Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Delayed thymocyte maturation in the trisomy 16 mouse fetus

Mouse fetuses with trisomy 16, an animal model for human trisomy 21 (Down syndrome), have severe defects in several hematopoietic stem cell populations and a marked reduction in thymocyte number. To determine whether there are other defects in the development of the trisomic thymus, the ontogeny of the cell surface antigenic determinants, Thy-1, Ly-1, CD3, CD4, CD8, and TCR v beta, was investigated. The trisomy 16 thymocytes were able to express all of determinants either during fetal life (days 14 to 19 of gestation) or in cultures of intact thymus lobes. However, in all instances (except for Thy-1, which already had a high proportion of expressing thymocytes by day 14), there was a delay in the time at which the determinants were first expressed, as manifested by reduced numbers of positively staining cells. Furthermore, there was also a delay in the rate at which the positively staining cells attained maximal Ag densities. Overall, there was an approximate 2 day lag in development of the fetal trisomic thymocytes. This lag permitted the identification of a large population of CD4-8+ cells prior to the appearance of CD4+8+ thymocytes. These findings are consistent with the identification of CD4-8+ as an intermediate stage between CD4-8- and CD4+8+ in fetal thymocyte ontogeny.     

Identification and distribution of the costimulatory receptor CD28 in the mouse.

T cell activation requires Ag-specific stimulation mediated by the TCR as well as an additional stimulus provided by Ag presenting cells. On human T cells, it has been shown that antibodies to the Ag CD28 can provide a potent amplification signal for cytokine production and proliferation. Here we describe the production of a mAb to the murine homologue of CD28, and the use of this antibody to examine the function and distribution of CD28 in the mouse. Anti-murine CD28 synergizes with TCR-mediated signals to greatly enhance lymphokine production and proliferation of T cells, and the CD28 signal is not blocked by cyclosporin A. In the peripheral lymphoid organs and in the blood of the mouse, all CD4+ and CD8+ T cells express CD28. In the thymus, CD28 expression is highest on immature CD3-, CD8+ and CD4+8+ cells, and on CD4-8- cells that express alpha beta and tau delta TCR. The level of CD28 on mature CD4+ and CD8+ alpha beta TCR+ thymocytes is two- to fourfold lower than on the immature cells. The potent costimulatory function of CD28 on mature T cells, together with the high level of expression on CD4+8+ thymocytes, suggest that this costimulatory receptor might play an important role in T cell development and activation.     

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.     

The regulated expression of a diverse set of genes during thymocyte positive selection in vivo

A signal initiated by the newly formed Ag receptor is integrated with microenvironmental cues during T cell development to ensure positive selection of CD4+CD8+ progenitors into functionally mature CD4+ or CD8+ T lymphocytes. During this transition, a survival program is initiated, TCR gene recombination ceases, cells migrate into a new thymic microenvironment, the responsiveness of the Ag receptor is tuned, and the cells commit to a specific T lineage. To determine potential regulators of these processes, we used mRNA microarray analysis to compare gene expression changes in CD4+CD8+ thymocytes from TCR transgenic mice that have received a TCR selection signal with those that had not received a signal. We found 129 genes with expression that changed significantly during positive selection, the majority of which were not previously appreciated. A large number of these changes were confirmed by real-time PCR or flow cytometry. We have combined our findings with gene changes reported in the literature to provide a comprehensive report of the genes regulated during positive selection, and we attempted to assign these genes to positive selection process categories.     

Expression of ets genes in mouse thymocyte subsets and T cells

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection

In the thymus, phenotypically and functionally mature single positive cells are generated from immature CD4+8+ precursors by a process known as positive selection. Although this event is known to involve alphabetaTCR ligation by peptide/MHC complexes expressed on thymic stromal cells, it is clear that positive selection is a multistage process involving transition through an intermediate CD4+8+69+ phase as well as subsequent postselection phases. By analyzing the development of preselection CD4+8+69- and intermediate CD4+8+69+ thymocytes in the presence of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient thymic stromal cells, we investigated the role of MHC molecules at three distinct points during positive selection. Although the initiation of positive selection is critically dependent upon MHC interactions, we find the that later stages of maturation, involving the differentiation of CD4+8- and CD4-8+ cells from CD4+8+69+ thymocytes, occur in the absence of MHC molecules. Moreover, an analysis of the postselection proliferation of newly generated CD4+8- and CD4-8+ thymocytes shows that this also occurs independently of MHC molecules. Thus, our data provide direct evidence that, although positive selection is a multistage process initiated by TCR-MHC interactions, continuation of this process and subsequent postselection events are independent of ongoing engagement of the TCR.     

