Altered allogeneic response in neonatally anti-MHC class I-treated mice.

Neonatal treatment of C3H mice (H-2k) with anti-Kk monoclonal antibodies results in altered cytotoxic responses against allogeneic targets. After 2-3 weeks of antibody treatment, no difference in the number of CD4+8- or CD4-8+ T cells was observed between the antibody- and saline-treated mice. However, antibody-treated mice had a significantly reduced cytotoxic response against various allogeneic major histocompatibility complex (MHC) class I-expressing targets. The strongest reduction was observed in very young mice (up to 2 weeks of age). As the mice got older, the allo MHC-specific responses reached control levels. No significant changes in T-cell receptor (TCR)-V-region usage was observed even in young antibody-treated mice. The results suggest that the reduction in the number of positively selecting elements reduces alloreactivity and most likely also the diversity of TCR-repertoire. However, the reduced alloresponsiveness was not restricted to either allogeneic K- or D-encoded molecules, suggesting that self MHC-D-region encoded molecules can mediate positive selection of T cells able to react against both K and D region-encoded allogeneic MHC class I molecules.

Increased number of CD4-CD8+ MHC class II-specific T cells in MHC class II-deficient mice.

Targeted disruption of the A beta-encoding gene of H2b mice abolishes major histocompatibility complex (MHC) class II expression and results in a failure to develop CD4+8- T cells. Besides this major effect, the lack of class II expression affects the level of T-cell receptor (TCR) and CD4 expression on differentiating thymocytes. Moreover, there is no class II-mediated negative selection of thymocytes. All this could result in TCR repertoire changes of the CD4-8+ T-cell subpopulation, which apparently develops normally in these mice. To test this hypothesis, the class II reactivity of CD4-8+ T cells from class II-deficient (class II0) mice was analysed. It was found that CD4-8+ T cells from class II0 but not from class II-expressing mice developed a significant level of cytotoxicity against class II-expressing target cells. These results demonstrate an influence of MHC class II molecules on the TCR repertoire of CD4-8+ T cells.

CD4+ and CD8+ alpha beta, and gamma delta T cells are cytotoxic effector cells of beta 2-microglobulin-deficient mice against cells having normal MHC class I expression.

Despite original reports that describe the absence of CD8+ CTLs in mice having homozygous deficiency in the beta 2-microglobulin gene, strong cytotoxic activities against cells with normal beta 2-microglobulin expression were observed in these animals. This cytotoxicity was reproducibly induced by immunizations with allogeneic or syngeneic cells. MHC class II expression by the stimulator cells was not required. The effector population contained alpha beta T cells with CD4+ or CD8+ phenotypes, as well as gamma delta T cells with CD8- and CD8+ phenotypes. Both alpha beta T cell types recognized MHC class I molecules. The gamma delta T cells seemed to respond to molecules that are affected by the absence of beta 2-microglobulin, but that are not encoded by classical MHC loci.

Increased number of cytotoxic T cells within CD4+8- T cells in beta 2-microglobulin, major histocompatibility complex class I-deficient mice.

Targeted disruption of beta 2-microglobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4-8+ T cells. Despite this, beta 2 M-/- mice reject skin grafts and cope with most viral infections tested. We asked whether CD4+8- cytotoxic T cells would play a role in compensating for the defect in CD4-8+ cytotoxic T cell function. We found that the cytotoxic activity against class II+ targets is significantly higher among CD4+8- T cells of beta 2M-/- than among those of beta 2M+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4+8-, class II-specific T cells is at least fivefold higher in beta 2M-/- than in beta 2M+/+ mice. These results suggest that CD4+8- cytotoxic T cells could play a major role in carrying out cytotoxic function in beta 2M-/- mice.

Humoral immune response to the antigen administered as an immune complex.

Antigen (HSA) bound in immune complexes at equivalence with syngeneic anti-HSA antibodies elicit much stronger humoral immune response then soluble HSA. On the other hand, administration of immune complexes formed with xenogeneic (rabbit) anti-HSA antibodies suppressed humoral immune response against HSA, but not against rabbit IgG in mice. We suggest that immunization with antigen bound in immune complex might represent a powerful tool in enhancing humoral immune responses.

Cellular immune response to the antigen administered as an immune complex in vivo.

Recently, it was shown that 10(2)- to 10(3)-fold lower doses of human serum albumin (HSA) are sufficient for the same T-cell response in vitro, if HSA is administered to the cultures bound in the immune complex rather than in the soluble form. In the present study, we analysed the capacity of HSA in the form of immune complexes to elicit specific cellular immune response in vivo. We found that antigen bound in the immune complex with murine, syngeneic polyclonal antibodies elicited the same T-cell response as fivefold higher doses of free antigen. On the other hand, HSA bound in the immune complexes with xenogeneic, rabbit polyclonal antibodies did not enhance anti-HSA cellular immune response. Our results indicate that binding of antigen in the immune complex could play an important role in enhancing an antigen-specific cellular immune response in vivo.

