Inhibition of antigen-specific T lymphocyte activation by structurally related Ir gene-controlled polymers. II. Competitive inhibition of I-E-restricted, antigen-specific T cell responses.

Our previous studies have defined a highly specific competitive inhibition between a pair of structurally related antigens (GT and GAT) for antigen presentation by accessory cells. The present report investigates this phenomenon in a second antigenic system, which is controlled by a distinct Ir gene product. Two GL phi-specific, I-Ed-restricted, interleukin 2-producing T cell hybridomas were constructed. The antigenic fine specificity of these two hybrid clones was distinct. One hybrid reacted solely with GL phi while the second cross-reacted with GLleu and GLT. These latter two copolymers, as well as the antigen GL, were found to inhibit the GL phi response of the non-cross-reactive hybrid. The structurally related antigen G phi was not inhibitory for this clone's response. The cross-reactive GL phi hybrid could also be inhibited, but, in this case, G phi and not GL caused the inhibition. Reciprocal inhibitions could be demonstrated between these and other hybrids (e.g., GAT responsive), indicating a very high degree of specificity to the inhibition. The inhibition caused by the various copolymers was reversible by increasing the concentration of GL phi, This effect was localized to the antigen-presenting cell and not the T cell hybridoma. Functionally, this competition did not appear to be for antigen uptake or general antigen processing. These findings generalize the phenomenon of antigen competition to a second antigen system in the context of a second Ia molecule. The possible mechanisms accounting for the complex pattern of specificities in this system are discussed.

Selective modification of a private I-A allo-stimulating determinant(s) upon association of antigen with an antigen-presenting cell.

A large panel of alloreactive, interleukin 2 (IL-2)-producing T cell hybridomas was constructed from B10 alpha BALB/c primary mixed lymphocyte cultures (MLC). Functional hybrids had specificity for either I-Ad or I-Ed. These cells were used to probe determinants on Ia molecules in an attempt to detect molecular association between a nominal antigen and an Ia molecule on an antigen-presenting cell (APC). The response of a small number of these clones was significantly blocked by the addition of the Ir gene-controlled copolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) to culture. A comparison of the inhibited and uninhibited hybrids revealed an identical dose response curve. Further, both types of hybrids were activated by the same stimulator cell and frequently recognized the identical Ia molecule on that cell. Nevertheless, the inhibitory effect of GAT was localized to the stimulator cell and not to the T cell hybrids. All of the hybrids whose stimulation was blocked had specificity for the I-A molecule, which is the gene product known to control and restrict responsiveness to GAT. Further, only GT, but not a number of other related antigens, was also specifically inhibitory, which correlates with the known associational specificity of these antigens on an APC. Finally, the same stimulator cell could be shown to coordinately lose an allostimulatory determinant(s), while it was gaining an I-Ad plus GAT determinant(s). The implications of these findings on the nature of antigen-Ia association and on the role of polymorphic Ia determinants are discussed.

Inhibition of antigen-specific T lymphocyte activation by structurally related Ir gene-controlled polymers. Evidence of specific competition for accessory cell antigen presentation.

The interaction of nominal Ag with major histocompatibility complex (MHC)-restricted T cells and accessory cells was studied by analyzing the effect of structurally related antigens on the response of antigen-specific MHC-restricted T cell hybridomas. The copolymer L-glutamic acid50-L-tyrosine50 (GT) completely inhibits the response of L-glutamic acid60-L-alanine40-L-tyrosine10 (GAT)-specific, I-Ad-restricted T cell hybridomas to GAT plus accessory cells. This inhibition is specific, as hybridomas of other specificities are not inhibited under identical conditions, and is unique to the GT antigen, as other similar copolymers are not inhibitory. The inhibitory effect is reversible by adding increasing amounts of GAT. Antigen-pulsing experiments localized the inhibition to the level of antigen-presenting cell (APC). GT-prepulsed APC are not inhibitory in cell-mixing experiments and can present other antigens. GT only inhibits the nominal antigen-directed component of a GAT-specific, autoreactive hybrid's response. Together these findings suggest that GT causes inhibition by competing for GAT association at the accessory cell. GT interferes with GAT presentation by an I-Adxb F1 APC to a BALB/c, I-Ad-restricted, but not B10, I-Ab-restricted, T cell hybridoma, and GT inhibits presentation by GAT-prepulsed APC. The implications of these findings for MHC-restricted presentation of antigen are discussed.

Cellular localization of anti-DNP-PLL and anticonveyor albumin antibodies in genetic nonresponder guinea pigs immunized with DNP-PLL albumin complexes.

