CD28-CD80/86 and CD40-CD40L Interactions Promote Thymic Tolerance by Regulating Medullary Epithelial Cell and Thymocyte Development.

Development and central tolerance of T lymphocytes in the thymus requires both TCR signals and collaboration with signals generated through costimulatory molecule interactions. In this review, we discuss the importance of CD28-CD80/86 and CD40-CD40L costimulatory interactions in promoting normal thymic development. This discussion includes roles in the generation of a normal thymic medulla, in the development of specific T-cells subsets, including iNKT and T regulatory cells, and in the generation of a tolerant mature T-cell repertoire. We discuss recent contributions to the understanding of CD28-CD80/86 and CD40-CD40L costimulatory interactions in thymic development, and we highlight the ways in which the many important roles mediated by these interactions collaborate to promote normal thymic development.

T cell-B cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction.

Thymic development requires bidirectional interaction or cross-talk between developing T cells and thymic stromal cells, a relationship that has been best characterized for the interaction between thymocytes and thymic epithelial cells. We have characterized in this article the requirement for similar cross-talk in the maintenance and function of thymic B cells, another population that plays a role in selection of developing thymic T cells. We found that maintenance of thymic B cells is strongly dependent on the presence of mature single-positive thymocytes and on the interactions of these T cells with specific Ag ligand. Maintenance of thymic B cell number is strongly dependent on B cell-autonomous expression of CD40, but not MHC class II, indicating that direct engagement of CD40 on thymic B cells is necessary to support their maintenance and proliferation. Thymic B cells can mediate negative selection of superantigen-specific, self-reactive, single-positive thymocytes, and we show that CD40 expression on B cells is critical for this negative selection. Cross-talk with thymic T cells is thus required to support the thymic B cell population through a pathway that requires cell-autonomous expression of CD40, and that reciprocally functions in negative selection of autoreactive T cells.

Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways.

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development.

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra(-/-) mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTßR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.

Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury.

Thymic epithelial cells (TECs) and dendritic cells are essential for the maintenance of thymopoiesis. Because these stromal elements define the progenitor niche, provide critical survival signals and growth factors, and direct positive and negative selection, detailed study of these populations is necessary to understand important elements for thymic renewal after cytotoxic injury. Study of TEC is currently hindered by lengthy enzymatic separation techniques with decreased viability. We present a new rapid separation technique that yields consistent viable TEC numbers in a quarter of the prior preparation time. Using this new procedure, we identify changes in stroma populations following total body irradiation (TBI). By flow cytometry, we show that TBI significantly depletes UEA+ medullary TEC, while sparing Ly51+ CD45- cells. Further characterization of the Ly51+ subset reveals enrichment of fibroblasts (CD45- Ly51+ MHCII-), while cortical TECs (CD45- Ly51+ MHCII+) were markedly reduced. Dendritic cells (CD11lc+ CD45+) were also decreased following TBI. These data suggest that cytotoxic preparative regimens may impair thymic renewal by reducing critical populations of cortical and medullary TEC, and that such thymic damage can be assessed by this new rapid separation technique, thereby providing a means of assessing optimal conditioning pretransplantfor enhancing thymic-dependent immune reconstitution posttranspiant.

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway.

Invariant NKT (iNKT) cells are a population of TCRalphabeta-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.

Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint.

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.

A novel role for CD28 in thymic selection: elimination of CD28/B7 interactions increases positive selection.

While the importance of the CD28/B7 costimulation pathway is well established for mature T cells, the role of CD28 in thymocyte selection is less well defined. The role of CD28 in both negative and positive selection was assessed using H-Y-specific TCR-transgenic (Tg) RAG-2-deficient (H-Yrag) mice. Negative selection in male H-Yrag mice was not affected by deficiency in CD28 or B7. Surprisingly, absence of CD28 or B7 in H-Yrag females resulted in increased numbers of CD8 single-positive (SP) thymocytes. The CD8 SP thymocytes found in these females were mature and functionally competent. Furthermore, double-positive (DP) thymocytes from CD28-knockout (CD28KO) or B7.1/B7.2 double-KO (B7DKO) females had higher levels of both CD5 and TCR than those from WT females, consistent with a stronger selecting signal. CD28KO H-Yrag fetal thymic organ cultures also had elevated numbers of thymic CD8 SP cells, reflecting increased thymic differentiation and not recirculation of peripheral T cells. Finally, increased selection of mature CD4 and CD8 SP T cells was observed in non-TCR-Tg CD28KO and B7DKO mice, indicating that this function of CD28-B7 interaction is not unique to a TCR-Tg model. Together these findings demonstrate a novel negative regulatory role for CD28 in inhibiting differentiation of SP thymocytes, probably through inhibition of thymic selection.

Dynamic regulation of IL-7 receptor expression is required for normal thymopoiesis.

Interleukin-7 receptor (IL-7R) levels are tightly controlled during ontogeny: high on double-negative (DN) cells, absent on double-positive (DP) cells, and high once again on thymocytes undergoing positive selection. To determine if loss of IL-7-mediated survival signals in DP cells is necessary for normal antigen-specific selection, we created T-lineage-specific IL-7R alpha chain (IL-7Ralpha) transgenic (Tg) mice in which IL-7R is expressed throughout ontogeny. There was no effect of the IL-7Ralpha Tg on negative selection. Surprisingly, however, although the thymi of IL-7Ralpha Tg mice were comparable at birth, there was a decrease in thymocyte number as the mice aged. This was found to be due to competition between DN and IL-7R-expressing DP cells for endogenous IL-7, which resulted in decreased levels of Bcl-2 in DN cells, increased DN apoptosis, and decreased DN cell number. Therefore, the down-regulation of IL-7R on DP cells is an "altruistic" act required for maintaining an adequate supply of local IL-7 for DN cells.

Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo.

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.

Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells.

Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.

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.