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.

Expression of the transcription factor cKrox in peripheral CD8 T cells reveals substantial postthymic plasticity in CD4-CD8 lineage differentiation.

Most T cells belong to either of two lineages defined by the mutually exclusive expression of CD4 and CD8 coreceptors: CD4 T cells are major histocompatibility complex (MHC) II restricted and have helper function, whereas CD8 T cells are MHC I restricted and have cytotoxic function. The divergence between these two lineages occurs during intrathymic selection and is thought to be irreversible in mature T cells. It is, however, unclear whether the CD4-CD8 differentiation of postthymic T cells retains some level of plasticity or is stably maintained by mechanisms distinct from those that set lineage choice in the thymus. To address this issue, we examined if coreceptor or effector gene expression in mature CD8 T cells remains sensitive to the zinc finger transcription factor cKrox, which promotes CD4 and inhibits CD8 differentiation when expressed in thymocytes. We show that cKrox transduction into CD8 T cells inhibits their expression of CD8 and cytotoxic effector genes and impairs their cytotoxic activity, and that it promotes expression of helper-specific genes, although not of CD4 itself. These observations reveal a persistent degree of plasticity in CD4-CD8 differentiation in mature T cells.

Classical and Alternative Activation and Metalloproteinase Expression Occurs in Foam Cell Macrophages in Male and Female ApoE Null Mice in the Absence of T and B Lymphocytes.

BACKGROUND: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. METHODS AND RESULTS: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice. CONCLUSION: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.

Loss of CTL function among high-avidity tumor-specific CD8+ T cells following tumor infiltration.

A major problem in generating effective antitumor CTL responses is that most tumors express self-antigens to which the immune system is rendered unresponsive due to mechanisms of self-tolerance induction. CTL precursors (CTLp) expressing high-affinity T-cell receptors (TCR) are often functionally deleted from the repertoire, leaving a residual repertoire of CTLp having only low-affinity TCR. Furthermore, even when unique antigens are expressed, their presentation by dendritic cells (DC) may predispose to peripheral tolerance induction rather than the establishment of CTL responses that kill tumor cells. In this study, we examined both high-avidity (CL4) and low-avidity (CL1) CD8(+) T-cell responses to a murine renal carcinoma expressing, as a neoantigen, high and low levels of the hemagglutinin (HA) protein from influenza virus A/PR/8 H1N1 (PR8; RencaHA(high) and RencaHA(low)). Our data show that, following encounter with K(d)HA epitopes cross-presented by bone marrow-derived DC, low-avidity CL1 cells become tolerized within tumor-draining lymph nodes (TDLN), and in mice bearing either RencaHA(high) or RencaHA(low) tumors, very few form tumor-infiltrating lymphocytes (TIL). In marked contrast, high-avidity CL4 cells differentiate into effector CTL within the TDLN of mice bearing either RencaHA(high) or RencaHA(low) tumors, and although they form TIL in both tumors, they lose CTL effector function. Critically, these results show that anticancer therapies involving either adoptive transfer of high-avidity tumor-specific CTL populations or targeting of preexisting tumor antigen-specific memory CD8(+) T cells could fail due to the fact that CTL effector function is lost following tumor infiltration.

The role of intercellular adhesion molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell responses.

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.

The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection.

The genetic programs directing CD4 or CD8 T cell differentiation in the thymus remain poorly understood. While analyzing gene expression during intrathymic T cell selection, we found that Zfp67, encoding the zinc finger transcription factor cKrox, was upregulated during the differentiation of CD4(+) but not CD8(+) T cells. Expression of a cKrox transgene impaired CD8 T cell development and caused major histocompatibility complex class I-restricted thymocytes to differentiate into CD4(+) T cells with helper properties rather than into cytotoxic CD8(+) T cells, as normally found. CD4 lineage differentiation mediated by cKrox required its N-terminal BTB (bric-a-brac, tramtrack, broad complex) domain. These findings identify cKrox as a chief CD4 differentiation factor during positive selection.