Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor beta chain repertoire of healthy macaques.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.

Pathogenesis of post-thymectomy autoimmune gastritis. Identification of anti-H/K adenosine triphosphatase-reactive T cells.

Autoimmune gastritis spontaneously develops following thymectomy of 3-day-old BALB/c mice (d3Tx). These mice develop autoantibodies to the gastric parietal cell proton pump, H/K ATPase, and aberrant expression of the H/K ATPase in the neonatal thymus prevents the induction of disease post-thymectomy. To characterize the effector cells mediating autoimmune gastritis, we isolated H/K ATPase-enriched preparations of parietal cell microsomes and further purified the enzyme by lectin affinity chromatography. Both preparations induced significant proliferative responses of gastric lymph node cells, which were mediated by CD4+, MHC class II-restricted T cells. Surprisingly, T cells reactive to the Ag could only be demonstrated in lymph nodes in the immediate proximity of the stomach; little or no response was seen when mesenteric or peripheral lymph nodes were tested. It is likely that the H/K ATPase-reactive T cells are actually the effector cells in this disease, as they could only be detected in mice that developed gastritis, as indicated by anti-parietal cell Ab, gastric inflammation, and the presence of cells capable of transferring disease into nu/nu mice. H/K ATPase-specific T cell proliferative responses could first be detected 5 wk post-thymectomy and were accompanied by high background responses at this time point. These latter responses may represent enhanced syngeneic MLRs, which we have previously shown to be elevated in d3Tx mice. Characterizations of the H/K ATPase-reactive and self-reactive T cell populations may reveal the factors that break peripheral T cell tolerance and lead to the development of organ-specific autoimmune disease.

Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity.

The third complementarity-determining region (CDR3) is the only nongermline-encoded hypervariable region of the T cell receptor beta (TCRB) chain, and it is the region that has been predicted to confer fine specificity of the TCR for peptide-MHC complexes. For this reason analysis of TCRB CDR3 heterogeneity may provide insight into immune mechanisms operative in infectious and autoimmune diseases. PBMC stimulated with either mitogen (PHA), superantigen (TSST-1), or nominal antigen (tetanus toxoid) have been compared with unstimulated PBMC using a two-dimensional approach. Analysis of the expressed TCRBV gene repertoire CDR3 length profile coupled with SSCP methodology enabled the discrimination of sequences with the same CDR3 length. For both freshly isolated and PHA stimulated PBMC, a normally distributed spectrum of CDR3 lengths (five or more products) was observed. These products differed by 3 bp (1 amino acid) due to the strict requirement for in-frame rearrangements in the CDR3 region of TCR. By contrast, tetanus toxoid stimulated PBMC had restricted profiles for most TCRBV families after as few as 7 days of incubation. The oligoclonal nature of samples showing CDR3 length restriction was revealed by SSCP analysis and confirmed by sequence determination. Superantigen stimulation resulted in unique patterns of diversity, which included polyclonal expansion of specific TCRBV families as well as oligoclonal expansion of most other TCRBV families. These data reveal complex yet distinct patterns of TCR diversity in response to different T cell activation stimuli.

Pathogenesis of post-thymectomy autoimmunity. Role of syngeneic MLR-reactive T cells.

Thymectomy of 3-day-old mice results in the development of multi-organ-specific autoimmune diseases. The disease process is mediated by CD4+ T cells and is characterized by an inflammatory infiltrate in the affected organ(s) and the presence of autoantibodies. Our analysis of the phenotype of the CD4+ T cells that remain in the 3-day thymectomized animal revealed that the majority (approximately 80%) of the CD4+ lymph node cells express an activated (MEL-14low) phenotype and a smaller percentage expressed the T cell activation Ag CD69 and IL-2R alpha-chain. Thymectomized animals also had an increase in the frequency of mitogen-induced CD4+ IL-4 producers and significantly higher levels of total serum IgG. Functional studies demonstrated that lymph node T cells from 3-day thymectomized mice had an enhanced response in the syngeneic MLR and appeared to preferentially respond to syngeneic dendritic cells. To determine whether the syngeneic MLR-reactive T cells were involved in the pathogenesis of the organ-specific disease, we developed a model that mimicked the 3dTx model by grafting neonatal thymi to adult nu/nu recipients followed by removal of the thymus graft on day 3 or 4. When compared with mice transplanted with an untreated thymus, nu/nu mice transplanted with adult APC-containing thymi demonstrated a decrease in the incidence and severity of gastritis, a marked decrease in the titer of anti-parietal cell Ab, and a decrease in total serum IgG. Thus, intrathymic tolerization to complexes of self-peptides and MHC class II on adult APC prevents organ-specific autoimmune disease.

Post-thymectomy autoimmunity: abnormal T-cell homeostasis.

Thymectomy of 3-day-old mice induces organ-specific autoimmune disease, which is characterized by the presence of autoantibodies and T-cell infiltrates in the affected organs. Here, Adriana Bonomo and colleagues propose a new model for the pathogenesis of this syndrome, which integrates many of the homeostatic mechanisms of the immune system, including central and peripheral tolerance, T-cell maturation and exportation from the thymus, as well as lymphocyte recirculation and homing.

