David, C S

Abstract The use of an anti-IL-2R antibody, 7D4, was investigated in vivo in the suppression of collagen-induced arthritis in mice. 7D4 was shown to reduce the incidence of arthritis in a group of mice immunized with type II collagen as compared with a group similarly immunized with collagen and treated with a control isotype matched anti-Forsmann antibody. 7D4 was also shown to reduce the severity of arthritis in the treated mice as compared to the mice in the group treated with the control antibody. Anti-IL-2R antibodies may be useful in the treatment of autoimmune diseases by selectively suppressing activated T cells.

Abstract The role of T cell-mediated and humoral immunity to type II collagen has been well documented in collagen-induced arthritis (CIA). Previous work from our laboratory has indicated that genomic deletions of TCR V beta genes may play a role in CIA resistance in mice. This indicated a selectivity of TCR usage by autoreactive T cells in CIA in mice. Certain strains of mice, although having a normal genomic V beta TCR repertoire, can show clonal deletion of peripheral T cells that bear specific V beta gene products in their TCR. These clonally deleted T cells are reactive with self-Ag such as minor lymphocyte stimulation (Mls) Ag. An Mls-congenic strain, BALB.D2.Mlsa, which differs only at the Mls-1 a locus from BALB/c (Mls-1b), was used to examine the effect of clonal deletion of Mls-1a-reactive T cells in CIA. These two strains were crossed to three CIA-susceptible strains, B10.RIII (H-2r, Mls-1b), DBA/1 (H-2q, Mls-1a), and B10.Q (H-2q, Mls-1b), and the crosses were injected with type II collagen. A significantly decreased incidence of arthritis was observed in the (BALB.D2.Mlsa x B10.Q)F1 hybrids, compared with (BALB/c x B10.Q)F1 hybrids, upon immunization with chick type II collagen. The BALB.D2.Mlsa cross mice also had significantly lower levels of antimouse collagen antibodies. Flow cytometric analysis confirmed the clonal deletion of Mls-1a-reactive V beta 8.1, V beta 6, V beta 7, and V beta 9 subsets in the (BALB.D2.Mlsa x B10.Q)F1 hybrids. The study of H-2q/d mice in (BALB.D2.Mlsa x B10.Q) x B10.Q back-crosses demonstrated a significant correlation between CIA resistance and Mls-1a locus. On the other hand, B10.RIII crosses showed only a modest decrease in CIA incidence in the presence of Mls-1a. As expected, all the DBA/1 crosses had an equal incidence of CIA, which was somewhat less than that seen in DBA/1 mice themselves. These studies point out that the Mls-1a locus could play a role in decreasing CIA incidence by clonal deletion of T cells bearing specific V beta TCR, which may be involved in the pathogenesis of CIA. The influence of the clonal deletion of T cells on CIA, and hence the usage of specific V beta TCR by autoreactive anti-type II collagen T cells, however, depends not only on the source of the type II collagen and the MHC class II molecules involved but also on other background genes in mice.

Abstract Mycoplasma arthritidis produces a soluble protein which is active for murine and human lymphocytes when presented by Ia-bearing accessory cells. By using fibroblasts transfected in vitro with various class II Ag, we demonstrated that presentation of the M. arthritidis mitogen (MAM) to T cells was mediated by E alpha-containing molecules. We also showed that splenocytes from transgenic mice expressing E alpha heterozygously (B10.TRG E alpha+) or homozygously (B10.E alpha TG +/+) underwent a similar proliferation in response to MAM as compared with the failure of control B10.TRG E alpha- splenocytes to respond to MAM. Although splenocytes from inbred C3H and CBA mice exhibited much higher proliferative responses to MAM than did those from B10.TRG.E alpha+ or B10.E alpha TG +/+ mice, flow cytometry showed similar levels of E alpha expression. Furthermore, gamma-irradiated splenocytes from B10.TRG E alpha + mice presented MAM to T hybridoma cells with a similar efficacy as did splenocytes from C3H mice. The lesser response to MAM of lymphocytes from the E alpha transgenic mice as compared with those from C3H and B10.K mice was likewise not due to differential expression of their V beta TCR. We conclude that presentation of MAM to T cells is accomplished by E alpha-containing molecules. The studies also suggest that the conserved, nonpolymorphic regions of class II molecules may play an important role in host immune response to microbial products.

