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Adaptive immunity plays central roles in the pathogenesis of rheumatoid arthritis (RA), as it is regarded as an autoimmune disease. Clinical investigations revealed infiltrations of B cells in the synovium, especially those with ectopic lymphoid neogenesis, associate with disease severity. While some B cells in the synovium differentiate into plasma cells producing autoantibodies such as anti-citrullinated protein antibody, others differentiate into effector B cells producing proinflammatory cytokines and expressing RANKL. Synovial B cells might also be important as antigen-presenting cells. Synovial T cells are implicated in the induction of antibody production as well as local inflammation. In the former, a recently identified CD4 T cell subset, peripheral helper T (Tph), which is characterized by the expression of PD-1 and production of CXCL13 and IL-21, is implicated, while the latter might be mediated by Th1-like CD4 T cell subsets that can produce multiple proinflammatory cytokines, including IFN-γ, TNF-α, and GM-CSF, and express cytotoxic molecules, such as perforin, granzymes and granulysin. CD8 T cells in the synovium are able to produce large amount of IFN-γ. However, the involvement of those lymphocytes in the pathogenesis of RA still awaits verification. Their antigen-specificity also needs to be clarified.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum–infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria–γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs. Junqueira et al. show that human γδ T cells control erythrocytic Plasmodium falciparum infection by multiple mechanisms: antibody-dependent phagocytosis, cytotoxic-granule-mediated erythrocyte lysis and direct parasite killing.

Natural killer (NK) cells are thought to play a key role in the successful establishment of a pregnancy by facilitating immunological adaptation of the semi-allogeneic developing embryo. The aim of this study was to explore the cell number, immunophenotypic characteristics, and activities of peripheral blood NK cells in women with repeated implantation failure (RIF). Peripheral blood was obtained from 27 women with RIF and 11 healthy, fertile controls during the middle luteal phase of the menstrual cycle. CD3- CD56+ NK cells were quantified and analyzed by flow cytometry for the expression of cytolytic molecules (granzyme B, granulysin, and perforin) as well as cell surface receptors responsible for NK cell activation or inhibition (NKG2D, NKp30, NKp46, CD158a, CD158b). NK cytotoxicity was measured at three effector-to-target cell ratios. Women with RIF and fertile controls did not differ significantly in the percentage of circulating CD3−CD56+ NK cells, or in the proportions of these cells that expressed granzyme B, granulysin, or perforin. The two groups also did not differ significantly in the proportions of NK cells expressing the receptors NKG2D, NKp30, NKp46, CD158a or CD158b. General linear model analysis showed that NK cytotoxicity increased with effector-to-target cell ratio. However, NK cytotoxicity did not differ significantly between patients with RIF and fertile controls. These results suggest that RIF is not associated with significant alterations in the number or function of peripheral blood NK cells. Keywords: Activating receptor, Inhibitory receptor, NK cytotoxicity, Peripheral blood NK cells, Repeated implantation failure

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain–like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum.

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease., Phenotypic markers that overlap between ILC1 and NK populations have impacted the robust and specific analysis of these immune cell populations. Employing scRNA sequencing here the authors identify CD127+ CD94+ innate lymphoid cells that express granulysin and perforin and are expanded in patients with Crohn’s disease.

Antigen-specific gamma-delta (γδ) T cells are important in exhibiting anti-mycobacterial immunity, but their role in latent tuberculosis (LTB) with diabetes mellitus (DM) or pre-DM (PDM) and non-DM comorbidities have not been studied. Thus, we have studied the baseline, mycobacterial (PPD, WCL), and positive control antigen-stimulated γδ T cells expressing Th1 (IFNγ, TNFα, IL-2) and Th17 (IL-17A, IL-17F, IL-22) cytokine as well as cytotoxic (perforin [PFN], granzyme [GZE B], granulysin [GNLSN]) and immune (GMCSF, PD-1, CD69) markers in LTB (DM, PDM, NDM) comorbidities by flow cytometry. In the unstimulated (UNS) condition, we did not observe any significant difference in the frequencies of γδ T cells expressing Th1 and Th17 cytokine, cytotoxic, and immune markers. In contrast, upon PPD antigen stimulation, the frequencies of γδ T cells expressing Th1 (IFNγ, TNFα) and Th17 (IL-17F, IL-22) cytokine, cytotoxic (PFN, GZE B, GNLSN), and immune (CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Similarly, upon WCL antigen stimulation, the frequencies of γδ T cells expressing Th1 (TNFα) and Th17 (IL-17A, IL-22) cytokine, cytotoxic (PFN), and immune (PD-1, CD69) markers were significantly diminished in LTB DM and/or PDM individuals compared to LTB NDM individuals. Finally, upon P/I stimulation we did not observe any significant difference in the γδ T cell frequencies expressing cytokine, cytotoxic, and immune markers between the study populations. The culture supernatant levels of IFNγ, TNFα, and IL-17A cytokines were significantly increased in LTB DM and PDM after stimulation with Mtb antigens compared to LTB NDM individuals. Therefore, diminished γδ T cells expressing cytokine, cytotoxic, and other immune markers and elevated levels of cytokines in the supernatants is a characteristic feature of LTB PDM/DM co-morbidities.