Genetic nonresponder guinea pigs incapable of an immune response to DNP-PLL alone were immunized with DNP-PLL complexed to ovalbumin or bovine serum albumin. Under these circumstances the animals produce both anti-DNP-PLL antibodies and antibodies directed against the conveyor albumin. Thus immune response to DNP-PLL complexed to conveyor albumin molecules can serve as a simple model of hapten-carrier relationships. To explore these relationships the question whether these two types of antibody are synthesized by the same or by different plasma cells was investigated by a combination of a double immunofluorescent technique and radioautographic localization of radioactive antigen. It was shown that the anti-DNP-PLL antibodies and the antibodies against the carrier albumin molecule were produced in separate cells. No cell-producing antibodies with both specificities were detected out of 526 cells studied.

Genetic control of immune responses in vitro. VI. Experimental conditions for the development of helper T-cell activity specific for the terpolymer L-glutamic aicd60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice.

Mice which are genetic nonresponders to the random terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) not only fail to develop GAT-specific antibody responses when stimulated with soluble GAT either in vivo or in vitro, but develop GAT-specific T cells which suppress the GAT-specific plaque-forming cell response of normal nonresponder mice stimulated with GAT complexed to methylated bovine serum albumin (MBSA).Thus, both responder and nonresponder mice have T cells which recognize GAT. However, nonresponder mice can develop GAT-specific helper T cells if immunized with GAT bound to MBSA or to macrophages. The relevance of Ir gene-controlled responses is discussed.

Mapping of the immune response genes in the major histocompatibility complex of the Rhesus monkey.

Interest in the Ir genes of rheus monkeys stems from their phylogenetic relationship to man and the extensive data already available on the major histocompatibility complex of the monkey. At least two independent dominant H-linked Ir genes have been identified in the rhesus. These genes control the ability of monkeys to respond to the random linear copolymer of glutamyl alanine (GA), or the dinitrophenyl conjugate of glutamyl lysine (DNP-GL). These synthetic polymers can elicit weak delayed-type skin reactions and strong humoral responses in some monkeys. In a series of unrelated monkeys phenotyped for the serologically defined RhL-A specificities of both segregant series, there were no correlations between any RhL-A specificity and responder status to the GA or DNP-GL polymers. However, segregation analysis of 21 rhesus families sired by 3 fathers indicated the capacity of the offspring to form antibodies was associated with genes coded for in the RhL-A complex. In three monkeys, verified recombination within the RhL-A complex between the genes coding for the serologically defined determinants (SD loci) and the gene(s) controlling the lymphocyte-activating determinants (Lad loci) responsible for mixed lymphocyte reactivity was established. In two of these monkeys the immune response genes controlling the DNP-GL response segregated with the Lad genes, while in the third case the Ir-GL gene segregated with the SD loci, tentatively localizing the Ir-GL gene between the SD and Lad loci. In addition, we have shown that genetically distinct genes control responsiveness to DNP-GL and GA. These genes were separated by recombination, thus one monkey inherited the Lad, Ir-GL, and SD loci from one paternal haplotype and by crossing over inherited the gene controlling GA responsiveness from the other paternal haplotype. The fine structure mapping of the RhL-A gene complex is compared with the H-2 and HL-A gene complexes. Several striking similarities were noted.

Genetic control of specific immune suppression. I. Experimental conditions for the stimulation of suppressor cells by the copolymer L-glutamic acid50-L-tyrosine50 (GT) in nonresponder BALB/c mice.

In the present studies we have confirmed that the random copolymer of L-glutamic acid50-L-tyrosine50 (GT) fails to induce an antibody response in a large number of inbred strains of mice. Nevertheless, GT complexed to methylated bovine serum albumin (MBSA) elicits a GT-specific IgG PFC response in vivo. Furthermore, injection of BALB/c mice with 10 to 100 mug of GT specifically decreases their ability to develop anti-GT PFC responses to a subsequent challenge with GT-MBSA. GT-specific tolerance can be transferred to normal, syngeneic recipients by spleen cells or thymocytes of GT-primed animals. These results indicate that the stimulation of suppressor cells can be observed in nonresponder mice with another synthetic polypeptide besides GAT. Various parameters of GT-specific immunosuppression in BALB/c mice are described. The application of these techniques to the study of the genetic factors controlling the stimulation of specific immune suppression is discussed.

Genetic control of immune responses in vitro. 3. Tolerogenic properties of the terpolymer L-glutamic acid 60-L-alanine30-L-tyrosine10 (GAT) for spleen cells from nonresponder (H-2s and H-2q) mice.