Abstract Spleen cells from a number of H-2 congenic mice were tested for their proliferative and/or cytolytic responses to partially purified products from CNBr cleavage of Sendai virus. In response to in vitro stimulation of Sendai-primed spleen cells with virus-infected cells, Sendai virus-specific cytolytic T lymphocytes restricted to H-2Kb, Kd, Kk, Kb, and Dd were generated. Of these, H-2Kb- and Kd-, but not H-2Kk-, Kb-, and Dd-, restricted cytolytic T cells were generated after stimulation with partially purified Sendai peptides. H-2Dd-restricted, virus-specific cytotoxicity was generated in response to unseparated products from CNBr cleavage of Sendai virus. Thus, the pool of partially purified peptides contains some but not all of the antigenic determinants that can stimulate a cytolytic response. These results suggest that each determinant on Sendai viral proteins may stimulate cytolytic T lymphocytes restricted to only a subset of the H-2K and D alleles which can be recognized by the sum of cytolytic T cells generated in response to all of the antigenic determinants on Sendai viral proteins. Thus, H-2K and D genes function as immune response genes regulating the cytolytic response to partially purified peptides and, by extension, possibly to individual antigenic determinants on Sendai viral proteins. Also, spleen cells from three mice differing only in the H-2I-S regions show differences in their proliferative responses to Sendai antigens. Only spleen cells from mice with the k haplotype in IA-IJ proliferate in response to partially purified peptides, whereas spleen cells from all three mice proliferate when virus is added to the cultures. These proliferating cells are sensitive to treatment with anti-Thy-1.2 and C, suggesting that immune response genes regulating T cell proliferative responses to individual viral determinants may also exist.

Abstract Mouse alloantiserum capable of detecting minor histocompatibility antigens has been produced. Cytotoxic antibody to products of the H-4 locus was produced by reciprocal immunization of minor H loci congenic strains B10 (H-4a) and B10.129(21M) (H-4b), with mitogen-stimulated lymphocytes and epidermal cells. Absorption studies suggested antibodies to more than 1 specificity. The antibodies cross-react with products of other minor H loci. Alloantiserum to the H-4 gene product precipitates a glycoprotein with a m.w. of approximately 45,000 from radiolabeled Con A-stimulated lymph node cells.

Abstract Recombinant D2.Gd (KdAdBbJbEbCbSbDb) was derived from parental strains B6 (H-2b) and DBA/2 H-2d). Recent peptide studies and 2-D electrophoresis studies from other laboratories have shown that the Aed (E beta) chain of D2.GD is structurally different from the parental-derived chain. An antiserum (D2.GD X A. TFR5)F1 anti-DBA/2 was produced that detects an Ia specificity (Ia.50) in the I-E molecule formed by the parent Aed chain and lacking in the I-E molecule formed by the variant Aed. chain. The variant Ae chain could have derived either from a mutational event or an intracistronic cross-over.

