Human cytotoxic lymphocyte differentiation in health and disease

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

Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria

How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum–infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum–infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8(+) Trm cells on a compartmental and functional basis. In human skin epithelia, CD8(+)CD49a(+) Trm cells produced interferon-γ, whereas CD8(+)CD49a(−) Trm cells produced interleukin-17 (IL-17). In addition, CD8(+)CD49a(+) Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8(+)CD49a(+) Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8(+)CD49a(–) Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8(+) Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

Unperturbed Cytotoxic Lymphocyte Phenotype and Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a debilitating disorder linked to diverse intracellular infections as well as physiological stress. Cytotoxic lymphocytes combat intracellular infections. Their function is attenuated by stress. Despite numerous studies, the role of cytotoxic lymphocytes in ME/CFS remains unclear. Prompted by advances in the understanding of defects in lymphocyte cytotoxicity, the discovery of adaptive natural killer (NK) cell subsets associated with certain viral infections, and compelling links between stress, adrenaline, and cytotoxic lymphocyte function, we reassessed the role of cytotoxic lymphocytes in ME/CFS. Forty-eight patients from two independent cohorts fulfilling the Canada 2003 criteria for ME/CFS were evaluated with respect to cytotoxic lymphocyte phenotype and function. Results were compared to values from matched healthy controls. Reproducible differences between patients and controls were not found in cytotoxic lymphocyte numbers, cytotoxic granule content, activation status, exocytotic capacity, target cell killing, or cytokine production. One patient expressed low levels of perforin, explained by homozygosity for the PRF1 p.A91V variant. However, overall, this variant was present in a heterozygous state at the expected population frequency among ME/CFS patients. No single patient displayed any pathological patterns of cellular responses. Increased expansions of adaptive NK cells or deviant cytotoxic lymphocyte adrenaline-mediated inhibition were not observed. In addition, supervised dimensionality reduction analyses of the full, multidimensional datasets did not reveal any reproducible patient/control discriminators. In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide useful biomarkers for the diagnosis of ME/CFS.publishedVersio