Although nonresponder, H-2(s) and H-2(q), mice fail to develop GAT-specific PFC responses to GAT, they do develop GAT-specific PFC responses when stimulated by GAT complexed to an immunogenic carrier such as methylated bovine serum albumin. The studies described in this paper show that injection of nonresponder mice with GAT specifically decreases their ability to develop anti-GAT PFC responses to a subsequent challenge with GAT-MBSA. Addition of GAT to cultures of spleen cells from nonresponder mice also prevents development of the GAT-specific PFC responses stimulated by GAT-MBSA. Thus, interaction of nonresponder spleen cells with GAT leads to the induction of unresponsiveness in vivo and in vitro. Various parameters of the tolerance induction have been investigated and described. A comparison of the effects of GAT on B cells indicates that nonresponder B cells are more readily rendered unresponsive by soluble GAT than are responder B cells. The significance of these data for our understanding of Ir gene regulation of the immune response is discussed.

Characterization of a new intra-H-2 recombinant separation of the Ir-RE and Ir-GLT genes.

This report characterizes the intra-H-2 crossover in the D2.GD mouse strain. Recombination occurred within the I region between the immune response (Ir) gene controlling immune responsiveness to ragweed extract (RE). Ir-RE, and the Ir gene governing responsiveness to the random linear terpolymers of glutamic acid and lysine with either tyrosine or pheylalanine (Ir-GLT and Ir-GLphi). The Ir-RE gene was tentatively located in the Ir-1A subregion and the Ir-GLT and Ir-GLphi gene(s) tentatively placed in the Ir-1B subregion. The importance of this recombinant strain to further studies on the fine structure of the I region is discussed.

Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors.

The present study examines the ability of hapten-specific murine splenic B lymphocytes to present hapten-proteins to carrier-specific T cell hybridomas. BALB/cB cells specific for 2,4,6-trinitrophenyl (TNP) were isolated from spleens of immune mice by elution from TNP-gelatin-coated dishes. Such cells presented the TNP-modified terpolymer, GL phi, at concentrations as low as 0.1 microgram/ml, to a GL phi-specific, I-Ed-restricted, interleukin 2-producing T cell hybridoma. In contrast, the same B lymphocytes required 1,000-fold higher concentrations of unmodified GL phi to stimulate the same T cell hybridoma. The presentation of low concentrations of TNP-GL phi by TNP-specific B lymphocytes was significantly or completely blocked by anti-Ig antibody or TNP-proteins, indicating that surface Ig receptors were critically involved in this phenomenon. Finally, binding of TNP-proteins did not alter the ability of the B cells to present unrelated, unhaptenated proteins or to stimulate alloreactive T cells. These results suggest that surface Ig receptors serve to focus antigens onto specific B lymphocytes and that such cells are highly efficient at presenting linked antigenic determinants to T cells. The implications of these findings for the mechanisms of physiologic, histocompatibility-restricted T-B collaboration are discussed.

Genetic control of immune responses in vitro. I. Development of primary and secondary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro.

In vivo, the antibody response in mice to the random terpolymer L-glutamic acid(50)-L-alanine(30)-L-tyrosine(10) (GAT) is controlled by a histocompatibility-linked immune response gene(s). We have studied antibody responses by spleen cells from responder and nonresponder mice to GAT and GAT complexed to methylated bovine serum albumin (GAT-MBSA) in vitro. Cells producing antibodies specific for GAT were enumerated in a modified Jerne plaque assay using GAT coupled to sheep erythrocytes as indicator cells. Soluble GAT stimulated development of IgG GAT-specific plaque-forming cell (PFC) responses in cultures of spleen cells from responder mice, C57Bl/6 (H-2(b)), F(1) (C57 x SJL) (H-2(b/s)), and A/J (H-2(a)). Soluble GAT did not stimulate development of GAT-specific PFC responses in cultures of spleen cells from nonresponder mice, SJL (H-2(s)), B10.S (H-2(s)), and A.SW (H-2(s)). GAT-MBSA stimulated development of IgG GAT-specific PFC responses in cultures of spleen cells from both responder and nonresponder strains of mice. These data correlate precisely with data obtained by measuring the in vivo responses of responder and nonresponder strains of mice to GAT and GAT-MBSA by serological techniques. Therefore, this in vitro system can effectively be used as a model to study the cellular events regulated by histocompatibility-linked immune response genes.

Genetic control of immune responses in vitro. II. Cellular requirements for the development of primary plaque-forming cell responses to the random terpolymer 1-glutamic acid 60-1-alanine30-1-tyrosine10 (GAT) by mouse spleen cells in vitro.

