Sophia Sarafova ^1,2* Gregory Swan ³ Chika Fujii ⁴ Mia E Guzinsky ⁵ Sheridan M Page ⁶ Isabelle V Meyers ⁶ Yordan P Penev ⁷ Sejiro Littleton ² Adinda Azzahra ⁶ Christine Richardson ⁸

• ¹ Davidson College, Davidson, United States
• ² Department of Integrative Immunobiology, Duke University, Durham, North Carolina, United States
• ³ Avantor, Radnor, PA, United States
• ⁴ Washington University in St. Louis, St. Louis, Missouri, United States
• ⁵ School of Medicine, Wake Forest University, Winston-Salem, North Carolina, United States
• ⁶ Department of Biology, Davidson College, Davidson, United States
• ⁷ College of Medicine, University of Florida, Gainesville, Florida, United States
• ⁸ Department of Biological Sciences, College of Liberal Arts and Sciences, University of North Carolina at Charlotte, Charlotte, North Carolina, United States

The regulation of Cd4 expression during T-cell development and immune responses is essential for proper lineage commitment and function in the periphery; however, the mechanisms of genetic and epigenetic regulation are complex, and their interplay not entirely understood.Previously, we demonstrated the need for CD4 upregulation during positive selection to ensure faithful commitment of MHC-II-restricted T cells to the CD4 lineage. In this study, we show that a region proximal to the silencer that contains E4m, here called NCE, has the required developmental-stage-specific canonical enhancer function and TCR responsiveness to mediate the CD4 upregulation required to prevent lineage errors. Our in vitro experiments demonstrate that NCE by itself can function as an enhancer at the intermediate (INT, CD4 + CD8 lo ) but not the double-positive (DP, CD4 + CD8 + ) stage of development by transient transfection of reporter plasmids in thymoma cell lines arrested at these stages of development. Furthermore, CRISPR/Cas9-mediated deletion of the coreNCE/E4m, in the same cell lines reveals reduced cell surface levels and re-expression rate of CD4, as well as reduced responsiveness to TCR signaling in NCE-deleted INT but not in NCE-deleted DP cells, consistent with the loss of a transcriptional enhancer. To avoid the developmental alterations that occur with direct manipulation of the endogenous Cd4 locus in vivo, we generated BAC-transgenic reporter mice with the Cd4 locus modified to express EGFP in the presence or absence of NCE. While the NCE sufficient transgenic mice exhibited upregulation of Cd4 reporter EGFP mRNA levels at the INT stage with a corresponding upregulation of EGFP fluorescence, in the absence of coreNCE/E4m there was a significant loss of Cd4 reporter EGFP mRNA by RT-qPCR and no detectable new EGFP production in any post-selection thymocytes, confirming that NCE is required for CD4 expression post selection in vivo. This work demonstrates that the canonical enhancer function of coreNCE/E4m combined with its known epigenetic capabilities is required for the upregulation 50 of CD4 following positive selection, and thus faithful lineage commitment.