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

Design of a Herringbone-Grooved Bearing for Application in an Electrically Driven Air Compressor

A turbo compressor was investigated to ensure the operational reliability of the charging of fuel cell systems. This study investigated air-lubricated herringbone bearings to support the high-speed rotating shaft. For reliable operation of the rotor bearing system, stable operation in the whole speed range (up to 120 krpm), as well as low lift-off speed, is an important issue. Some publications containing guidelines for an optimized design in terms of stability and lift-off behavior date back to the 1970s, with some simplifying assumptions (such as narrow groove theory and small eccentricity analysis). Many publications have addressed the calculations, as well as the optimization of herringbone-grooved bearings; however, general design guidelines are still missing in the view of the authors. Although the investigations related to bearings for the support of a lightweight rotor for a special compressor of a fuel cell unit, this study could also indicate favorable bearing designs for other high-speed applications. Here, the compressible Reynolds equation was solved in the whole solution domain using a conservative finite difference scheme, and the corresponding bearing characteristics were determined. In a perturbation analysis, the linearized dynamic coefficients of the herringbone bearing are calculated. To compare the suitability and performance of the various herringbone-grooved bearing designs, especially at high speed, the simple model of a Jeffcott rotor airborne with two identical herringbone-grooved journal bearings (HGJBs) was used. The geometrical parameters of the HGJBs were varied, and their effects on bearing characteristics and stability were evaluated. Recommendations concerning favorable geometrical bearing parameters for a sufficiently high stability threshold speed and reasonable low lift-off speed were the result of the parameter study

Towards a realistic microscopic description of highway traffic

Simple cellular automata models are able to reproduce the basic properties of highway traffic. The comparison with empirical data for microscopic quantities requires a more detailed description of the elementary dynamics. Based on existing cellular automata models we propose an improved discrete model incorporating anticipation effects, reduced acceleration capabilities and an enhanced interaction horizon for braking. The modified model is able to reproduce the three phases (free-flow, synchronized, and stop-and-go) observed in real traffic. Furthermore we find a good agreement with detailed empirical single-vehicle data in all phases.Comment: 7 pages, 7 figure

An empirical test for cellular automaton models of traffic flow

Based on a detailed microscopic test scenario motivated by recent empirical studies of single-vehicle data, several cellular automaton models for traffic flow are compared. We find three levels of agreement with the empirical data: 1) models that do not reproduce even qualitatively the most important empirical observations, 2) models that are on a macroscopic level in reasonable agreement with the empirics, and 3) models that reproduce the empirical data on a microscopic level as well. Our results are not only relevant for applications, but also shed new light on the relevant interactions in traffic flow.Comment: 28 pages, 36 figures, accepted for publication in PR

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

Enhancement of cytokine-driven NK cell IFN-γ production after vaccination of HCMV infected Africans

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine-induced responses, we studied NK-cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK-cell responses (IFN-γ, CD107a, CD25) occurred after in vitro re-stimulation of post-vaccination NK cells with TIV or DTPiP antigens compared to pre-vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN-γ in response to exogenous IL-12 with IL-18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN-γ+ cells observed within CD56bright CD57- , CD56dim CD57- NKG2C- and CD56dim CD57- NKG2C+ NK-cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN-γ function in HCMV infected donors and raise the potential for further exploitation of NK cell "pre-activation" to improve vaccine effectiveness

Asymptomatic CMV infections in long-term renal transplant recipients are associated with the loss of FcRγ from LIR-1+ NK cells

While it is established that cytomegalovirus (CMV) disease affects NK-cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age-matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR-1, NKp30, NKp46, and FcRγ, a signaling adaptor molecule, on CD56dim NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK-cell function as assessed by TNF-α and CD107a expression. The most active NK cells were FcRγ–LIR-1+NKG2C– and displayed high antibody-dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom-free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcRγ but express LIR-1