The cellular requirements for the development of primary IgG GAT-specific PFC responses in cultures of spleen cells from responder, C57Bl/6, mice stimulated with GAT and GAT-MBSA and in cultures of spleen cells from nonresponder, SJL and B10.S, mice stimulated with GAT-MBSA were investigated. Macrophages were required for development of responses to GAT and GAT-MBSA in cultures of spleen cells from responder mice and for responses to GAT-MBSA in cultures of spleen cells from nonresponder mice. Macrophages from nonresponder mice supported the development of responses to GAT by nonadherent responder spleen cells, indicating that the failure of nonresponder mice to respond to GAT is not due to a macrophage defect. Furthermore, responder macrophages supported the responses of nonadherent, nonresponder spleen cells to SRBC and GAT-MBSA, but not to GAT. This indicates that the capacity to respond to GAT is a function of the nonadherent population which is composed of thymus-derived (T) helper cells and precursors of antibody-producing cells. Treatment of spleen cells with anti-theta serum and complement before culture initiation abolished PFC responses to GAT and GAT-MBSA thus establishing the requirement for T cells in the development of PFC responses to these antigens. Since precursors of antibody-producing cells in nonresponder mice are capable of synthesizing antibody specific for GAT after stimulation with GAT-MBSA and since the response to GAT is thymus-dependent, it appears that nonresponder mice lack GAT-specific helper T cell function.

Specific immune response genes of the guinea pig. II. Relationship between the poly-L-lysine gene and the genes controlling immune responsiveness to copolymers of L-glutamic acid and L-alanine and L-glutamic acid and L-tyrosine in random-bred Hartley guinea pigs.

The ability of guinea pigs to make immune responses to GA, a linear random copolymer of L-glutamic acid and L-alanine, GT, a random linear copolymer of L-glutamic acid and L-tyrosine, and PLL, a linear homopolymer of L-lysine, is controlled by different autosomal dominant genes specific for each of those polymers. We have investigated the relationship between the PLL gene and the GA and GT immune response genes by simultaneously immunizing random-bred Hartley strain guinea pigs with GA and PLL, GT and PLL, or GA and GT. In most Hartley guinea pigs the ability to respond immunologically to GA and to PLL is inherited together; that is, most animals responding to GA respond to PLL and vice versa. However, a few animals respond to either GA or to PLL but not both, demonstrating that the GA and PLL immune response genes are not identical but linked in most Hartley animals. Conversely, when simultaneously immunized with GT and PLL, most Hartley guinea pigs respond to either PLL or GT but not both, indicating that GT and PLL responsiveness tends to segregate away from each other. Thus, the GT and PLL immune response genes also are not inherited independently but, rather, behave as alleles or pseudoalleles. Similar results are observed when Hartley guinea pigs are simultaneously immunized with GA and GT. The ability to respond to GA segregates away from the ability to respond to GT. Our studies demonstrated that the specific immune response genes thus far identified in guinea pigs controlling the ability to respond to GA, GT, and PLL, respectively, are found on the same chromosome. In most Hartley animals, the GA and PLL immune response genes are often linked, i.e. occur on the same chromosome strand, and tend to behave as alleles or pseudoalleles to the GT immune response gene.

Specific immune response genes of the guinea pig. I. Dominant genetic control of immune responsiveness to copolymers of L-glutamic acid and L-alanine and L-glutamic acid and L-tyrosine.

The immunogenicity of three random copolymers of amino acids with L-glutamic acid and L-alanine (GA), L-glutamic acid and L-tyrosine (GT), or L-glutamic acid, L-alanine, and L-tyrosine (GAT), administered in complete Freund's adjuvant, was studied in several inbred and random-bred guinea pig strains. The animals were tested for delayed sensitivity and their sera were assayed for the presence of antibody directed against the immunizing polymer. All of the guinea pigs developing delayed hypersensitivity also had significant antibody levels in their sera. Inbred strain 2 guinea pigs responded to immunization with GA, but failed to form detectable responses to GT. Inbred strain 13 animals, on the other hand, responded to GT, but not to GA. The (2 x 13)F(1) hybrids responded to both GA and GT with both delayed hypersensitivity and circulating antibody. Thus, the ability of these inbred guinea pigs to respond immunologically to GA or GT is controlled by distinct autosomal dominant genes. A variable percentage of random-bred guinea pigs, depending on their source as well as their strain, responded to immunization with GA and with GT. All guinea pigs, both inbred and random bred, responded to immunization with GAT. The ability to respond immunologically to GAT, therefore, does not seem to be under simple genetic control. However, the levels of anti-GAT antibody found in the sera of animals lacking the ability to respond to GA were much lower than those detected in GA responder animals.

