Browsing by Subject "T cell development"

T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.

The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.

Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, Tcrb alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However Tcrb alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of Tcrb alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.

In search of the mechanism by which recombination is suppressed on peripheral Tcrb alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral Tcrb alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.

T cells are a critical arm of the adaptive immune system which function to coordinate and orchestrate complex immune reactions, as well as, kill damaged and infected cells. Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood.

E proteins are transcription factors which have been shown to play essential non-redundant roles throughout T cell development. The functions of E proteins in T cell development include, enforcing T lineage commitment, promoting proper TCR rearrangements, regulating developmental progression and functional checkpoints, and coordinating complex transcriptional networks underpinning developmental progression. Due to the large number of genome-wide binding sites and massive number of genes regulated by E proteins, their numerous functions are poorly understood. The goal of this dissertation is to determine the role of the E protein-regulated transcription factor Zfp335 in T cell development.

We utilized conditional deletion models to determine the role of Zfp335 in early and late stages of conventional and unconventional T cell development. Through these efforts we uncovered we uncover an unexpected link between the transcription factor Zfp335 and control of the cGAS/STING pathway for sensing cytosolic DNA in post-β-selection DN4 thymocytes. The absence of Zfp335 drives cGAS/STING-dependent death of DN4 cells. Zfp335 controls survival by sustaining expression of Ankle2, which in turn regulates the activity of Baf to suppress cGAS/STING-dependent cell death. Additionally, genetic ablation of Zfp335 precludes the development of unconventional iNKT cells due to STING-independent cell death following lineage commitment along with preventing effector differentiation of surviving cells.

Our studies also uncovered an additional cGAS/STING-independent role in the terminal maturation of conventional αβ T cells. The absence of Zfp335 prevents the establishment of a naïve T cell compartment, inhibits differentiation of CD4 T cells and promotes the developmental acquisition of an effector program in CD8 T cells. Using in vivo and ex vivo genetic manipulation combined with detailed bioinformatic analyses we show that Zfp335 functions to promote T cell development, maturation, and effector differentiation through the regulation of a small but essential set of genes.

To our knowledge, these studies detail the first described role for cGAS/STING in T cell development and strongly suggest a transcriptional mechanism downstream of Zfp335 which coordinates genome-wide alterations to chromatin compaction required for proper establishment of conventional and unconventional T cell pools. Together, these studies will provide a novel framework for understanding the life and death of developing T cells and may uncover novel pathways for enhancing the efficacy of T cell-based therapeutics.