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T cell antigen receptors (TCR) are composed of an antigen-recognizing unit, the TCRα β heterodimer, and a signal transduction ensemble, the CD3 complex. Whereas mammals possess three CD3 dimers (δ ϵ , γ ϵ , and ζ 2), birds and amphibians have only two (δ /γ -ϵ and ζ 2). To understand evolutionary changes in TCR/CD3 assembly,a phylogenetic approach was employed to dissect the interaction of TCRα β heterodimers with the CD3 components. While sheep and mouse TCRα and TCRβ chains could replace the corresponding human chains in mutant human T cells to restore surface TCR/CD3 expression and function, chicken TCRα , TCRβ and CD3δ /γ chains were unable to replace the corresponding human chains in forming a chimeric TCR/CD3 complex. The inability of chicken TCR/CD3 components to replace the human molecules in T cells was found to result from the lack of interaction between chicken TCRα β heterodimers and the human CD3 complex. In contrast, if no CD3 molecules are present (non-T cells), TCRα -TCRβ chain pairing can take place in an apparently non-controlled way. Thus, the TCR-CD3 interactions have changed with the evolutionary divergence of two mammalian CD3γ and CD3δ genes from a single prototypic chicken δ /γ gene. Our data suggest that the structures in mammalian TCR.C regions, which distinguish between CD3δ and CD3γ chains, have evolved with the appearance of two separate CD3δ and CD3γ functions.

A candidate avian CD6 homolog is identified by the S3 monoclonal antibody. The S3 antigen exists in a phosphorylated glycoprotein form of 130 kDa and a nonphosphorylated form of 110 kDa. Removal of phosphate groups and N-linked carbohydrates indicates a 78-kDa protein core. During thymocyte differentiation, the gamma delta T cells do not express S3, whereas mature CD4+ and CD8+ cells of alpha beta lineage acquire S3 antigen. All alpha beta T cells in the blood and spleen express the S3 antigen at relatively high levels. In contrast, only the CD8+ subpopulation of gamma delta T cells in the spleen expresses the antigen and neither alpha beta nor gamma delta T cells in the intestinal epithelium express the S3 antigen. The S3 antigen is also found on embryonic splenocytes with a phenotypic profile characteristic of avian natural killer cells. The biochemical characteristics and this cellular expression pattern imply that the S3 antigen is the chicken CD6 homolog.

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, gamma delta T cells and alpha beta T cells expressing one of the two prototypic V beta families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4+ alpha beta T cells expressing either V beta 1 or V beta 2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that alpha beta T cell alloreactivity is determined by CDR3 variability. By themselves, the gamma delta T cells were incapable of inducing GVH reactions. However, host gamma delta T cells were recruited into the donor alpha beta T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that gamma delta T cells may play a secondary role in GVH reactions.

Thymocyte development was monitored in an embryonic thymus organ culture system to establish a model in the chicken in which the functional nature of the thymic microenvironment could be assessed. Thymus lobes were removed from 10-day-old embryos and cultured for 2-10 days. Cell yield increased to a maximum in 4-8 days of culture with a corresponding decrease in average cell size. An initial thymocyte population of predominantly CD3-CD4-CD8- cells gave rise to all CD3/CD4/CD8-defined subpopulations in vitro, maintaining high levels of CD3-CD4-CD8+ and CD3+CD4-CD8+ cells and a low representation of CD3-CD4+CD8-, CD3+CD4+CD8-, CD3-CD4+CD8+ and CD3+CD4+CD8+ thymocytes. This is the first observation of a CD3-CD4+CD8- population in the chicken. Developmental kinetics of CD3+ cells were similar to that in the embryo, suggesting that the in vitro environment is sufficient to promote and maintain thymocyte maturation. Thymocytes of both the gamma delta and alpha beta T cell receptor (TcR) lineages developed in that order, confirming in ovo data and the lineage potential of the first wave of thymocyte precursors. One unusual finding was a relative accumulation of gamma delta TcR+ thymocytes in culture, incorporating all CD4/CD8 subsets, including a previously undetected population, CD4+CD8-. This may indicate a favorable developmental environment or simply a lack of normal cellular emigration. A detailed comparison with T cell development in the embryo demonstrated that the chicken thymus organ culture system reflects thymic events in ovo during a limited time period and thus should prove useful in the identification of functionally relevant thymic molecules.