Restriction of primary responses to the IgG class and dependency of IgM responses on secondary immunization for the copolymers of L-glutamic acid, L-tyrosine, and L-alanine.

Primary responses to the linear polymers of L-glutamic acid, L-tyrosine, and L-alanine are restricted to the IgG class of antibodies. The appearance of specific IgM antibodies against these antigens is dependent upon secondary immunization, in contrast to many classical antigenic systems. The presence of an IgM response was verified by a direct plaque-forming cell assay, the inhibition of direct plaques by an antiserum specific for mouse micron-chain, and the physical separation of IgM and IgG GAT-specific antibodies by gel filtration. Preimmunization of the appropriate nonresponder strain with GAT or GT inhibits both the secondary IgM and IgG responses to GAT-MBSA and GT-MBSA, respectively. The tolerance observed is due to the induction of suppressor cells as demonstrated by cell transfer experiments.

Genetic control of immune responses in vitro. IV. Conditions for cooperative interactions between nonresponder parental B cells and primed (responder plus nonresponder) F1 T cells in the development of an antibody response under Ir gene control in vitro.

The conditions for cooperative interactions between nonresponder B10.S B cells and GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells in the response by cultures of mouse spleen cells to GAT were investigated. GAT-specific antibody responses could be elicited by soluble GAT in cultures of GAT-primed irradiated (C57BL/6 x SJL)F(1) T cells with C57BL/6 B cells but not with B10.S B cells. In contrast, when GAT was presented to the cultures on F(1) macrophages or as aggregates of GAT with MBSA, GAT-specific PFC responses were observed with both B10.S or C57BL/6 B cells. Irradiated GAT-primed T cells were nevertheless essential for the development of these responses. The GAT-specific response of B10.S B cells in these cultures was inhibited by the addition of soluble GAT at culture initiation. These results indicate that genetic disparity at Ir loci is not an absolute barrier to T-B-cell cooperative interactions in the response to antigens under Ir gene control. The significance of these data for the function of Ir gene products in immunocompetent cells is discussed.

Genetic control of specific immune suppression. II. H-2-linked dominant genetic control of immune suppression by the random copolymer L-glutamic acid50-L-tyrosine50 (GT).

Several inbred as well as congenic resistant strains of mice, which fail to respond to the random copolymer of L-glutamic acid50-L-tyrosine50 (GT), were shown to develop specific PFC responses when stimulated by GT complexed to an immunogenic carrier such as methylated bovine serum albumin (MBSA). In these studies we have found that GT preimmunization has a tolerogenic effect on the response to GT-MBSA in some mouse strains; whereas in other strains of mice, GT fails to inhibit the GT-MBSA response. We may, therefore, conclude that immune suppression cannot account for nonresponsiveness in all cases. The development of specific immune suppression in response to GT was shown to be inherited as a dominant trait in F1 hybrids resulting from the mating of suppressor with nonsuppressor strains. This trait is, therefore, under the control of a gene or genes that we have designated as specific immune suppression gene(s) Is genes. The strain distribution of GT induced suppression demonstrates that Is genes are coded for in the H-2 complex. Furthermore, immune suppression by the two related copolymers, GT and GAT, are distinct in different strains of mice. The significance of these data for our understanding of the regulation of the immune response is discussed.

Specific immune response genes of the guinea pig. V. Influence of the GA and GT immune response genes on the specificity of cellular and humoral immune responses to a terpolymer of L-glutamic acid, L-alanine, and L-tyrosine.

The ability of guinea pigs to make immune responses to the random linear copolymer of L-glutamic acid and L-alanine, GA, and to L-glutamic acid and L-tyrosine, GT, is each controlled by a different immune response gene. On the other hand, the random linear terpolymer of L-glutamic acid, L-alanine, and L-tyrosine, GAT, which contains both GA and GT antigenic determinants, is immunogenic in all guinea pigs. After GAT immunization, all animals develop delayed hvpersensitivity and serum antibody specific for GAT. However, only those guinea pigs possessing the GA immune response gene demonstrate cross-reactive delayed hypersensitivity when challenged with GA. In addition, the anti-GAT antisera produced by those animals having the GA gene contain cross-reacting anti-GA antibodies. The sera from guinea pigs lacking the GA gene have no anti-GA antibody activity. Thus, we have demonstrated that a specific immune response gene controlling responsiveness to a "simple" antigen can determine the specificity of both cellular and humoral immune responses to a more complex antigen.