Sarah J. Potter

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D’s influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8⁺ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4⁺ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.

Objective: There is a clinical need to understand how dysregulated thymocyte development, caused by pathogenic variants in the gene encoding the histone-modifying enzyme, lysine methyltransferase 2D ( KMT2D ), contributes to immune dysfunction, including immune deficiency, autoimmunity, and lymphoproliferative sequela, and immune-driven mortality in individuals with Kabuki syndrome type 1 (KS1). Methods: We studied peripheral T cells and thymocytes in both individuals with KS1 and murine constitutive and conditional targeted Kmt2d in T cells and hematopoietic lineages. KMT2D target genes, identified by RNA-sequencing of murine Kmt2d -knockout single-positive thymocytes, were validated with H3K4me3 ChIP-PCR and flow cytometry. Results: Recent thymic emigrant (RTE) and naïve T cells were reduced, and memory and double-negative (DN)-T cells were expanded in human KS1 and murine models. Kmt2d loss led to Mature 1 CD8 ⁺ -single positive (SP) thymocyte accumulation and a decrease in SP thymocyte egress licensing expression (normally associated with the Mature 2 phenotype). Splenomegaly is associated with hematopoietic-driven Kmt2d loss and brings to light potential overlapping phenotypes with lymphoproliferative syndromes. Finally, we identified a KMT2D-regulated cluster of integrins which likely mediates aspects of the T cell egression. Conclusions: Single-positive thymocyte populations deficient in Kmt2d display less integrin, less maturation, and less egress licensing gene expression; thereby, altering the downstream peripheral T cell composition that contribute to the observed KS1-associated immune deficiency. T cell intrinsic Kmt2d loss increases the percentage of peripheral DNT cells potentially through dysregulated apoptotic signaling, while hematopoietic-driven Kmt2d loss predisposes to splenomegaly; therefore, loss of Kmt2d recapitulates several distinct features of lymphoproliferative syndromes.