CD8 surface levels alter the fate of alpha/beta T cell receptor-expressing thymocytes in transgenic mice.

The mature T cell receptor (TCR) repertoire is established on the basis of discriminative events involving binding of the TCR alpha and beta chains and CD4 or CD8 on immature thymocytes to major histocompatibility complex (MHC)/self-peptide complexes expressed in the thymus. To ask whether the strength of the interaction between a CD8/TCR complex and a MHC/self-peptide ligand plays a pivotal role in deciding the fate of a maturing thymocyte, we generated lines of transgenic mice that express distinct and elevated levels of CD8 alpha, approximately 2, 3, and 6-10 times. These lines were then crossed to a transgenic line expressing the class I-restricted TCR, 2C. We found that thymocytes expressing the 2C TCR in combination with the highest levels of CD8 were deleted on the H-2 Kb background that is normally positively selecting for the 2C TCR. In contrast, thymocytes coexpressing the 2C TCR and moderately elevated levels of CD8 were selected for maturation. These results demonstrate directly that CD8 levels can affect the developmental fate of a maturing thymocyte and argue in support of an affinity model for thymocyte selection.

Identification and characterization of new murine T cell receptor beta chain variable region (V beta) genes.

By screening previously isolated genomic clones spanning the mouse TCR V beta locus with V beta-specific oligonucleotides, we have isolated one new functional V beta gene and six V beta pseudogenes. Because this method of identifying new genes does not depend on expression levels, we conclude that most, if not all, V beta genes in the mouse have been identified. The newly identified pseudogenes increase the frequency of mouse TCR V beta pseudogenes to 28%, a frequency similar to that estimated for mouse Ig VH pseudogenes (24). Three of the newly discovered pseudogenes are clustered in a region around another pseudogene (V beta 17b). The extensive DNA diversity, as reflected in both the nucleotide sequence and the RFLP, indicates that this genomic region is a possible hotspot of recombination. The new functional gene, V beta 19a, is expressed at very low levels, which explains why it has not been isolated earlier. V beta 19 shows expression patterns that correlate with the previously described Va beta and Vb beta haplotypes.

T-B cell interaction inhibits spontaneous apoptosis of mature lymphocytes in Bcl-2-deficient mice.

Bcl-2 expression is tightly regulated during lymphocyte development. Mature lymphocytes in Bcl-2-deficient mice show accelerated spontaneous apoptosis in vivo and in vitro. Stimulation of Bcl-2-deficient lymphocytes by anti-CD3 antibody inhibited the spontaneous apoptosis not only in T cells but also in B cells. The rescue of B cells was dependent on the presence of T cells, mainly through CD40L and interleukin (IL)-4. Furthermore, we generated Bcl-2-deficient mice transgenic for a T cell receptor or an immunoglobulin, both specific for chicken ovalbumin, to test for antigen-specific T-B cell interaction in the inhibition of the spontaneous apoptosis. The initial T cell activation by antigenic peptides presented by B cells suppressed apoptosis in T cells. Subsequently, T cells expressed CD40L and released ILs, leading to the protection of B cells from spontaneous apoptosis. These results suggest that the antiapoptotic signaling via CD40 or IL-4 may be largely independent of Bcl-2. Engagement of the Ig alone was not sufficient for the inhibition of B cell apoptosis. Thus, the physiological role of Bcl-2 in mature lymphocytes may be to protect cells from spontaneous apoptosis and to extend their lifespans to increase the opportunity for T cells and B cells to interact with each other and specific antigens in secondary lymphoid tissues. Bcl-2, however, appears to be dispensable for survival once mature lymphocytes are activated by antigen-specific T-B cell collaboration.

Identification of a T cell receptor beta chain variable region, V beta 20, that is differentially expressed in various strains of mice.

