Natural killer (NK)1 cells are large granular lymphocytes that provide a first innate immune defense. They are able to kill virus-infected and transformed cells and furthermore release cytokines and chemokines to activate adaptive immune cells (1, 2).The balance of signals from activating and inhibitory NK cell surface receptors tightly regulates NK cell activity. Activated NK cells release lytic granules through a process called degranulation. Therefore, NK cell cytotoxicity requires the formation of the F-actin-rich NK immune synapse (NKIS) and the transport of Perforin-containing lytic granules to the NKIS. Furthermore, this process requires granule-associated MYH9 protein (non-muscle Myosin IIa) mediating the interaction of granules with F-actin at the NKIS (3-5), leading to lytic granule exocytosis. Whereas related phenotypes and functional properties are well characterized, the underlying regulatory protein network mediating differentiation, cytokine release, and cytotoxicity, is still incomplete.NK cells are defined by the expression of the surface molecule CD56 (NCAM1) and the absence of the T cell receptor (TCR) associated protein CD3 and can be further subdivided into subsets (6, 7). CD56 expressing cells originate from CD34 ϩ HSCs. Notably, the commitment to the NK lineage includes discrete steps from HSC to cells, expressing high CD56 levels (CD56 bright ) (8, 9), which act immune regulatory by the release of various cytokines. NK cells with low CD56-expression (CD56 dim ) predominantly constitute cytotoxic responses (10, 11). Contact of CD56 (NCAM1) with fibroblasts (12) and neutrophils (13) supports the differentiation process from CD56 bright to CD56 dim NK cells. The progression of early differentiation steps is proven by telomere length investigation (14 ) and early presence in blood after HSC transplantation (HSCT) (14, 15). Indeed, CD56 dim NK cells are able to change their phenotypic properties, which can be correlated with continued differentiation throughout their whole lifespan (15-18). CD57 was determined to be a senescence marker in T cells (19 1 The abbreviations used are: NK, natural killer; CD56, NK cell marker; NCAM1, neural cell adhesion molecule; CD57, senescence marker in T and NK cells (HNK-1 or Leu-7); CMV, cytomegalovirus; CPDA-1, anticoagulant , containing citric acid, sodium citrate, monobasic sodium phosphate and dextrose; CTLs, cytotoxic T lymphocytes; FDR, false discovery rate; HLA, self-human leukocyte antigen; HSC, hematopoietic stem cell; iTRAQ, isobaric tags for relative and absolute quantification in mass spectrometry; KIR, killer immunoglobulin-related receptors in NK cells; LC-MS/MS, liquid-chromatography coupled with peptide sequencing (mass spectrometry); MAD, median absolute deviation from the median; NKIS, NK cell immune synapse; RF, regulation factor. Research
Mucosal-associated invariant T cells (MAIT) constitute the most abundant anti-bacterial CD8 T-cell population in humans. MR1/TCR-activated MAIT cells were reported to organize cytotoxic and innate-like responses but knowledge about their molecular effector phenotype is still fragmentary. Here, we have examined the functional inventory of human MAIT cells (CD3 Vα7.2 CD161 ) in comparison with those from conventional non-MAIT CD8 T cells (cCD8 ) and NK cells. Quantitative mass spectrometry characterized 5500 proteins of primary MAIT cells and identified 160 and 135 proteins that discriminate them from cCD8 T cells and NK cells donor-independently. Most notably, MAIT cells showed a unique exocytosis machinery in parallel to a proinflammatory granzyme profile with high levels of the granzymes A, K, and M. Furthermore, 24 proteins were identified with highest abundances in MAIT cells, including CD26, CD98, and L-amino-oxidase (LAAO). Among those, expression of granzyme K and CD98 were validated as MAIT-specific with respect to non-MAIT CD8 effector subsets and LAAO was found to be recruited together with granzymes, perforin, and CD107a at the immunological synapse of activated MAIT cells. In conclusion, this study complements knowledge on the molecular effector phenotype of MAIT cells and suggest novel immune regulatory functions as part of their cytotoxic responses.
Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans.
Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.
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