Influence of kappa and IgH genes on the T helper cell response to p-azobenzenearsonate tyrosine.

Immunization of mice with the p-azobenzenearsonate-L-tyrosine conjugate (ABA-Tyr) leads to the activation of ABA-specific T helper cells capable of proliferating in vitro in the presence of the corresponding antigen. This response is under a dual genetic regulation by H-2 and non-H-2 linked genes, and H-2d mice are high-responder. We demonstrate here, using strains congenic to BALB/c for chromosomes (Chr.) 6 or 12 that this T cell response is also influenced by kappa and IgH linked genes. Double congenic mice indicate that Chr.6 and Chr.12 genes have a complementary effect on the response which cannot be predicted solely by the alleles expressed on either of the two chromosomes. In addition, responses in Bailey's inbred recombinant mice allow a possible mapping of the Chr.12 gene at the 5' end of the IgH complex and of the VH-dextran gene family. The mechanisms which may account for the influence of immunoglobulin gene products on the ABA-specific T cell repertoire are discussed.

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones.

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.

Cell-cell interaction responsible for the induction of first order suppressor T cells in hapten-specific contact sensitivity reactions.

C3H/He mice were found to be low responders in the contact sensitivity response to ABA. Intravenous injection of ABA-coupled syngeneic spleen cells induced hapten-specific Ts in C3H/He mice. These cells were Ts1 because they acted on the inductive phase of the contact sensitivity. They could suppress the contact sensitivity in H-2-compatible CBA mice which were known to be high responders to ABA. Using in vivo and in vitro systems for the induction of Ts, it was shown that I-A-I-J+Thy-1- adherent cells were necessary as APC for the induction of Ts1.

Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host.

Suppressor T-cell factor(s) (TsF1) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF1 is restricted by genes linked to Igh-1 allotypic markers. TsF1 obtained from B6.Igh-1n mice was unable to suppress the immune response in B6.Igh-1b mice and vice versa. However, TsF1 prepared from B6.Igh-1n T cells "parked" in an Igh-congeneic B6.Igh-1b environment displays an additional restriction specificity of the host. Thus, TsF1 prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1n (donor) and B6.Igh-1b (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1a. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response.

Analysis of the mode of antigen presentation required for triggering of T cell proliferation by using various azobenzenearsonate-tyrosine derivatives.

Various azobenzenearsonate-tyrosine (ABA-Tyr) derivatives were synthesized by modifying amino and carboxyl groups at the alpha-carbon of tyrosine, with preservation of most of the ABA-Tyr moiety (ABA plus hydroxyphenyl portion of tyrosine). These derivatives were tested for the ability to stimulate ABA-L-Tyr specific T cell lines derived from B10.BR and B10.S mice. ABA-acetyltyramine, ABA-hydroxyphenylpropionic acid (ABA-PPr), and ABA-propylphenol, which lack either the carboxyl or amino group or both, could not induce T cell proliferation. The lack of stimulation by these derivatives was not due to their cytotoxic effects. A similar pattern of proliferation was obtained on stimulating lymph node T cells from B10.BR and B10.S mice primed with ABA-L-Tyr. Some differences were observed, however, between B10.BR and B10.S mice. ABA-L-Tyr-specific T cells from B10.BR mice could not respond well to ABA-D-Tyr in contrast to B10.S T cells. Furthermore, B10.BR mice primed with ABA-acetyltyramine or ABA-PPr in complete Freund's adjuvant could not induce ABA-L-Tyr-reactive T cells, whereas T cells from B10.S mice primed with these derivatives could proliferate in the presence of ABA-L-Tyr. The differences between B10.BR and B10.S mice were further investigated by using (B10.S X B10.BR)F1 mice. T cells from ABA-L-Tyr-immunized F1 mice responded poorly to ABA-D-Tyr when presented with B10.BR antigen-presenting cells (APC), but responded well when presented with B10.S APC. Similarly, T cells from ABA-PPr-primed F1 mice did not proliferate to ABA-L-Tyr in the presence of B10.BR APC, but could proliferate in the presence of B10.S APC. Our results clearly indicate that the presence of charged groups at the alpha-carbon of tyrosine plays a critical role in the triggering of ABA-L-Tyr-specific T cell proliferation. The significance of these results is discussed.

Binding of antigen in the absence of histocompatibility proteins by arsonate-reactive T-cell clones.