Cellular immune response to the antigen administered as an immune complex.

Immune complexes are specifically bound to the Fc receptors on the surface of various types of cells capable of presenting antigens. It was therefore determined if human serum albumin (HSA), bound in an immune complex, is presented more efficiently to the HSA-specific T cells than HSA alone. Primed, polyclonal murine T cells were stimulated in vitro with either HSA alone or HSA bound at equivalence to syngeneic, polyclonal anti-HSA antibodies of IgG class. The stoichiometric composition of the insoluble immune complex was AgAb2.91. The present study shows that 10(2)-10(3)-fold lower doses of HSA are sufficient for the same T-cell response in vitro, if HSA is administered to the cultures in the form of the immune complex rather than in the soluble form. The enhancement of T-cell proliferation in the presence of immune complex was blocked with monoclonal antibody (mAb) against Fc receptor. Our results indicate that binding of antigen in the immune complex could play an important role in enhancing an antigen-specific cellular immune response.

Dynamics of positive and negative selection in the thymus: review and hypothesis.

T cells recognize with a single receptor both a product of antigens processed by antigen presenting cells (APC1) and a self-marker molecule, encoded by the major histocompatibility complex (MHC, a property termed MHC-restricted recognition of antigen). During their differentiation in the thymus, T cells "learn" what to regard as self-MHC molecules, and only the cells once able to recognize antigen in the context of self-MHC will be "positively selected" to exit the thymus. The cells, once capable of reacting to self molecules, do not exit the thymus. They are "negatively selected" (deleted). Both "positive" and "negative" selection depends on the T-cell-receptor (TCR) specificity. Furthermore, the TCR specificity determines the final phenotype of the mature T cells; namely, the cells with receptors specific for the MHC-class I molecule will acquire the CD4-CD8+ phenotype, while the cells with receptors specific for the MHC-class II molecule will acquire the CD4+CD8- phenotype. However, a few mature T cells in the periphery do not follow the rule: CD4 expression class II restriction and CD8 expression class I restriction. We believe that these T lymphocytes have a receptor with very high affinity for one class of MHC molecules and cross-react with another class of MHC molecules (with somewhat lower affinity). The majority of T lymphocytes with such receptors bind the thymic MHC molecule, for which they have the highest affinity. Since this affinity is too high for further differentiation, such clones are deleted in the thymus. However, a small fraction of these cells bind the alternative class of MHC molecules, due to cross-reactivity of their receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential differentiation of T cell receptor specificities based on the MHC glycoproteins encountered during development. Evidence for positive selection.

T cells recognize foreign antigens together with those MHC glycoproteins they have encountered during their development in the thymus. How the repertoire of antigen-specific TCRs is selected has not yet been fully defined. We have investigated the T cell repertoire specificities of CD4-CD8+ cytotoxic T cells developing under conditions where one of the class I MHC-encoded molecules is blocked, while other class I-MHC glycoproteins are still expressed. We show that antigen-specific T cells restricted to the blocked class I fail to develop, while generation of other class I-specific T cell proceeds undisturbed. This highly selective perturbation of the T cell receptor repertoire demonstrates that development of CD4-CD8+ T cells with a certain TCR specificity requires expression of particular alleles of class I MHC. Thus, TCR-MHC interactions provide signals essential to the differentiation of precursor T cells.

A novel gamma delta T cell receptor for antigen adds limited diversity to the gamma delta repertoire in adult thymus.

The surface expression of CD3-associated TCR chains on hybridoma cell lines derived from adult gamma delta thymocytes was analyzed. These cell lines were unusual, in that a) they expressed a surface heterodimer consisting of a 40- and a 42-kDa chain, i.e., comprised of chains different from any previously reported gamma delta-TCR all of which express C gamma 1- or C gamma 2-encoded gamma-chains; b) their CD3-associated TCR could not be categorized as alpha beta-TCR dimers, despite the similarities in m.w. of the TCR chains, because full size 1.3-kb beta-chain mRNA capable of encoding a functional beta-chain could not be detected in these cells; c) neither of the receptor chains could be precipitated with anti-C gamma 1C gamma 2-peptide antisera. Biochemical analysis demonstrated that the 42-kDa delta-chain is a novel chain, which differs from any reported delta-chains in size, charge and number of glycosylation sites. Collectively, the data on analysis of the 40-kDa chain strongly suggest that it represents a gamma-chain encoded for by the C gamma 4 locus, protein products of which have not yet been reported in the thymus. This gamma-chain was also unique, in that its isoelectric point was much lower than that of other gamma-chains. The gamma- and delta-chains on these C gamma 4-expressing hybridomas were indistinguishable from one another in size and charge (as determined by nonequivalent pH gradient electrophoresis/SDS-PAGE analysis and analysis after endoglycosidase treatment). Because the cell lines were randomly chosen from large panels of hybridomas, these results may well imply strikingly nonrandom pairing of thymocyte-derived C gamma 4 chains and the delta-chains reported here. Thus, only limited additional gamma delta repertoire diversity may be generated by availability of this gamma delta-TCR in the thymus.

