Although it was previously demonstrated that T cell receptor-mediated interaction is necessary to thymic stromal development, the role of costimulatory signals in thymic development has not been assessed. Studies were therefore undertaken to determine whether costimulation influences cortico-medullary organogenesis and thymic repertoire selection. Mice with genetically altered expression of costimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, or of the costimulatory receptors CD28 and CD154 (CD40 ligand) have been analyzed for thymic development and expressed T cell repertoire. Disruption of the CD28-B7 pathway or of the CD40-CD154 pathway had minimal effect on development of normal thymic cortico-medullary architecture. In contrast, disruption of both pathways resulted in a profound failure of thymic development. These findings identify a previously unappreciated role of redundant costimulatory pathways mediating an essential function in thymic development. The effect of these costimulatory pathways on selection of the T cell repertoire was studied in parallel. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. Together, these findings have elucidated the roles of costimulatory pathways in thymic T cell repertoire selection and maintenance of self tolerance. The role of costimulation in development of functionally unique populations of lymphocytes has been analyzed. NKT cells are a subset of T cells with demonstrated importance in responses to tumors and infectious agents. We have observed that, in contrast to conventional T cells, intra-thymic development of NKT cells is highly sensitive to costimulaton through the CD28/B7 pathway. Either ablation of CD28 or B7 or altered expression of B7 in knockout or transgenic mice severely impairs NKT development. It thus appears that discrete T cell populations, such as NKT cells and regulatory T cells, differentiate through distinct pathways characterized by differing requirements for costimulation. The expression of both CD28 and B7 is highly regulated during thymic development, such that the most immature thymocytes are protected from exposure to costimulatory signaling. We hypothesized that costimulation of immature thymocytes might disrupt normal differentiation, and addressed this hypothesis by genetically engineering ectopic expression of costimulatory receptor/ligand molecules. The transgenic over-expression of CD28 and CD86 had dramatic effects on thymic maturation, leading to differentiation of CD4+CD8+ thymocytes in the absence of TCR expression, suggesting a previously undescribed ability of costimulatory signaling to bypass pre-TCR function in T cell development. This observation has provided an opportunity to further define the requirements for pre-TCR in T cell development. Suppression of TCR beta chain rearrangement (beta suppression or allelic exclusion), regarded as a pre-TCR dependent function, is efficiently mediated by CD28 signaling in the absence of a pre-TCR. Ongoing studies are characterizing the mechanisms mediating these critical parameters of T cell development. Effects on expression of RAG recombinase as well as effects on chromatin accessibility and germline gene transcription have been identified and are being characterized. The ataxia telangiectasia mutated (ATM) protein plays a central role in sensing and responding to chromatin changes including DNA double strand breaks (dsb) generated by insults such as ionizing radiation. Lymphoid cells are unique in undergoing physiologic dsb in the processes of Ig class switch recombination (CSR) and T or B cell receptor V(D)J recombination, and a role for ATM in these processes has been suggested by clinical observations in ataxia telangiectasia (AT) patients with mutations in the Atm gene as well as by studies of mice deficient in ATM. We have characterize a defect in T cell development in ATM-deficient mice that is associated with decreased efficiency in V-J rearrangement of the endogenous T cell receptor (TCR)-alpha locus, with consequent reduction in the number of mature TCR-expressing CD4+ and CD8+ thymic T cells. CD4+CD8+ thymocytes from ATM-deficient mice exhibit a reduction in TCRalpha V-J rearrangement as measured by real-time genomic PCR, while containing an increased frequency of unresolved TCR Jbeta coding ends (CE), indicating a delay in TCRalpha V-J coding joint formation. The absence of ATM thus results in reduced frequency of successfully completed TCRalpha recombination, leading to decreased TCRalpha/beta expression and decreased positive selection of mature CD4+ and CD8+ T cells, defining a pivotal role for ATM in T cell development. Ongoing studies are analyzing the role of ATM in earlier stages of T cell development, including TCR beta rearrangement, and in protection from oncogenic events underlying B cell lymphomas as well as thymic T cell lymphomas. Costimulatory pathways play critical roles during lymphocyte development as well as during differentiaion of mature immune responses. The dissection of specific cell interaction pathways mediating these effects is, however, incomplete. Two of the most critical costimulatory pathways ar those mediated by CD28-B7 and CD40-CD40L interactions. To dissect in detail the nature of these interactions, we have initiated the generation of conditional knock-out and mutant transgenic mouse models, which have not previously existed. To date, we have generated conditioal CD80 (B7-1) knockout mice and have begun the charactrization of cell-specificB7 expression during T cell development, and in functional properties of T cells, B cells, dendritic cells, andthymic epithelium.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC009265-29
Application #
8348923
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2011
Total Cost
$1,500,071
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Chiang, Y Jeffrey; Hodes, Richard J (2016) T-cell development is regulated by the coordinated function of proximal and distal Lck promoters active at different developmental stages. Eur J Immunol 46:2401-2408
Williams, Joy A; Tai, Xuguang; Hodes, Richard J (2015) CD28-CD80/86 and CD40-CD40L Interactions Promote Thymic Tolerance by Regulating Medullary Epithelial Cell and Thymocyte Development. Crit Rev Immunol 35:59-76
Williams, Joy A; Zhang, Jingjing; Jeon, Hyein et al. (2014) Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways. J Immunol 192:630-40
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