A cDNA library of TCR beta chain transcripts from BALB/c thymocytes was constructed using anchored polymerase chain reaction (PCR). Screening of this library led to the identification of a V beta gene segment, V beta 20, structurally related to V beta 3 and V beta 17. Genomic analysis of mice displaying deletions in their V beta loci, together with mapping of cosmid clones, situated V beta 20 2.5 kb beside V beta 17. The expression of V beta 20 was estimated by PCR in mice of different H-2 and Mls types. Peripheral T cells from H-2k and H-2d mice did not express V beta 20, whereas in I-E-negative mice (C57Bl/6 and SJL), V beta 20 transcripts were detected. The lack of V beta 20 transcripts in (C57Bl/6 x CBA/J)F1, (C57Bl/6 x BALB/c)F1, and in congenic B6.H-2k mice suggests that the differential use of V beta 20 is due to an I-E-mediated clonal deletion process. The involvement of the Mls super antigens was excluded by analysis of all Mls type combinations. The nature of the V beta 20-deleting element(s) is discussed in the context of the I-E/superantigen systems controlling the expression of V beta 11 and V beta 17.

CD45RA and CD45RBhigh expression induced by thymic selection events.

CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice.

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin-associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP-1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development.

Expression of a murine polyclonal T cell receptor marker correlates with the use of specific members of the V beta 8 gene segment subfamily.

A series of murine T lymphocyte clones were examined for reactivity with the KJ16-133 and F23.1 mAbs. Clones that were KJ16-133+,F23.1+ and KJ16-133-,F23.1+ were identified, but no KJ16-133+,F23.1- clones were observed. Within our panel of clones, therefore, the KJ16-133 antibody identifies a subset of F23.1+ cells. All F23.1+ clones examined express members of the V beta 8 subfamily of beta chain variable region genes; clones expressing V beta 8.1 or V beta 8.2 reacted with both KJ16-133 and F23.1, while clones expressing V beta 8.3 reacted only with F23.1. Thus, the differential reactivity of the KJ16-133 and F23.1 antibodies with cloned T cells correlates with the V beta gene expression of each clone. Reactivity with these antibodies should therefore be of utility for predicting the V beta gene expression in some T cell clones.

No requirement for p56lck in the antigen-stimulated clonal deletion of thymocytes.

Activation of protein-tyrosine kinases (PTKs) is required for signal transduction during T cell activation, although the pathway used during thymic selection is unknown. An in vitro system was established in which T cell receptor transgenic thymocytes underwent clonal deletion in response to peptide antigen. The effects of two PTK-specific inhibitors, herbimycin A and genistein, on the clonal deletion of immature thymocytes and the activation of mature thymocytes were examined. Clonal deletion occurred while T cell activation was inhibited and when no p56lck activity was evident. Thus, p56lck is not required for the antigen-stimulated step of clonal deletion of immature thymocytes, and negative selection proceeds via a distinct pathway.

Structure and diversity of the human T-cell receptor beta-chain variable region genes.

In order to characterize the variability of the expressed human T-cell receptor (TCR) beta-chain repertoire and contrast this variability to the known murine beta-chain repertoire, 15 independent complementary DNA (cDNA) clones containing TCR beta-chain variable region (V beta) genes were isolated from a human tonsil cDNA library. The nucleotide and derived amino acid sequences of these 15 V beta genes were analyzed together with 7 previously defined sequences. Fifteen different human V beta genes could be identified from 22 independent sequences. By means of DNA hybridization and sequence homology comparisons, it was possible to group these 15 genes into ten distinct V beta subfamilies, each containing from one to seven members. Minimal polymorphism was noted between individuals, except in multimember subfamilies. The amino acid sequences of these genes contain conserved amino acids that are also shared by murine TCR V beta genes and immunoglobulins; no features were found that distinguish human V beta genes from their murine counterparts. Evaluation of secondary structure showed that maximum variability coincides with generally hydrophilic portions of the amino acid sequence, while specific hydrophobic regions were conserved in all V beta genes examined.

Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.

In order to examine the mechanisms by which clonal deletion of autoreactive T cells occurs, a peptide antigen was used to induce deletion of antigen-reactive thymocytes in vivo. Mice transgenic for a T cell receptor (TCR) that reacts to this peptide contain thymocytes that progress from the immature to the mature phenotype. Intraperitoneal administration of the peptide antigen to transgenic mice results in a rapid deletion of the immature CD4+ CD8+ TCRlo thymocytes. Apoptosis of cortical thymocytes can be seen within 20 hours of treatment. These results provide direct evidence for the in vivo role of apoptosis in the development of antigen-induced tolerance.

