The role of V(D)J recombination and CD45 isotype expression in human natural killer cell development

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In contrast to T and B lymphocytes in which recombination of V(D)J elements is a crucial process for the generation of diversified T cell (TCR) and immunoglobulin (Ig) receptors, natural killer (NK) cells are considered as innate lymphocytes without clonotypic receptors. However, non-productive genetic rearrangements on TCR and Ig loci have also been observed in NK cells suggesting a multilineage differentiation potential of lymphoid precursors. The process of V(D)J recombination is initiated by RAG1/2 (“recombination activation genes”) endonucleases targeting recognition signal sequences (RSS) to cleave adjacent DNA. Subsequently, DNA ends are processed and joined by factors of the non-homologous end-joining (NHEJ) pathway. Differentiation of human induced pluripotent stem cells (hiPSCs) engineered by RAG1/2-fate mapping reporter into NK cells in vitro revealed an ontogeny of RAG1/2 expression in 3-6% of NK cells, which was confirmed by genomic rearrangements in the IGH locus. NK cells with RAG ontogeny expressed predominantly CD45dim/CD45RB and displayed a memory-like and terminally differentiated phenotype with impaired DNA damage response (DDR). In contrast, NK lymphocytes without traceable V(D)J recombination were found to be CD45bright due to the predominant expression of CD45RA. The isotype CD45RB has been reported on distinct memory populations of CD4+ and CD8+ T cells, as well as on B cells. Although CD45dim/CD45RO+ NK cells have been described in peripheral blood of individuals with malignancies or severe infections, CD45 isotypes and their impact on development and function have not been investigated on NK cells so far. We intend to study the differentiation potential of iPSC-derived NK cell precursors related to the occurrence of V(D)J recombination and CD45 isotype expression in vitro. With these investigations we aim to elucidate whether V(D)J recombination is a random process on the rather variable road of NK cell differentiation, or if NK cells with RAG-fate ontogeny contain specific functional properties. In addition, NK cell populations obtained from patients with combined immunodeficiencies due to V(D)J recombination defects, patients with blood cancers, and patients with CMV infection following allogeneic stem cell transplantation will be investigated in terms of maturation, CD45 isotype expression and function.