Premature escape of double-positive thymocytes to the periphery of young mice. Possible role in autoimmunity.

Thymectomy of 3-day-old mice induces organ-specific autoimmune disease. To define a relationship between the development of T cells early in the neonatal period and autoimmunity, we studied the thymus and peripheral lymphoid tissues of 3-day-old mice. Lymph nodes, but not spleens, of 3- to 4-day-old mice contained a significant number of thymus-derived CD4+CD8+ cells that are phenotypically similar to CD4+CD8+ thymocytes in their level of expression of CD3 and HSA. It is likely that the prematurely exported cells are the progenitors of autoreactive T cells because the lymph nodes from 3- to 4-day-old male, but not female, mice which express a transgenic TCR specific for the H-Y Ag contained a large number of CD4+CD8+Tg+ as well as CD4-CD8+Tg+ T cells. Thus, the neonatal thymus is capable of exporting immature T cells that in the absence of a thymus may differentiate into autoimmune effector cells.

The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells.

Constitutive production of cytokines was observed in 3 of 12 gamma/delta T cell lines derived from murine epidermis and correlated with the expression of the C gamma 4, V delta 6 T cell receptor (TCR). After adaptation of one of the lines (T195/BW) to serum-free culture conditions, cessation of the "spontaneous" production of interleukin 4 (IL-4) was observed and IL-4 production could then by induced by the addition of RGD-containing extracellular matrix (ECM) proteins to the culture. The response to the ECM proteins could be completely inhibited by a mAb to the murine vitronectin receptor (VNR). However, the induction of IL-4 production could also be inhibited by anti-CD3 and by an anti-clonotypic mAb to the TCR-gamma/delta of T195/BW. As TCR-gamma/delta loss mutants of T195/BW also failed to respond to ECM proteins, these data demonstrate that engagement of the VNR by its ligand is necessary, but not sufficient, for the induction of IL-4 production. Furthermore, the VNR is expressed by many other T cell clones (both gamma/delta and alpha/beta), none of which produce lymphokines constitutively. Taken together, these observations strongly favor the view that not only is coexpression of the VNR and TCR required for the induction of IL-4 production, but that the TCR must also be engaged by its ligand, most likely a cell surface antigen expressed by the hybridoma itself.

Chromosomal location of the Ly-49 (A1, YE1/48) multigene family. Genetic association with the NK 1.1 antigen.

The Ly-49 (A1, YE1/48) Ag is a disulfide-linked dimer with 44-kDa subunits, and is expressed on the cell surface of rare T cell tumors of C57BL/6 origin. Although this Ag is undetectable by flow microfluorimetry analysis, normal cells have been shown to express the A1 Ag by immunoprecipitation experiments performed on surface radioiodinated spleen and thymus cells. We (J. Immunol. 143:1379, 1989) and others (J. Immunol. 142:1727, 1989) have recently isolated cDNA encoding the Ly-49 Ag. Southern blots with an Ly-49 cDNA probe revealed multiple bands, consistent with cross-hybridization to other members of a multigene family, and significant RFLP between the C57BL/6 and BALB/c strains. When the RFLP patterns displayed by other common laboratory strains as well as informative recombinant inbred strains were examined, the Ly-49 gene family displayed five RFLP patterns and the entire family was found to reside on a contiguous stretch of the distal portion of mouse chromosome 6, the same region to which the NK1.1 Ag has been mapped. Although tissue distribution studies and transfection analysis ruled out the possibility that Ly-49 was identical to NK1.1 Ag, approximately 20% of NK1.1 cells isolated from normal spleen coexpressed Ly-49 and all Ly-49+ cells were CD3-. Although spleen cells cultured in high doses of rIL-2 demonstrated similar coexpression of NK1.1 and Ly-49, approximately 10% of CD3+ cells coexpressed Ly-49. The chromosomal mapping data and the expression of the Ly-49 and NK1.1 Ag suggest that the NK1.1 Ag may be a member of the Ly-49 multigene family.

Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation.

We have produced a hamster mAb, H1.2F3, which was derived by immunization with a murine TCR-gamma delta + epidermal T cell line. H1.2F3 immunoprecipitates a cell surface-expressed disulfide-linked dimer that has a m.w. of 85,000 under non-reducing conditions and consists of subunits of 35,000 to 39,000 m.w. This dimer is distinct from the CD3-associated TCR-gamma delta complex (CD3/TCR), inasmuch as H1.2F3 does not co-precipitate or co-modulate with the CD3/TCR complex and recognizes an Ag with a single-peptide backbone of 22,000 m.w. after N-Glycanase treatment. H1.2F3 is weakly reactive with a small percentage of cells from unfractionated thymus, spleen, or lymph node, but reactivity with both T and B lymphocytes is markedly enhanced by a brief period of stimulation with Con A or PMA in vitro. This enhancement requires de novo protein synthesis. Enhanced expression of the H1.2F3 Ag can also be induced in vivo by injection of Con A or anti-CD3. H1.2F3 is a potent stimulator of T, but not B, cell proliferation in the presence of PMA and FcR-bearing accessory cells. These functional and biochemical studies strongly suggest that the Ag recognized by H1.2F3 is the murine homologue of the human CD28 Ag recognized by mAb 9.3.