Human cytotoxic lymphocyte differentiation in health and disease

Abstract

Cytotoxic lymphocytes, comprising natural killer (NK) cells and CD8+ cytotoxic T cells, eradicate infected or malignant cells by release of lytic granules and alarm the immune system through production of pro-inflammatory cytokines and chemokines. NK cells and CD8+ T cells belong to different arms of the immune system, employing complementary strategies for target cell recognition. As part of the innate arm, NK cells sense missing or induced self-molecules by an array of germline-encoded activating and inhibitory cell surface receptors. In contrast, adaptive CD8+ T cells depend on somatically recombined, clonally distributed T cell receptors (TCR) that recognize unique foreign peptides presented by MHC class I on target cells. Importantly, while mature NK cells readily kill target cells without prior sensitization, naive CD8+ T cells require antigen priming to differentiate into cytotoxic effector and long-lived memory cells, providing long-term protection against re-infection. Recently, memory features including longevity and recall responses have also been ascribed to differentiated NK cell subsets. The work presented in my thesis contributes to our molecular understanding of cytotoxic lymphocyte differentiation processes in health and disease.

Exocytosis of lytic granules containing cytotoxic cargo is tightly controlled, but the transcriptional regulation of the factors governing degranulation is poorly understood. In paper I, we found that expression of one of those factors, Munc13-4, was induced upon cytotoxic lymphocyte maturation and required cooperative binding of the transcription factors ELF1 and STAT4 to an evolutionary conserved region in intron 1. Transcription factor-binding facilitated chromatin remodeling and DNA accessibility, allowing for enhanced transcription of the conventional as well as induction of a newly identified, alternative Munc13-4 isoform that is likely to play a central role in lymphocyte cytotoxicity.

Infection with cytomegalovirus (CMV) in mice and man is associated with expansion and persistence of NK cell subsets with enhanced effector function. In paper II, we show that such adaptive NK cells display previously unappreciated phenotypic and functional heterogeneity, and provide a molecular definition of such diverse subsets. Human adaptive NK cells lacked expression of the intracellular signaling molecules FcεRγ, SYK and EAT-2 as well as the transcription factor PLZF, thereby altering the signaling properties of key NK cell surface receptors and the responsiveness to innate cytokines, respectively. Silencing of signaling protein expression correlated with promoter DNA methylation and global DNA methylation patterns of adaptive NK cells approximated those of differentiated CD8+ cytotoxic effector T cells. Importantly, adaptive NK cells failed to kill activated, autologous T cells, implying a functional specialization towards immunosurveillance of infected cells. Moreover, utilizing samples from patients with bone marrow disorders associated with GATA2 haploinsufficiency (paper III) or acquired PIGA mutations in hematopoietic stem cells (paper IV), we demonstrate that adaptive NK cells are long-lived and can persist in settings of hematopoietic stem and progenitor cell attrition where canonical NK cells are lost.

Tissue-resident memory T (TRM) cells provide early, localized adaptive immunity in nonlymphoid tissues. In paper V, we discovered a functional dichotomy of CD8+ skin TRM cells based on expression of the marker CD49a. Upon stimulation, CD49a+ cells produced IFN-γ and acquired cytotoxic potential by induction of the lytic granule constituents perforin and granzyme B. Primed CD49a+ T cells accumulated in the dermis and epidermis of vitiligo lesions, an autoimmune condition characterized by local depigmentation as a result of melanocyte destruction. In contrast, CD49a– T cells produced IL-17 and were enriched in lesional skin from psoriasis patients, promoting local inflammation. These insights shed light on novel mechanisms controlling human cytotoxic lymphocyte differentiation and may thus be of potential benefit to health.