Inducer T-cell clones reactive to the p-azobenzenearsonate (arsonate) hapten possess binding sites for radioactive arsanylated proteins, which are not present on clones with other antigen specificities. Binding occurred in the absence of histocompatibility proteins. Binding was specific for the p-azobenzenearsonate hapten, since unconjugated proteins and proteins conjugated to the nonactivating o-azobenzenearsonate hapten neither bound to the clones nor competed binding of radioactive antigen. One of the clones was studied in more detail, using a panel of structural analogs of arsonate conjugated to the carrier protein ovalbumin. All conjugates that activated the clone in the presence of antigen-presenting cells also competed binding of radioactive antigen in the absence of antigen-presenting cells. Nonactivating conjugates did not compete binding. Based on evidence in this and the succeeding paper (Rao et al., accompanying paper), we suggest that these arsonate-binding sites may include the physiological antigen receptors of arsonate-reactive T-cell clones.

Analogs that compete for antigen binding to an arsonate-reactive T-cell clone inhibit the functional response to arsonate.

We have tested several structurally related haptens, conjugated to ovalbumin, for their effect on activation of an inducer T-cell clone reactive to the p-azobenzenearsonate (arsonate) hapten. Low concentrations of some analogs inhibited DNA synthesis and lympkokine production by the clone in response to arsanylated antigen, but not in response to the lectin concanavalin A. Inhibition was specific for this clone, since the response of clones reactive to other antigens was not blocked. Inhibition may result from competition of these analogs with arsonate at a site on the T cell. The effectiveness of blocking by arsonate analogs parallels their ability to bind to a previously described arsonate-binding site on the clone (Rao et al., accompanying paper). We suggest that the binding and blocking assays detect the same physiological arsonate-recognition site on the clone, and hence that the cell-surface arsonate-binding sites we have described mediate its physiological response to antigen.

Suppressor factor from a T cell hybrid inhibits delayed-type hypersensitivity responses to azobenzenearsonate.

By using polyethylene glycol 1540, BW5147 AKR T lymphoma cells were fused with splenocytes from A/J mice treated so as to induce suppressor T cells specific for azobenzenearsonate (ABA). Of 576 microwells originally seeded, 132 demonstrated growing cell clones, 4 of which produced an ABA-binding supernatant factor. When tested in vivo for suppression of delayed-type hypersensitivity to ABA, two of these cell lines, A4 and F12, were shown to produce suppressive supernatant factors. Fluorescence analysis of the F12 cells with appropriate antisera demonstrated this T cell hybrid to be Thy 1.2+, Lyt 1+,2-, and surface immunoglobulin negative, the surface marker phenotype of conventional ABA-specific suppressor T cells. This cloned suppressor cell line, F12, produces a culture supernatant factor that is suppressive at dilutions up to 1:100 and has provided material for genetic and immunochemical analysis.

Allorestriction and xenorestriction phenomena in the induction of delayed-type hypersensitivity (DTH) responses in the guinea pig against azobenzenearsonate- (ABA) modified peritoneal exudate cells.

The role of MHC products in the induction of DTH responses Ia antigens play a crucial role in these cells in the recognition of ABA determinants by lymphocytes. Second, JY-1 guinea pigs were employed as responder animals. A clear allorestriction phenomenon was observed again when weakly modified ABA-PEC were used. On the other hand, the allorestriction phenomenon was apparently abrogated when heavily modified ABA-PEC were employed: allogeneic as well as syngeneic ABA-PEC induced strong and comparable DTH responses. Even in this condition, xenogeneic (mouse) ABA-PEC were barely immunogeneic, On the basis of these observations, the cross-reactivity of Ia antigens in the conjunctive recognition of haptenic determinants by T cells is discussed.

Requirements for inducing tolerance of hapten-specific delayed hypersensitivity: epitope density.

Tolerance to hapten-specific antibody formation and delayed hypersensitivity was examined in adult rabbits. The azobenzenearsonate (ABA) or sulfonate-specific antibody response to hapten-hemocyanin immunogens was suppressed by deaggregated hapten-rabbit IgG conjugates given 21 and 14 days before challenge. High affinity antibody was preferentially suppressed. Delayed hypersensitivity to ABA-tyrosine was suppressed by deaggregated ABA-rabbit IgG conjugates injected 17 and 10 days before challenge. Conjugates with a high hapten density, ABA15-23-rabbit IgG were effective tolerogens. Conjugates with four to six ABA groups per carrier molecule were very poor tolerogens. Increasing the amount of low substituted conjugate injected did not improve tolerogenicity. It appears that a high epitope density is required for effective induction of tolerance to ABA-specific delayed hypersensitivity in the rabbit.