Surface expression of only gamma delta and/or alpha beta T cell receptor heterodimers by cells with four (alpha, beta, gamma, delta) functional receptor chains.

Surface expression of TCR dimers by cells synthesizing three or four distinct types of receptor chains was analyzed. Cells containing intact gamma, alpha, and beta chains had only gamma delta dimers on the cell surface. In human PEER cells, addition of a functional alpha chain led to the loss of gamma delta dimer expression and expression of only alpha beta dimers. This result was not due to transcriptional down-regulation of the gamma or delta loci. In murine cells expressing all four chains, both gamma delta and alpha beta dimers could be demonstrated on a single cell. No other chain combinations (alpha gamma, alpha delta, beta gamma, or beta delta) were detected. Thus, there is stringent control of assembly and/or transport of TCR heterodimers, such that functional receptors consist only of alpha beta and gamma delta pairs, and no additional repertoire diversity is generated by cross pairing.

Predominant variable region gene usage by gamma/delta T cell receptor-bearing cells in the adult thymus.

Previous studies have indicated that the diversity of gamma genes expressed by gamma/delta-bearing murine T cells is limited, but comparable information concerning the expressed diversity of delta genes is lacking. In this study, we have investigated the rearrangement and expression of delta and gamma genes in T cell hybridomas that express gamma/delta T cell receptors. Three productive delta chain cDNA clones were isolated (delta 7.3, delta 7.1, and delta 2.3) that encode new variable region sequences. Two of the delta cDNAs differ significantly from those observed in the V alpha repertoire. In addition, one cDNA expressed a new J delta region (J delta 2), which was localized between J delta 1 and C delta genes. Using these and other delta gene probes and gamma gene probes, we found that five independent hybridomas expressed four different V delta s and three different V gamma s. However, analysis of an enriched population of gamma/delta-expressing cells from the adult thymus suggests that only a few V delta genes and one V gamma gene are used by the majority of the cells. These results suggest that important components of receptor chain that contribute to specificity (i.e., the germline V gene sequences) are relatively nondiverse in the thymic gamma/delta population.

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants.

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.

T cell receptor gamma/delta chain diversity.

The TCR-gamma and -delta chains of six murine hybridomas were compared by one-dimensional SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analysis. This allowed the identification of three distinct gamma chains (gamma a, gamma b, and gamma c) and three distinct delta chains (delta a, delta b, and delta c). Four gamma/delta chain combinations (gamma a delta a, gamma b delta b, gamma b delta c, and gamma c delta a) were observed. These results indicate that multiple forms of the delta chain are expressed and suggest that the delta chains are encoded for by an Ig-like rearranging gene. This delta chain polymorphism significantly enhances the potential diversity of TCR-gamma/delta, which may be of importance for a better understanding of the putative ligand(s) recognized by this receptor.

Activation properties of T cell receptor-gamma delta hybridomas expressing diversity in both gamma- and delta-chains.

To elucidate the structure, diversity, and activation properties of the murine T3-associated gamma delta-receptor, examination was made of the gamma delta and T3 components, T cell receptor (TCR) gene transcription, activation properties, and lymphokine production in a panel of four cloned T cell hybridomas expressing a TCR-gamma delta. Biochemical analysis of the gamma and delta proteins expressed on these hybridomas reveals new gamma and delta species not observed in whole populations of dLy-1 Lyt-2-L3T4-thymocytes from which these hybridomas were derived. Thus, analysis of expression of the TCR-gamma delta complex at the clonal level indicates that both gamma and delta appear to be expressed as multiple distinct gene products within a homozygous inbred mouse strain. Northern blot analysis reveals that, whereas all gamma delta hybridomas had mature 1.5-kb TCR-alpha-chain and mature TCR-gamma-chain transcripts, none had mature 1.3-kb TCR-beta-chain transcripts, thus indicating that the type of TCR heterodimer expressed reflects of the state of TCR gene transcription in these hybridomas. Our results also reveal striking similarities between TCR-gamma delta and TCR-alpha beta cells with respect to their activation properties. First, all five of the T3 components associated with the gamma delta-complex are of the same size and have the same glycosylation patterns as those associated with alpha beta-heterodimers. Second, induction of function in these gamma delta cells (assayed by lymphokine production) can be achieved with a variety of stimuli known to elicit activation signals in alpha beta cells as well: direct receptor-engagement (i.e., through anti-Thy-1), and phorbol 12-myristate 13-acetate-plus-ionomycin-mediated. Collectively, these findings suggest that gamma delta T cells express a receptor of at least limited diversity and use T3-mediated activation pathways very similar to those employed by TCR-alpha beta-bearing T cells.