In a study of T cell ontogeny using monoclonal antibodies specific for chicken T cell receptors (TcR) and associated cell surface molecules, we found a subset of lymphocytes that express cytoplasmic CD3 epitopes in the absence of cell surface CD3/TcR complexes. Approximately half of these cells, which were present in the spleen, bursa and intestine of young chick embryos, expressed surface CD8. None expressed CD4, TcR 1 (gamma/delta), TcR 2 (alpha/beta) or TcR 3 (a third CD3-associated heterodimer in the chicken). These cytoplasmic CD3+CD8 +/- cells, tentatively named TcR 0 cells to denote their lack of surface TcR, appeared first in the spleen of 8-day embryos, 4 days before TcR expression begins in the thymus, and reached a peak frequency of approximately 10% of the splenic cell in 14-day embryos. The TcR 0 cells were also present in adult birds, where they comprised only about 1% of the CD3+ spleen cells and approximately 40% of the lymphocytes in the intestinal epithelium. We conclude that the avian TcR 0 cells represent a thymus-independent lineage of lymphocytes which, like natural killer cells in mammals, may play an important role in body defense.

Since the two major theories assume that either a hypersensitivity to plaque immunogens or a direct tissue damage caused by plaque constituents plays the dominant role in the etiology of periodontitis, we first investigated the nature of the immune response to and the endotoxin content of human plaque. It was found that pooled human plaque samples contained a very low but significant level of endotoxin, as determined by phenol-water extraction, by pyrogenicity in New Zealand white rabbits, by local Shwartzman skin test, and by the Limulus lysate assay. A measurable humoral immune response could be elicited by human plaque material if injected into guinea pigs intraperitoneally. Intragingival application of the same material was a less efficient route of immunization. This humoral immune response was measured by passive hemagglutination and immune counterelectrophoresis. Cell-mediated immune response, as measured by skin sensitivity, lymphoblast transformation and rosette formation assays in guinea pigs, sensitized either intragingivally or intraperitoneally, gave diverse results. No cell-mediated immune response could be detected by using the lymphoblast transformation assay. A weak but positive reaction could be elicited in one-third to one-half of the sensitized animals by injecting them intradermally or by testing rosette formation of their spleen cells with plaque-coated sheep erythrocytes. The weak and inconsistent results do not rule out the possibility that cell-mediated immune responses and their mediators cause human periodontitis, but they make us cautious in considering such events as the sole factors in the development of the disease. As of now, we have even less reason to exclude the possibility that certain types of immune responses to plaque protect the patient from periodontitis.

Two monoclonal antibodies, CT-1 (IgG1, kappa) and CT-1a (IgG3, kappa) were prepared against chicken thymocytes. The antigen identified by these antibodies was found to be a glycoprotein with a major polypeptide component having an apparent molecular weight of 63 000 and a minor polypeptide component of approximately 103 000. Immunoprecipitation and blocking experiments revealed that the two antibodies react with different antigenic determinants of the molecule. Ontogenic studies employing immunofluorescence failed to reveal the antigenic determinants on cells from the embryo or embryonic yolk sac on days 3 and 6 of incubation. The number of embryonic thymocytes bearing the molecules detected by these antibodies increased from 6% on day 12 to 54% on day 13; the frequency of thymocytes expressing this glycoprotein reached adult levels (greater than 90%) by the 15th day of embryonic age. In contrast, the CT-1 and CT-1a antibodies reacted with only 2-5% of blood and splenic cells and less than 0.05% of cells from bursa or bone marrow of young adult chickens. In the quail CT-1 reacted with cortical, but not medullary, thymocytes, while the CT-1a antibody was unreactive with quail thymocytes. The surface glycoproteins detected by these discriminating monoclonal antibodies may provide an important discriminating marker for thymic lymphocytes in the chicken and the quail.