombinant B10 strains map theH-2-linked gene to theI-E subregion. Evidence is presented for complementation betweenH-2 and non-H-2 genes in the determination of the low responder phenotype. Low responsiveness appears to be inherited as a dominant trait. Possible functions of the two series of genes are discussed in relation to suppressor mechanisms.     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

Ir gene control of immune responses to insulins. II. Phenotypic differences in T cell activity among nonresponder strains of mice

Immune responses by mice to heterologous insulins are controlled by H-2 linked Ir genes. In studies to determine the mechanisms responsible for nonresponsiveness, we found that although pork insulin failed to stimulate antibody or proliferative responses in H-2b mice, it did prime T cells that can express helper activity in adoptive recipient mice. This helper activity was insulin-specific in both elicitation and expression. In studies presented in this paper, we have extended this analysis to the response patterns of helper T cells stimulated by sheep, horse, and rat insulins in mice bearing different H-2 haplotypes. The results demonstrate that nonresponder forms of insulin, including rat insulin, prime T cells in H-2b and H-2d, but not H-2k, mice. These results suggest that regulation of nonresponsiveness to insulin appears to be through different pathways in mice bearing different H-2 haplotypes.     

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.     

Genetic control of the humoral response to an H-2 public specificity.

The humoral response of mice to an H-2 public specificity, termed H-2. '28' was found to be under genetic control. The genes determining this specificity were mapped to both the H-2K and H-2D regions, suggesting possible structural homologies between the products determined by these two regions. Genetic analyses indicated that a single non-H-2-linked gene regulates the anti-H-2. '28' response. In a backcross study, no linkage was detected between this putative H-2. '28' Ir gene and either the V H region or the Ly-2 locus to which the K-light chain locus is linked. Thus, a regulatory rather than a structural genetic locus seems a more likely basis for differences in response to this antigen. Our data further indicate that control of the humoral response to H-2. '28' is determinant specific since responses of the same backcross mice to other K and D alloantigens were not found to be subject to the same control.     

Monoclonal T cell responses to two epitopes on a single immunogen controlled by two distinct genes

The fine specificities of immune T cells were studied in a system in which the response to the antigen can involve two immune response (Ir) genes and two epitopes on a single synthetic polypeptide immunogen. The (BALB/c X SJL)F1 (H-2d X H-2s) mice can respond to the random terpolymer poly(Glu55, Lys36, Phe9) (GLPhe) through the H-2d-linked Ir gene (Ir-d) or through the complementing Ir gene (Ir-dxs), which controls the immune response to poly(Glu, Phe), epitopes that are present in GLPhe. Nine groups of monoclonal T cells were obtained from (H-2d X H-2s)F1 mice immunized with GLPhe. These groups were delineated by the differences in major histocompatibility (MHC) restriction on antigen-presenting cells (APC) and the cross-reactions with GPhe or GLT. A unique T cell line was discovered that can react to the three polymers (GLPhe, GLT, GPhe) even though GLT and GPhe immune T cells do not normally show reciprocal cross-reactions. The monoclonal T cells retain helper activities in the Mishell-Dutton culture. Although the activation of T cells is antigen specific and MHC restricted, the subsequent B cell response is nonspecific.     

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.     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): Subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship betweenIr genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu⁴⁰ala⁶⁰) (GA) and poly(glu⁵¹lys³⁴tyr¹⁵) (GLT¹⁵). The response to GA was found to be controlled by anIr gene in theI-A subregion, whereas the anti-GLT¹⁵ response was shown to be under dual control, oneIr gene mapping probably in theI-A subregion, and the other in theI-E subregion. We obtained two different lines of evidence suggesting identity ofIr and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derivedH-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT¹⁵ response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation ofIr genes.