Characterizing Roles for PD-1 and Capicua in Thymocyte Tolerance and Selection

• Author / Creator

  May, Julia F.

• The stochastic diversification of the T cell receptor (TCR) carries with it both the potential for recognizing virtually infinite foreign peptides, but also the potential for the erroneous recognition of self-peptides, which represents a threat of autoimmunity. During development in the thymus, T cells are positively selected based on weak self-reactivity and negatively selected when they demonstrate strong self-reactivity. Thymocytes are negatively selected when they recognize with high affinity self-peptides expressed ubiquitously (UbA, encountered in the thymic cortex) or in a tissue-restricted manner (TRA, encountered in the thymic medulla). To model negative selection to these differentially expressed antigens, here we used the MHCI-restricted HYcd4 (UbA) and OT-I (TRA) models. Negative selection serves to censor or silence self-reactive clones by instructed apoptosis (clonal deletion), diversion into nonconventional lineages (e.g. Treg), or functional inactivation (tolerance).
  We previously showed that impairment in clonal deletion in a model for negative selection to TRA did not lead to a spontaneous loss of tolerance. In this study, we investigated the non-deletional tolerance established in cells that escaped clonal deletion in this model. Tolerance was established in a T cell-intrinsic manner in the thymus, and we provide the first epigenetic profile for thymocytes in this state. PD-1 is a co-inhibitory receptor that functions to depress the TCR signal. It has been negatively associated with T cell function under various contexts, but it has received the most attention for its role in maintaining T cell dysfunction during anti-tumour or anti-viral responses. We determined that PD-1 and PD-L1 were required for the establishment of thymic tolerance in our model. Tolerance was largely maintained following adoptive transfer of either mature thymocytes or splenic CD8+ T cells into PD-L1-/- recipients, but CRISPR-mediated ablation of the Pdcd1 locus resulted in broken tolerance. Overall, these data suggest that the requirements for PD-1-mediated tolerance differ between the establishment versus the maintenance of tolerance.
  Capicua (CIC) is an evolutionarily conserved transcriptional repressor that has recently been implicated for its role in early T cell development and expansion, and as a suppressor of autoimmunity. CIC has been postulated to participate in a negative feedback network for ERK signaling. Such a network was described in a study using human cell lines; ERK signaling attenuated CIC activity, while CIC repressed the transcription of a negative ERK regulator, DUSP6. A similar regulatory loop may also function in thymocytes, as known ERK regulators (DUSP6 among them) have been observed to be upregulated in the absence of CIC. In this study, we investigated the role of CIC in positive and negative selection using a CICFL/FL CD4-cre knock-out model, which allows for the separation of early development events and selection proper. CIC deficiency resulted in impaired positive selection in mixed bone marrow chimeras for polyclonal thymocytes and in two MCHI-restricted TCR transgenic models. In contrast, negative selection in these transgenic models was largely unperturbed, suggesting CIC plays a unique role in the integration of the weak TCR signals associated with positive selection. Finally, CRISPR-mediated deletion of Dusp6 was unable to rescue impaired positive selection in the CIC deficient thymocytes in HYcd4 mixed bone marrow chimeras, suggesting that this negative ERK regulator is not required for CIC’s influence on selection. However, whether DUSP6 participates in this process redundantly alongside other regulators remains an unanswered question.

• Subjects / Keywords

  • Immunology
  • Tolerance
  • PD-1
  • programmed cell death protein 1
  • Capicua
  • Thymocyte Selection

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-2mps-zs45

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.