Variations in thymocyte susceptibility to clonal deletion during ontogeny. Implications for neonatal tolerance.

Activation of immature thymocytes via the TCR results in programmed cell death and clonal deletion. We have examined thymocytes from mice of different ages and observed that, whereas TCR-mediated signaling caused deletion of thymocytes from newborn and 3-week-old mice, it failed to delete thymocytes from mice of 1 week of age. This could not be attributed to differences in cell surface TCR expression, TCR-mediated phosphoinositide hydrolysis or Ca2+ mobilization, or total cellular levels of TCR zeta- and eta-chains. Moreover, thymocytes of all ages were equally susceptible to corticosteroid- and Ca2+ ionophore-induced programmed cell death. These data are consistent with the notion that fetal and neonatal thymocytes represent a relatively synchronous wave of cells passing through phases in which they are susceptible and then resistant to TCR-induced programmed cell death. They also support the notion that the classical phenomenon of neonatal tolerance is due to clonal deletion and that the inability of allogeneic cells to tolerize mice at 1 week of age is because the thymocytes are refractory to TCR-alpha beta-mediated clonal deletion.     

Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.     

The expression of T cell receptor-associated proteins during T cell ontogeny in man

The expression of TCR-associated molecules was examined in human fetal and postnatal tissues. From gestational wk 7 onward in the fetal liver, putative prothymocytes have been identified with cytoplasmic CD3 positivity (cCD3+). These immature cells are TdT- and do not express membrane CD3 (mCD3-) or TCR beta identified by beta F1, but show CD7 and CD45 positivity without CD1, CD2, CD5, CD4, CD8, CD10, and class II Ag. Their high proliferative activity is indicated by greater than 85% Ki67 positivity. After the 10th wk, beta F1+, mCD3+ cells also appear in the liver and these are mostly Ki67- but no TCR gamma delta-bearing cells can be identified at such an early stage of extrathymic development. In the mCD3- TdT-fetal thymus (10 1/2 to 18th wk) cCD3+, mCD3- CD1-blasts proliferate (Ki67+) and lack TCR-beta or TCR-gamma delta. The TdT-, CD1+ cortical thymocytes develop into TCR-beta + and WT31-positive (TCR-alpha beta +) cells. Subsequently TdT-positive thymocytes become detectable around 19 to 20 wk, and in such glands the peak of proliferative activity is seen among TdT+, cCD3+ cells which appear to acquire, in a regular sequence, cytoplasmic beta F1 (TCR-beta), mCD3, and TCR-alpha beta (WT31 positivity) together with the loss of TdT and Ki67 positivity. A newly described transitional population of cells is TdT-, beta F1+ but exhibits no detectable WT31 positivity. These cells correspond to the CD1+, mCD3+ thymocytes and are probably the targets of thymic selection. The cells of the TCR-gamma delta lineage, detected by mAb TCR-delta-1 and delta TCS1, are rare (0.02 to 0.5%) among thymocytes from gestational wk 10 1/2 onward through the whole span of thymic development, but these cells include a proportion (18 to 59%) of cells expressing CD1 Ag, suggesting that these TCR-gamma delta cells differentiate in the thymus. Among the CD1+, TCR-gamma delta + thymocytes, no TdT positivity can be detected.     

