This review summarizes our current knowledge of T‐cell maturation and repertoire selection in the rat thymus. Some unique features of early thymocyte development and of CD4/CD8 lineage decision are ...described. A detailed analysis of lineage progression through the CD4, CD8 “double positive” compartment and T‐cell receptor‐induced CD8 T‐cell maturation in cell culture is provided. A second emphasis is placed on interactions between germline‐encoded T‐cell receptor elements with MHC molecules in thymic repertoire selection and alloreactivity
Different rat Tcrb haplotypes express either TCR β variable segment (Tcrb-V) 8.2l or 8.4a. Both V segments bind the mAb R78 but differ by one conservative substitution (L14V) and clusters of two and ...four substitutions in the complementarity-determining region (CDR) 2 and CDR4 hypervariable loop 4 (HV4). Independently of MHC alleles numbers of R78+CD4+ cells are lower in Tcrb-V8.2l-expressing than in Tcrb-V8.4a-expressing strains. Expression of R78+ TCR during T cell development, analysis of backcross populations and generation of a Tcrb congenic strain LEW.TCRB(AS) define two mechanisms how Tcrb haplotypes affect the frequency of R78+ cells, one acting prior to thymic selection leading to up to 2-fold higher frequency of Tcrb-V8.4a versus Tcrb-V8.2l in unselected thymocytes and another occurring between the TCRlow and the CD4/CD8 single-positive stage. The latter leads to a 50% reduction of frequency of Tcrb-V8.4a CD8+ cells but not CD4+ cells and does not affect either subset of Tcrb-V8.2l cells. A comparison of rat classical class I MHC (RT1.A) sequences and current models of TCR–MHC–peptide interaction suggests that this reduction in frequency of Tcrb-V8.4a CD8 cells may be a consequence of differential selection of Tcrb-V8.2l versus Tcrb-V8.4a TCR by differential binding of CDR2β to highly conserved areas of C-terminal parts of the α helices of class I MHC molecules.
TCRAV segments contribute significantly to MHC restriction as illustrated by their general preference for either the CD4 or CD8 T cell subset and additional, MHC allele-specific overselection during ...T cell differentiation. The 10-fold over-representation of the TCRAV8S2 (VA8S2) segment in CD8 over CD4 T cells by the RT1f haplotype of LEW.1F rats provides the most striking example of MHC allele-specific overselection of a VA segment reported so far. Also in alloreactivity, VA8S2+ CD8 cells from RT1f– rats are preferentially expanded by RT1f+ stimulators. We have identified the class I molecule, Af, mediating VA8S2 overselection and report that it differs only in four amino acids at the MHC–TCR interface from the class I molecule Aa, which is neutral with regard to selection of VA8S2. We also provide an extensive survey of the TCRAV8 family and show that among 14 functional VA8 segments in LEW rats, the dramatic Af-dependent overselection is unique for VA8S2. Surprisingly, VA8S2 expression in CD8 T cells of RT1f+ rats derived from a Sprague-Dawley stock was only 3% as compared to the 12% observed in LEW.1F. The VA8S2 segment of Sprague-Dawley (VA8S2SD) differs from VA8S2 of the LEW background (VA8S2l) in only two amino acids, one of which is located in CDR2 and could thus participate in allele-specific recognition of Af. However, analysis of the pre- and postselection thymic repertoires of Sprague-Dawley and LEW.1F rats and of the repertoire of CD8 cells from both strains expanded in the alloreactive response to RT1f revealed that the difference in VA8S2 representation between the two backgrounds is explained by differential availability in the preselection repertoires and not by a difference in overselection. Sequence comparisons of Af and Aa and of both VA8S2 segments suggest a predominant role of CDR1 in hyper-reactivity to Af. Thus, the VA composition of the mature TCR repertoire is influenced by Tcra locus polymorphisms at two levels: the regulation of VA usage in the preselection repertoire and the composition of structural elements which contribute to specific VA–MHC interactions during thymic selection.