Abstract Epidermal cells (EC) of AKR and CBA (H-2k) mouse strains share non-H-2 alloantigens that induce potent cytotoxic T lymphocytes (CTL) in H-2 compatible C3H hosts, and these CTL lyse EC targets in preference to lymphoid cell (LC) targets in H-2-restricted fashion in short-term cell-mediated cytotoxicity (CMC) assays. In contrast, C3H EC are not immunogenic in this regard in AKR and CBA hosts. (C3H × CBA)F1 hybrid hosts are nonresponders to AKR EC, exactly like the CBA parent strain hosts, and their own EC are immunogenic in C3H hosts and susceptible to lysis by the CTL so generated. Hence, CBA mice express a non–H-2 CMC determinant on EC that is absent on C3H EC. Offspring produced by backcrossing (C3H × CBA)F1 hybrids to the antigen-negative C3H parent strain were tested individually for their responsiveness to AKR EC immunogens and for the susceptibility of their own EC to lysis by C3H anti-AKR EC effectors. There was a clear 1: 1 distribution of the backcross offspring into 2 populations in both test systems. Moreover, responsiveness and antigenicity were inversely correlated in individual mice. Thus, the expression of EC alloantigens in the segregating offspring depends on the activity of genes at a single Mendelian locus. These results are most compatible with the hypothesis that a loss mutation occurred early in the history of the C3H strain at a non-H-2 locus that specifies a system of alloantigens expressed preferentially on EC. The finding that CTL generated by AKR EC immunogens in C3H/Heston and C3H/Andervont mice exhibit the same preferential lysis of EC as opposed to LC targets is consistent with this hypothesis because these C3H strains separated over 40 yr ago. Because this locus does not appear to correspond to any known locus, we have provisionally designated it the Epa (epidermal alloantigen) locus.

Abstract Epidermal cells (EC) obtained by trypsinization of tail skin of H-2k haplotype mouse strains AKR, B10.BR, B10.K, CBA, C3H, and RF were used as stimulators and targets of cell-mediated cytotoxicity (CMC) in 3-hour chromium-release assays. Host splenic effector cells were generated by priming mice with an injection of EC and boosting in mixed culture with irradiated donor-strain EC stimulators; then assayed for CMC towards donorstrain EC and lymphoid cell (LC) targets. When C3H is the donor strain and any of the other strains the host, little CMC is registered with EC targets. However, when C3H is the host and any of the other strains the donor, the CMC registered with donor-strain EC targets invariably is greater than that registered with LC targets, even with mitogen-induced lymphoblasts, indicating that C3H mice respond to non-H-2 alloantigens preferentially expressed by EC. The CMC evoked in C3H hosts by EC from any of the other other H-2k strains cross-reacts with EC targets from all of them, indicating extensive sharing of target-cell determinants. However, the CMC is H-2 restricted, and mapping tests with recombinant lines on the C57BL/10 background proved that the restriction depends on antigens mapping in the K region of the H-2 complex. T cell elimination tests indicate that the effector cells in these reactions are cytotoxic T lymphocytes. Moreover, tests conducted with medium supplemented with adult mouse serum instead of fetal calf serum indicate that the reactions are not dependent on xenogeneic serum but represent true examples of H-2-restricted CMC directed towards non-H-2 alloantigens expressed preferentially on EC.

Abstract Pretreatment of mice genetically susceptible to type II collagen-induced arthritis (CIA) with monoclonal or polyclonal antisera specific for I region gene products (Ia antigens) suppressed or delayed the onset of CIA, whereas pretreatment with anti-Ia to an irrelevant haplotype was without effect. The humoral response to type II collagen was transiently depressed 14 days after immunization but antibody levels did not differ significantly after 28 days. The peak delayed-type hypersensitivity to type II collagen was unaffected by anti-Ia treatment. Monoclonal antibody of one anti-Ia specificity enhanced both the antibody response and the arthritis incidence in one mouse strain.

Abstract Ia specificities 22 and 23 were initially identified by using conventional alloantisera and were mapped to the I-E subregion of k and d haplotypes on the basis of their reactivity with selected recombinants. Recently we found that Ia 22 and 23 are hybrid determinants on the basis of their expression on selected F1 cells, but absence from both parental cells. Initial attempts to detect hybrid Ia antigens by immunizing parents with F1 cells were unsuccessful. By utilizing lipopolysaccharide (LPS)-stimulated F1 spleen lymphoblasts as immunogens, 1 of the parents as recipient and the other parent cells for absorption of antisera, specific anti-Ia.22 and 23 antibodies were produced. The specificity of these parent anti-F1 sera was confirmed by cytotoxic and immunoprecipitation analyses.