Effect of cyclosporin A on the ontogeny of different T cell sublineages in chickens

We have used a panel of murine mAb against chicken TCR and associated molecules to study the effect of cyclosporin A (CsA) on the ontogeny of the different sublineages of T cells. After injection of CsA (20 mg/kg/day from day 0 to 20) we observed a significant suppression of the normal maturation of the TCR2 (alpha beta TCR) cells in their transition from cortical CD4+CD8+ thymocytes to the mature single positive cells in the thymus medulla. The TCR3 subpopulation, a distinct form of alpha beta-like TCR in chickens, was inhibited from initially developing within the cortex by CsA, indicating that the TCR3 subpopulation is functionally distinct from the TCR2+ cells. In contrast, the maturation and peripheral emigration of TCR1 (gamma delta TCR) cells was unaffected by CsA treatment. Mature splenic T cells sorted for either TCR1+ or TCR2+ subsets were equally sensitive to CsA blockade of Con A-stimulated mitogenesis, indicating that there is no inherent difference in CsA sensitivity between these sublineages. Furthermore, no difference was detected in the expression of class II MHC Ag in thymi of birds treated with olive oil vs CsA. Inasmuch as the mechanism of CsA action appears to involve inhibition of TCR initiated signal transduction for lymphokine synthesis, these data indicate that a similar signaling is involved in thymic repertoire selection for TCR2. The lack of an effect on TCR1 cell maturation suggests that the TCR1 repertoire may not undergo selection in the thymus as do TCR2+ cells.     

Effects of cyclosporine A on thymocyte differentiation in fetal thymus organ culture

During the course of differentiation in the thymus, precursor T cells are negatively selected by a self-tolerance mechanism or positively selected to acquire restriction specificity to self major histocompatibility complexes. We investigated the process of T cell differentiation and those selections using a fetal thymus organ culture with or without cyclosporine A. The agent blocked the maturation step from CD4+8+ double positive cells to mature CD4-8+ or CD4+8- single positive cells. On the other hand, the agent did not inhibit the development of CD3+4-8- T cell receptor (TCR)alpha beta- cells, which were supposed to be T cells bearing gamma delta-TCR chains. These results suggest that the development of thymocytes bearing alpha beta- or gamma delta-TCR chains differ in requirement for thymocyte-stromal cell interaction.     

Characterization of cells comprising murine thymocyte multicellular complexes

Characterization of complexes between thymocytes and thymic stromal cells was facilitated in the present study by demonstrating that complexes would reform if cells were incubated for 1.5 to 2 h in vitro at ambient temperature. Several immunologic approaches were used to determine the phenotype of complexed cells. Bound T cells were 97% double-positive (CD4 and CD8), 3% double-negative and greater than 99% CD3 positive by using immunoperoxidase immunohistology on cytospins. Five percent expressed the TCR beta-chain and 1 to 2% were IL-2R positive. The percentages were the same whether complexes were preformed in vivo or formed in vitro. Despite the apparent absence of single positive cells in complexes, when isolated CD4 or CD8 positive cells were tested for their ability to bind to adherent thymic macrophages, each subpopulation contained some cells which were capable of complex formation. When thymocytes were fractionated by density, steroid sensitivity or peanut agglutinin positivity then allowed to form complexes, cells with an immature phenotype had a greater propensity for complex formation. Central stromal cells all were class II MHC gene product (I-A and I-E) positive, expressed macrophage-associated Ag (B23.1 and MAC-1), were negative for cytokeratin but positive for vimentin, were reactive with a polyclonal antimacrophage serum, but did not express dendritic cell Ag (33D1). The data demonstrate that immature thymocytes bind exclusively to class II MHC gene product positive thymic macrophages. This binding step may play a role in the acquisition of T cell function in the thymus.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

CD4-CD8- thymocytes from MRL-lpr/lpr mice exhibit abnormal proportions of alpha beta- and gamma delta-TCR+ cells and demonstrate defective responsiveness when activated through the TCR.