Expression of the rat CD8 molecule was studied using five novel monoclonal antibodies (mAb), four of which are specific for the V-like domain of CD8 alpha, whereas one reacts either with the beta ...chain or with a determinant only expressed on the CD8 alpha/beta heterodimer. mAb to both chains effectively blocked purified lymph node CD8 T cells in mixed lymphocyte reaction and in cell-mediated cytotoxicity. Flow cytometric analysis showed that CD8 T cells from lymph nodes or spleen of normal rats almost exclusively express the alpha/beta isoform, regardless of the T cell receptor isotype (alpha/beta or gamma/delta). In contrast, natural killer (NK) cells carry only CD8 alpha chains. This CD8 alpha + beta - phenotype was also prominent among CD8 T cells from athymic rats and from intestinal epithelium of normal rats. CD8 alpha homodimers can also be expressed as a result of activation, as shown by analysis of CD4 CD8 double-positive T cells obtained from highly purified lymph node CD4 T cells by in vitrok stimulation. Such CD4+CD8 alpha + beta - cells also represent a major subset among adult intestinal intraepithelial lymphocytes (IEL), suggesting local activation. Taken together, the difference in CD8 isoform expression among T cells from athymic rats, NK cells, and gut IEL versus CD8 T cells from peripheral lymphatic organs of euthymic animals suggests that like in mice, expression of the CD8 heterodimer is more dependent on intrathymic maturation than that of the homodimer. Since the more stringent thymus dependence of CD8 alpha + beta + T cells may be due to a requirement for thymic selection on self major histocompatibility complex class I antigens, the virtually exclusive CD8 alpha + beta + phenotype of peripheral rat gamma/delta T cells could mean that antigen recognition by this subset is also restricted by MHC class I molecules.
Unselected CD4+8+ rat thymocytes, generated in vitro from their direct precursors, are readily converted to functional TCRhigh T cells by stimulation with immobilized TCR-specific mAb plus IL-2. ...Lineage decision invariably occurs toward CD4-8+, regardless of the timing of TCR stimulation after entry into the CD4+8+ compartment or the concentration of TCR-specific mAb used for stimulation. CD4-specific mAb synergizes with suboptimal TCR-specific mAb in inducing T cell maturation, but lineage decision remains exclusively CD4-8+. These results contrast with those obtained in mice, in which Abs to the TCR complex were shown to promote CD4+8- T cell maturation from CD4+8+ thymocytes. Surprisingly, when rat and mouse CD4+8+ thymocytes were stimulated with PMA/ionomycin under identical conditions, the opposite lineage commitment was observed, i.e., mouse thymocytes responded with the generation of CD4+8- and rat thymocytes with the generation of CD4-8+ cells. It thus seems that CD4+8+ thymocytes of the two species respond with opposite lineage decisions to strong activating signals such as given by TCR-specific mAb or PMA/ionomycin. A possible key to this difference lies in the availability of p56lck for coreceptor. supported signaling. We show that in contrast to mouse CD4+8+ thymocytes, which express both a complete and a truncated CD8 alpha-chain (CD8 alpha') unable to bind p56lck, rat thymocytes only express full-length CD8 alpha molecules. Mice, but not rats, therefore may use CD8 alpha' as a "dominant negative" coreceptor chain to attenuate the CD8 signal, thereby facilitating MHC class II recognition through the higher amount of p56lck delivered, and rats may use a different mechanism for MHC class distinction during positive selection.
The in vitro response of unprimed rat T cells to retroviral and bacterial superantigens (SAg) was analyzed with TCR V beta 8.2-, 8.5-, 10-, and 16-specific mAbs. Specific stimulation of V beta 8.2 ...and 8.5 CD4 cells was observed in the response to Mls1a, the retroviral SAg encoded by integrated provirus Mtv-7 (Mtv-7 SAg), which was presented by mouse B cells or mouse fibroblasts transfected with DR1 genes and the Mtv-7 SAg. Additionally, a strong response of V beta 16 CD4 cells to an as yet unidentified mouse SAg was found. Only some of the bacterial SAg known to stimulate mouse and human T cells also activated rat lymph node cells. SEA, SEE, and TSST-1 stimulated rat T cells well; SEB, SEC1, and SED did not. This defect was apparently a result of weak binding to rat MHC class II molecules because presentation by human MHC class II molecules restored T cell activation. Under these conditions, SEB stimulated V beta 8.2+ and 8.5+ CD4 and CD8 cells from Lewis rats. A comparison of several rat strains revealed an unresponsiveness to SEB or Mtv-7 SAg for V beta 8.2 cells from F344 and DA rats. Determination of the nucleotide sequences of the Tcrb-V8.2 of these strains revealed differences between SAg-responsive and SAg-unresponsive Tcrb-V8.2 in seven amino acids, four of them located in the putative SAg contact site. The significance of these findings for the evolution of TCR-SAg interactions is discussed.
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