Requirement for CD8 beta chain in positive selection of CD8-lineage T cells.

CD8 is either an alpha alpha homodimer or an alpha beta heterodimer, although most peripheral CD8-lineage T cells express only the CD8 alpha beta heterodimer. The physiological function of CD8 beta was elucidated with mice that were chimeric for the homozygous disruption of the CD8 beta gene. The CD8 beta-1- T cells developed normally to CD4+CD8+ stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8 beta gene into CD8 beta-deficient T cells. Thus, CD8 beta is necessary for the maturation of CD8+ T cells.

Germline organization of the murine T cell receptor beta-chain genes.

The complete germline organization of the beta-chain genes of the murine T cell receptor was elucidated in order to obtain the structural basis for understanding the mechanisms of somatic DNA rearrangements. Twenty of the 22 known variable (V beta) genes are clustered within 250 kilobases of DNA 5' to the constant region (C beta) genes. These V beta genes share the same transcriptional orientation as the diversity (D beta), joining (J beta), and C beta genes, which implies that chromosomal deletion is the mechanism for most V beta to D beta-J beta rearrangements. Within this V beta cluster, the distance between the most proximal V beta gene and the D beta-J beta-C beta cluster is 320 kilobases, as determined by field-inversion gel electrophoresis. The large distance between V beta and D beta, relative to that between D beta and J beta, may have significant implications for the ordered rearrangement of the T cell receptor beta-chain genes.

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

T-cell receptor beta-chain expression: dependence on relatively few variable region genes.

Fifteen independently isolated complementary DNA clones that contain T-cell receptor (TCR) V beta genes were sequenced and found to represent 11 different V beta genes. When compared with known sequences, 14 different V beta genes could be defined from a total of 25 complementary DNA's; 11 clones therefore involved repeated usage of previously identified V beta's. Based on these data, we calculate a maximum likelihood estimate of the number of expressed germline V beta genes to be 18 with an upper 95 percent confidence bound of 30 genes. Southern blot analysis has shown that most of these genes belong to single element subfamilies which show very limited interstrain polymorphism. The TCR beta-chain diversity appears to be generated from a limited V beta gene pool primarily by extensive variability at the variable-diversity-joining (V-D-J) junctional site, with no evidence for the involvement of somatic hypermutation.

Thymic selection of cytotoxic T cells independent of CD8 alpha-Lck association.

The CD8 alpha cytoplasmic domain associates with p56lck, a nonreceptor protein-tyrosine kinase. The biological relevance of CD8 alpha-Lck association in T cell development was tested with transgenic mice generated to express a CD8 alpha molecule with two amino acid substitutions in its cytoplasmic domain, which abolishes the association of CD8 alpha with Lck. The CD8 alpha mutant was analyzed in a CD8-/- background and in the context of the transgenic 2C T cell receptor. The development and function of CD8+ T cells in these mice were apparently normal. Thus, CD8 alpha-Lck association is not necessary for positive selection, negative selection, or CD8-dependent cytotoxic function.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development.

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

Molecular requirements for cell fate determination during T-lymphocyte development.

Antigen-specific T lymphocytes recognize peptide antigens in conjunction with the products of the self major histocompatibility complex (MHC). In addition, they are immunologically self-tolerant. To acquire these characteristics, thymocytes undergo a stringent cellular selection process during development. The study of thymocyte development at the molecular level is impeded in mammalian systems by the heterogeneity of the thymocyte population in each individual. However, the use of mice transgenic for the T-cell receptor successfully circumvented this problem and made it possible to elucidate some of the requirements for positive selection, which leads to thymocyte differentiation, survival, and MHC restriction, and negative selection, which leads to programmed cell death, clonal deletion, and self-tolerance. T-cell fate is determined primarily by the nature of the interaction between a complex composed of the T-cell receptor and CD4 or CD8 molecules on the T-cell surface and the peptide antigens that are bound to MHC products and are displayed by other nonlymphoid cells present in the thymus. The molecular analysis of the receptor-ligand interactions involved in this process in transgenic mice provides opportunities to dissect cell fate determination in an intact mammalian system and to understand the molecular basis for immunological self-tolerance and MHC-restriction.