MRL-lpr/lpr (lpr) mice develop profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in the peripheral lymphoid organs. Earlier studies from our laboratory demonstrated an increased proportion of DN cells in the thymus of lpr mice with age. Inasmuch as the DN thymocytes constitute a heterogenous population of cells, in the present study, we investigated the TCR phenotype of DN thymocytes and their responsiveness to activation through the TCR. The DN thymocytes of young (1 month of age) lpr mice contained approximately 65% CD3+ cells of which approximately 60% were alpha beta-TCR+ and approximately 39% were gamma delta-TCR+ as detected by using pan anti-TCR mAbs. In old (4-6 months of age) or young MRL-(+/+) mice, similar proportions of CD3+, alpha beta- or gamma delta-TCR+ DN thymocytes were detected. Interestingly, however, in old (4-6 months of age) lpr mice, the CD3+ T cells increased to approximately 86% and the majority of these (approximately 81%) were alpha beta-TCR+ and only approximately 3% were gamma delta-TCR+. Also, in old lpr mice, there was a 10-fold increase in the absolute number of alpha beta-TCR+ DN cells in the thymus, whereas, the absolute number of gamma delta-TCR+ DN cells in the thymus did not alter significantly. Furthermore, a majority (approximately 84%) of the old lpr DN thymocytes expressed CD45R, similar to the peripheral DN T cells. In contrast, only a small number (approximately 1%) of DN thymocytes from young lpr or MRL-(+/+) mice expressed CD45R. The DN thymocytes from young lpr or MRL-(+/+) mice demonstrated strong and similar proliferative responsiveness to stimulation with PMA + calcium ionophore or PMA + IL-2, or to immobilized mAb directed against the TCRs (CD3, alpha beta and gamma delta). In contrast, the DN thymocytes and the DN peripheral T cells from old lpr mice demonstrated marked defect in responding to the above stimuli. The present study suggests that with the onset of lymphadenopathy, the DN cells in the thymus of old lpr mice are increasingly skewed toward the alpha beta-TCR repertoire, the majority of which express CD45R and respond poorly to mitogenic stimuli or when activated through the TCR. It is suggested that migration of such cells continuously to the periphery may result in severe lymphadenopathy seen in old MRL-lpr/lpr mice.     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

A role for CD8 in limiting degeneracy of thymocyte selection.

The introduction of a soluble TCR (sTCR) recognizing class I major histocompatibility complex (MHC) in the fetal thymic microenvironment in vitro produces the selection of thymocytes with enhanced avidity for self class I MHC (8). The sTCR was supposed to impose enhanced avidity for self MHC at an early degenerate phase of TCR-driven selection. This could determine increased reactivity to self at later stages of differentiation when specificity of TCR-ligand interaction augments and the effect of sTCR vanishes. This hypothesis was based on the observed deletion of CD4+8+ thymocytes upon upregulation of TCR and the increase in cell size of some CD8+ cells which are expanded in long-term fetal thymus organ cultures (FTOC) as well as in the periphery of adoptively transferred nude mice. Here we show that the developing alphabeta thymocyte which does not express CD8 at the cell surface has a selective advantage in FTOC with sTCR, thus suggesting that participation of CD8 in self peptide/MHC recognition confers specificity to T-cell selection and results in excessive signaling in thymocytes in spite of the presence of sTCR.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

Elimination of CD8+ thymocytes in transgenic mice expressing an anti-Lyt2.2 immunoglobulin heavy chain gene.

Individual T cell populations are characterized by specific surface proteins, namely by the T cell receptor complex (TCR) and by two accessory molecules, CD8 (Lyt2) and CD4 (L3T4). CD8 and CD4 are required for T cell interactions with class I or class II major histocompatibility complex molecules. In the thymus, immature CD8(-4)-TCR- cells differentiate, possibly via a short stage of CD8+4- thymocytes, into CD8+4+ TCR+ T cells and mature further into the main T cell populations, the CD8+4- TCR+ cytotoxic T lymphocytes and the CD4+8- TCR+ T helper cells. In order to analyse the differentiation steps involving CD8, we generated transgenic mice expressing mu heavy chain genes from an anti-Lyt2.2 hybridoma. Transgenic lines expressing either the complete (mu sm) or only the secreted mu protein (mu s) suffer from a severe depletion of their CD8+4+ thymocytes affecting also the mature CD8+4- and CD4+8- populations. The depletion is correlated to the expression of transgenic mu-chain proteins within thymocytes. This intrathymocyte expression of the mu chain prevents CD8-4- thymocytes from further differentiation, most probably via intracellular interactions between mu heavy chain and CD8 proteins. These results show that CD8 plays an important role during thymocyte maturation.