E Protein Regulation of the T Cell Receptor Alpha – T Cell Receptor Delta Locus

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2023

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Abstract

The B and T lymphocytes utilize B and T cell receptors, respectively, for the specific recognition of pathogen. Somatic recombination of the antigen receptor loci imparts the immense diversity and specificity of the adaptive immune system’s antigen recognition. The recombination activating gene (RAG) 1 and 2 mediated V(D)J recombination process is a highly developmentally regulated process, occurring at strict stages of B and T lymphocyte development. Both the expression of RAG1/2 and the accessibility of the recombination substrates come under strict developmental stage-specific regulation. E protein transcription factors and the inhibitory Id proteins mediate many of the signaling and transcriptional changes that occur during lymphocyte development. While E protein binding has been identified at antigen receptor loci, the understanding of their cis-regulatory function is lacking.

Recombination of the Tcra-Tcrd locus occurs during two temporally separate stages of T lymphocyte development. Tcrd, nested with Tcra, undergoes recombination first, during the double negative (DN) stage. Cells that commit to the αβ T lymphocyte lineage will then undergo Tcra recombination during the double positive (DP) stage. These processes are highly dependent on cis-acting enhancers termed Eδ and Eα. E proteins have long been known to bind at Eα, while recent DN cell E protein ChIP-seq allowed identification of a vast number of additional targets within the Tcra-Tcrd locus. A series of mutagenesis studies were pursued to better understand E protein cis-regulatory function during Tcra-Tcrd locus recombination.

Eα mutants lacking one or two of the three E protein binding sites were generated. The mutant lacking two binding sites displayed a partial block in αβ T lymphocyte development at the positive selection stage. Loss of transcription at the Jα array reflected a loss in accessibility, which was mirrored by dysregulated primary and impaired secondary Vα-Jα rearrangement. Therefore, Eα E protein binding increases Tcra locus accessibility, which is required for efficient Tcra recombination and the resulting Tcra repertoire diversity.

Analysis of available E protein ChIP-seq revealed strong E protein binding downstream of Trav15-dv6 family V segments. Mutagenesis of the E protein binding region directly downstream of Trav15d-1-dv6d-1 resulted in a loss of Vγ1.1Vδ6.3 T lymphocytes, whose development relies on Tcrd recombination of Trav15d-1-dv6d-1. This has established a new method of E-Id protein axis regulation of Vγ1.1Vδ6.3 T lymphocyte development. Furthermore, analysis suggested that the Vγ1.1Vδ6.1 and Vγ1.1Vδ6.3 T lymphocyte subsets compete during development, with expansion of Vγ1.1Vδ6.1 T lymphocytes only possible in the absence of Vγ1.1Vδ6.3 T lymphocytes. Competition and affinity for ligand likely play a tremendous role in this process. Analysis of two Vγ1.1Vδ6.3 TCR transgenic lines revealed that small differences in CDR3γ and CDR3δ are sufficient to significantly modulate Vγ1.1Vδ6.3 T lymphocyte development and immune profile. These findings and the generated genetic models can form the basis of elucidating the ligand of the Vγ1.1Vδ6.3 TCR.

The findings presented here have advanced the understanding of E protein regulation of the Tcra-Tcrd locus, and by extension the lineage-defining potential of the Vγ1.1Vδ6.3 TCR. This is the first report of E protein cis-regulation of the Tcra locus and of Tcrd recombination of Trav15-dv6 family V segments (specifically Trav15d-1-dv6d-1). Extensive E protein binding of the Vα/Vδ array suggests further regulatory mechanisms and action.

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Mihai, Ariana (2023). E Protein Regulation of the T Cell Receptor Alpha – T Cell Receptor Delta Locus. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/29179.

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