Elimination of latent HIV reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is ‘shock and kill’. This strategy is based on the transcriptional activation of latent HIV with a Latency-Reversing Agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) act as an LRA by increasing Signal Transducers and Activators of Transcription (STAT) activation mediated by IL-15 in cells isolated from aviremic participants. The IL-15 superagonist (N-803) is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanism of action by promoting the activation STATs and have shown some promise in pre-clinical models directed towards HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in NK cells and the biological consequences associated with increased STAT activation in NK effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increased secretion of CXCL10 and IFN-γ and expression of cytotoxic proteins including Granzyme B, Granzyme A, Perforin, Granulysin, FASL and TRAIL. This increased cytotoxic profile results in an increase cytotoxicity against different tumor cell lines and HIV-infected cells. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and targeting this pathway could be further explored for HIV cure interventions.Author SummarySeveral clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit of these trials, they have been crucial to elucidate hurdles that ‘shock and kill’ strategies have to overcome to promote an effective reduction of the latent reservoir leading. These hurdles include low reactivation potential mediated by LRAs; the negative influence of some LRAs on the activity of Natural Killer and CD8T effector cells; an increased resistance to apoptosis of latently infected cells; and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of ‘shock and kill’ strategies aimed towards HIV eradication. In here, we showed that the LRA HODHBt also improves IL-15 mediated NK effector and memory-like functions. As such, pharmacological enhancement of IL-15 mediated STAT activation can open new therapeutic venues towards an HIV cure.

Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria.One Sentence SummaryA population of cytolytic memory CD4+ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.

Introduction Zoonotic cutaneous leishmaniasis (ZCL), due to infection by Leishmania (L). major, is characterized by polymorphic clinical manifestations which could be attributed to the host's immune response. In this study we investigated the involvement of cytotoxic cells on the outcome of the disease. Methods Expression of granzyme B (GrB), granulysine (Grly), and interferon (IFN)‐γ was evaluated within ZCL lesion specimens using the technique of real‐time quantitative polymerase chain reaction (RT‐qPCR). Immunohistochemical staining was performed using anti‐CD3, CD4, CD8, CD56, GrB, and IFN‐γ antibodies to identify the phenotype of GrB and IFN‐γ‐producing cells. Results GrB and Grly mRNA was detected within 75% and 80% of ZCL lesions, respectively. Statistical analysis demonstrated a significant correlation between levels of GrB and Grly. Interestingly, expression of these molecules correlates negatively with the lesion's age. The highest levels were measured in early lesions (E‐ZCL) (lesion age ≤1 month) comparing to late lesions (L‐ZCL) (lesion age >1 month). Otherwise, IFN‐γ mRNA was detected only within 56% and a positive correlation was found between levels of this cytokine and those of GrB. Immunohistochemical analysis showed that GrB is produced essentially by CD8+T cells whereas IFN‐γ is produced by both CD4+ and CD8+T cells. Conclusion Together our results demonstrate the presence of cytotoxic cells producing GrB and Grly within leishmaniasis cutaneous lesions.

