p53 prevents maturation of T cell development to the immature CD4-CD8+ stage in Bcl11b-/- mice

Signaling pathways such as the pre-TCR and Wnt pathways regulate alpha/beta T cell differentiation in thymus. Mice lacking an essential component of the pre-TCR exhibit arrest at the (CD4(-)CD8(-)) (CD44(-)CD25(+)) stage (DN3) of thymocyte development, and introduction of p53 deficiency into those mice abrogates this arrest, resulting in transition to the (CD4(+)CD8(+)) double-positive (DP) stage. This paper examines the effect of inactivation of p53 on thymocyte development in Bcl11b(-/-) mice that exhibit arrest at the DN3 or (CD4(-)CD8(+)) immature single-positive (ISP) stage. No DP thymocytes were detected in thymocytes of adoptive transfer experiments in scid mice that were derived from p53(-/-)Bcl11b(-/-) precursors but ISP thymocytes increased in the proportion and in the cell number approximately three times higher than those from Bcl11b(-/-) precursors. Consistently, the level of apoptosis decreased to the level of wild-type precursors. These results suggest that inactivation of p53 is sufficient for DN3 thymocytes to differentiate into the ISP, but not to DP, stage of thymocyte development in Bcl11b(-/-) mice. This provides evidence for a novel p53-mediated checkpoint that regulates the transition from the DN3 to ISP stage of thymocyte development.     

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.     

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.     

CD83 expression influences CD4+ T cell development in the thymus

T lymphocyte selection and lineage commitment in the thymus requires multiple signals. Herein, CD4+ T cell generation required engagement of CD83, a surface molecule expressed by thymic epithelial and dendritic cells. CD83-deficient (CD83-/-) mice had a specific block in CD4+ single-positive thymocyte development without increased CD4+CD8+ double- or CD8+ single-positive thymocytes. This resulted in a selective 75%-90% reduction in peripheral CD4+ T cells, predominantly within the naive subset. Wild-type thymocytes and bone marrow stem cells failed to differentiate into mature CD4+ T cells when transferred into CD83-/- mice, while CD83-/- thymocytes and stem cells developed normally in wild-type mice. Thereby, CD83 expression represents an additional regulatory component for CD4+ T cell development in the thymus.     

A critical role for CD2 in both thymic selection events and mature T cell function

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2(-/-) H-2(b) mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2K(b) molecule were compared on a wild-type or CD2(-/-) background. In N15tg RAG-2(-/-) CD2(-/-) mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4(-)CD8(-) double-negative thymocytes at the CD25(+)CD44(-) stage. Moreover, mature N15tg RAG-2(-/-) CD2(-/-) T cells are approximately 100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-gamma production. Repertoire analysis shows substantial differences in Valpha usage between non-tg C57BL/6 (B6) and B6 CD2(-/-) mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

IL-12 inhibits thymic involution by enhancing IL-7- and IL-2-induced thymocyte proliferation

IL-12 has been reported to affect thymic T cell selection, but the role of IL-12 in thymic involution has not been studied. We found that in vivo, IL-12b knockout (IL-12b(-/-)) mice exhibited accelerated thymic involution compared with wild-type (WT) B6 mice. This is characterized by an increase in thymocytes with the early development stage phenotype of CD25(-)CD44(+)CD4(-)CD8(-) in aged IL-12b(-/-) mice. Histologically, there were accelerated degeneration of thymic extracellular matrix and blood vessels, a significantly decreased thymic cortex/medulla ratio, and increased apoptotic cells in aged IL-12b(-/-) mice compared with WT mice. There was, however, no apparent defect in thymic structure and thymocyte development in young IL-12(-/-) mice. These results suggest the importance of IL-12 in maintaining thymic integrity and function during the aging process. Surprisingly, in WT B6 mice, there was no age-related decrease in the levels of IL-12 produced from thymic dendritic cells. Stimulation of thymocytes with IL-12 alone also did not enhance the thymocyte proliferative response in vitro. IL-12, however, provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in aged WT and IL-12b(-/-) mice. Our data strongly support the role of IL-12 as an enhancement cytokine, which acts through its interactions with other cytokines to maintain thymic T cell function and development during aging.     

Compensatory anion currents in Kv1.3 channel-deficient thymocytes

Kv1.3 is a voltage-gated potassium channel with roles in human T cell activation/proliferation, cell-mediated cytotoxicity, and volume regulation and is thus a target for therapeutic control of T cell responses. Kv1.3 is also present in some mouse thymocyte subsets and splenocytes, but its role in the mouse is less well understood. We report the generation and characterization of Kv1.3-deficient (Kv1.3-/-) mice. In contrast to wild-type cells, the majority of Kv1.3-/- thymocytes had no detectable voltage-dependent potassium current, although RNA and protein for several potassium channel subunits were found in the thymocyte population. Surprisingly, the level of chloride current in the Kv1.3-/- thymocytes was increased approximately 50-fold over that in wild-type cells. There were no abnormalities in lymphocyte types or absolute numbers in thymus, spleen, and lymph nodes and no obvious defect in thymocyte apoptosis or T cell proliferation in the Kv1.3-/- animals. The compensatory effects of the enhanced chloride current may account for the apparent lack of immune system defects in Kv1.3-/-mice.     

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.     

T cell development in PU.1-deficient mice.