HTLV-1 infection causes a chronic progressive debilitating neuroinflammatory disease which is called, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the host defense mechanisms against viral infection is apoptosis which may control HTLV-1 infection. Therefore, we aimed to investigate this process and its interaction with viral factors in HTLV-1-infected asymptomatic carriers (ACs) compared to HAM/TSP patients. Fas, FasL, TRAIL, perforin, granzyme A, granzyme B, and granulysin gene expression and serum levels of Fas, FasL, TRAIL, and granulysin in the peripheral blood of 21 sex- and age-matched healthy controls (HCs), ACs, and HAM/TSP patients were evaluated. Also, the level of granulysin secretion in the cell culture supernatant was measured. Finally, the correlation of the expression of these molecules with HTLV-1 proviral load (PVL), Tax, and HBZ mRNA expression was analyzed. ACs compared to HAM/TSP patients significantly over-expressed the Fas, FasL, TRAIL, perforin, and granzyme B molecules. Fas, FasL, TRAIL, and granulysin serum levels were not different among studied groups; whereas, the secretion of granulysin was significantly decreased in ACs and HAM/TSP patients compared to HCs. Also, HAM/TSP patients expressed higher levels of HTLV-1 PVL, Tax, and HBZ mRNA. In addition, in ACs, inverse correlations between the Fas, FasL, TRAIL, perforin, granzyme B, and granulysin levels with HBZ mRNA expression were seen. ACs compared to HAM/TSP patients over-expressed the apoptosis- and cytotoxicity-related molecules. It could be concluded that successful control of the HTLV-1 infection and suppression of HAM/TSP development stem from the strong apoptosis and cytotoxic activity in the peripheral blood of ACs.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αβ+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3−CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αβ+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αβ+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

The intrinsic immunosuppressive properties of regulatory T (Treg) cells can be harnessed for therapeutic approaches aiming at down-modulating harmful immune reactions. In this context, expanded type 1 Treg cells (Tr1 cells) specific for ovalbumin (ova-Tr1 cells) have been tested for clinical efficacy in the treatment of autoimmune disorders such as refractory Crohn’s disease (CD). The clinical use of these therapeutic products warrants exploration of their mechanism of action. Here, we identified a relationship between the CD activity index and the expression of lytic molecules by the ova-Tr1 cells administered in the previously reported First-in-Man study [Crohn’s And Treg cells Study 1 (CATS1) study]. Accordingly, ova-Tr1 cells were found to carry granules containing high levels of lytic molecules, including multiple granzymes and granulysin. These cells displayed a T-cell receptor (TCR)-independent cytotoxic activity, which was preferentially directed toward myeloid cell lines and monocyte-derived dendritic cells. Upon contact with myeloid cells, ova-Tr1 cells induced their apoptosis via a perforin-independent and a granulysin/granzyme-dependent mechanism. As compared to CD8+ cytotoxic T cells, ova-Tr1 cells required more time to lyse target cells and displayed a more gradual lytic activity over time. Notably, this activity was sustained over days resulting in the control of myeloid cell populations at a relatively low ratio. Our study reveals that ova-Tr1 cells are endowed with a sustained cytotoxic activity that relies on a unique combination of granulysin and granzymes and that preferentially eliminates myeloid target cells in a TCR-independent manner.

Heparin-binding hemagglutinin (HBHA), a surface protein of Mycobacterium tuberculosis, is an attractive vaccine candidate and marker of protective immunity against tuberculosis, although the mechanisms underlying this protective immunity are not fully understood. Comparisons of the immune responses of latently M. tuberculosis–infected (LTBI) subjects to those of patients with active tuberculosis (aTB) may help to identify surrogate markers of protection, as LTBI subjects are most often lifelong protected against the disease. HBHA was shown to induce strong Th1 responses and cytotoxic CD8+ responses in LTBI subjects, but additional mechanisms of control of M. tuberculosis infection remain to be identified. In this study, using HBHA-induced blast formation as a readout of specific T lymphocyte activation, we report the presence in M. tuberculosis–infected subjects of HBHA-induced CD4+ T cell blasts that degranulate, as measured by surface capture of CD107a. This suggests the induction by HBHA of a CD4+ T cell subset with cytolytic function, and as nearly half of these cells also contained IFN-g, they had both Th1 and cytotoxic characteristics. We further identified a CD4+ T lymphocyte subset producing IFN-g together with a combination of mediators of cytotoxicity, i.e. perforin, granzymes, and granulysin, and we called them polycytotoxic CD4+ T lymphocytes. Interestingly, whereas purified protein derivative induced such cells in both LTBI subjects and patients with aTB, HBHA-specific polycytotoxic CD4+ T lymphocytes were detected in LTBI subjects and not in patients with pulmonary aTB. To our knowledge, we thus identified a new HBHA-induced CD4+ T cell subset that may contribute to the control of M. tuberculosis infection., SCOPUS: ar.j, info:eu-repo/semantics/published