These studies address the role of PU.1 in T cell development through the analysis of PU.1-/- mice. We show that the majority of PU.1-/- thymocytes are blocked in differentiation prior to T cell commitment, and contain a population of thymocyte progenitors with the cell surface phenotype of CD44+, HSAbright, c-kitint, Thy-1-, CD25-, Sca-1-, CD4-, and CD8-. These cells correspond in both number and cell surface phenotype with uncommitted thymocyte progenitors found in wild-type fetal thymus. RT-PCR analysis demonstrated that PU.1 is normally expressed in this early progenitor population, but is down-regulated during T cell commitment. Rare PU.1-/- thymi, however, contained small numbers of thymocytes expressing markers of T cell commitment. Furthermore, almost 40% of PU.1-/- thymi placed in fetal thymic organ culture are capable of T cell development. Mature PU. 1-/- thymocytes generated during organ culture proliferated and produced IL-2 in response to stimulation through the TCR. These data demonstrate that PU.1 is not absolutely required for T cell development, but does play a role in efficient commitment and/or early differentiation of most T progenitors.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.     

Selective support of a mouse thymus epithelial cell line （MTEC1） to the viability and proliferation of CD4＋CD8—,CD4^—CD8^—,and CD4^＋CD8^＋thymocytes in vitro

The MTEC1 cell line,established in our laboratory,is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constituteively produce multiple cytokines.The selection of thymic microenvironment on developing T cells was investigated in an in vitro system.Unseparated fresh thymocytes from Balb/c mice were cocultured with MTEC1 cells or/and MTEC1-SN,then,the viability,proliferation and phenotypes of cultured thymocytes were assessed.Without any exogenous stimulus,both MTEC1 cells and MTEC1-SN were able to maintain the viability of thymocytes,while only the MTEC1 cells,not the MTEC1-SN,could directly activate thymocytes to exhibit moderate proliferation,indicating that the proliferative signal is delivered through cell surface interatcions of MTEC1 cells and thymocytes.Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTEC1 cells preferentially activate the subsets of CD4^ CD8^-,CD4^ CD^8 and CD^4- CD^8- thymocytes;whereas MTEC1-SN preferentially maintained the viability of CD4^ CD^8- and CD4^-CD8^ thymocyte subsets.For the Con A-activated thymocytes.both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency,phenotyped as CD4^ CD8^-,CD4^-CD8^ ,and CD^4-CD8^-subests,In summary,MTEC1 cells displayed Selection of thymic epithelial cells on thymocyte subsets. selective support to the different thymocyte subsets,and the selectivity is dependent on the status of thymocytes.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Lysosomal localization of murine CD1d mediated by AP-3 is necessary for NK T cell development

The presentation of lipid and glycolipid Ags to T cells is mediated through CD1 molecules. In the mouse and rat only a single isoform, CD1d, performs these functions, while humans and all other mammals studied have members of both group I (CD1a, -b, and -c) and group II (CD1d) isoforms. Murine CD1d contains a cytoplasmic tyrosine-based sorting motif that is similar to motifs recognized by adaptor protein complexes that sort transmembrane proteins. Here we show that the adaptor protein complex, AP-3, directly interacts with murine CD1d and controls its targeting to lysosomes. AP-3 deficiency results in a redistribution of CD1d from lysosomes to the cell surface of thymocytes, B cell-depleted splenocytes, and dendritic cells. The altered trafficking of CD1d in AP-3-deficient mice results in a significant reduction of NK1.1(+)TCR-beta(+) and CD1d tetramer-positive cells, consistent with a defect in CD1d self-Ag presentation and thymocyte-positive selection. The AP-3 complex has recently been shown to associate with the human CD1b isoform, which has an intracellular distribution pattern similar to that of murine CD1d. We propose that lysosomal sampling may be so critical for efficient host defense that mice have evolved mechanisms to target their single CD1 isoform to lysosomes for sampling lipid Ags. Here we show the dominant mechanism for this trafficking is mediated by AP-3.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.     

CD40 ligand functions non-cell autonomously to promote deletion of self-reactive thymocytes

CD40 ligand (CD40L)-deficient mice have been shown to have a defect in negative selection of self-reactive T cells during thymic development. However, the mechanism by which CD40L promotes deletion of autoreactive thymocytes has not yet been elucidated. We have studied negative selection in response to endogenous superantigens in CD40L-deficient mice and, consistent with previous reports, have found a defect in negative selection in these mice. To test the requirement for expression of CD40L on T cells undergoing negative selection, we have generated chimeric mice in which CD40L wild-type and CD40L-deficient thymocytes coexist. We find that both CD40L wild-type and CD40L-deficient thymocytes undergo equivalent and efficient negative selection when these populations coexist in chimeric mice. These results indicate that CD40L can function in a non-cell-autonomous manner during negative selection. Deletion of superantigen-reactive thymocytes was normal in B7-1/B7-2 double-knockout mice, indicating that CD40-CD40L-dependent negative selection is not solely mediated by B7 up-regulation and facilitation of B7-dependent T cell signaling. Finally, although the absence of CD40-CD40L interactions impairs negative selection of autoreactive CD4(+) and CD8(+) cells during thymic development, we find that self-reactive T cells are deleted in the mature CD4(+) population through a CD40L-independent pathway.     

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.