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

PROBLEM Unexplained recurrent miscarriage (uRM) is defined as two or more spontaneous abortions prior to 20 weeks of gestation with unknown etiology. Peripheral blood natural killer (pNK) cells contact with the villus and exert important role in normal pregnancy. However, it is still controversial about the association between pNK cytotoxicity and uRM, and the underlying mechanism remains unknown so far. METHOD OF STUDY In this study, we aim to compare the percentage, immunophenotype, and function of pNK cells between patients with uRM and fertile controls. The peripheral blood was collected from 49 patients with uRM and 11 fertile women in their middle luteal phase of the menstrual cycle. pNK cells were co-cultured with K562 cells at different cell ratios to measure the cytotoxicity. The percentage of CD3- CD56+ , CD3- CD56bright , and CD3- CD56dim pNK was analyzed by flow cytometry and quantified to evaluate the expression of cytotoxic granules (granzyme B, granulysin, and perforin), and the cell surface receptors related to pNK cell cytotoxicity (NKG2D, NKp30, NKp46, CD158a, and CD158b) were also detected. RESULTS The general linear model analysis showed that pNK cell cytotoxicity in patients with uRM was significantly lower than that in fertile controls. In addition, the ratios of NKG2D/CD158a, NKp30/CD158a, and NKp46/CD158a in CD3- CD56bright pNK subsets were significantly lower in uRM group than that in fertile control. The logistical regression analysis showed that the reduced NKp30/CD158a, NKp46/CD158a ratios in CD3- CD56bright pNK subsets were significantly associated with uRM. CONCLUSION Our results suggested that a low pNK cytotoxicity, which is mediated by inhibitory signals, might be associated with uRM.

T(H)17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of T(H)17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the T(H)17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known T(H)17-secreted protein with direct antimicrobial activity. We generated C. acnes–specific antimicrobial T(H)17 clones ((AM)T(H)17) with varying antimicrobial activity against C. acnes, which we correlated by RNA sequencing to the expression of transcripts encoding proteins that contribute to antimicrobial activity. Additionally, we validated that (AM)T(H)17-mediated killing of C. acnes and bacterial pathogens was dependent on the secretion of granulysin, granzyme B, perforin, and histone H2B. We found that (AM)T(H)17 cells can release fibrous structures composed of DNA decorated with histone H2B that entangle C. acnes that we call T cell extracellular traps (TETs). Within acne lesions, H2B and IL-17 colocalized in CD4(+) T cells, in proximity to TETs in the extracellular space composed of DNA decorated with H2B. This study identifies a functionally distinct subpopulation of T(H)17 cells with an ability to form TETs containing secreted antimicrobial proteins that capture and kill bacteria.

Our understanding of innate immune responses in human rectal mucosal tissues (RM) and their contributions to promoting or restricting HIV transmission is limited. We defined the RM composition of innate and innate-like cell subsets, including plasmacytoid dendritic cells; CD1c + myeloid DCs; neutrophils; macrophages; natural killer cells (NK); Marginal Zone-like B cells (MZB); γδ T cells; and mucosal-associated invariant T cells in RM from 69 HIV-negative men by flow cytometry. Associations between these cell subsets and HIV-1 replication in ex vivo RM explant challenge experiments revealed an inverse correlation between RM-NK and p24 production, in contrast to a positive association between RM-MZB and HIV replication. Comparison of RM and blood-derived MZB and NK illustrated qualitative and quantitative differences between tissue compartments. Additionally, 22 soluble molecules were measured in a subset of explant cultures (n = 26). Higher production of IL-17A, IFN-γ, IL-10, IP-10, GM-CSF, sFasL, Granzyme A, Granzyme B, Granulysin, and Perforin following infection positively correlated with HIV replication. These data show novel associations between MZB and NK cells and p24 production in RM and underscore the importance of inflammatory cytokines in mucosal HIV infection, demonstrating the likely critical role these innate immune responses play in early mucosal HIV replication in humans.

Background Adoptive cell therapy (ACT) is an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. Methods In this study, we explored the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. Results We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1000U/ml interleukin (IL)-2 and (b) 100U/ml IL-2+100U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in phenotype, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells harbor increased amounts of perforin, granzyme B and granulysin in a resting state and release more upon activation. IL-2/IL-15-expanded Vγ9Vδ2 T cells also showed higher levels of transcription factor T-bet, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. Conclusions These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Cancer immunotherapy has shown great advances during recent years, but it has yet to reach its full potential in all cancer types. Adoptive cell therapy (ACT) is now an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. In this study, we aimed to explore the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1,000 U/ml interleukin (IL)-2 and (b) 100 U/ml IL-2 + 100 U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in natural killer (NK) cell marker or activation marker expression, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells were characterized by an increased expression of perforin, granzyme B, and granulysin compared to IL-2-expanded cells. These cytotoxic molecules were not only increased in a resting state, but also released to a greater extent upon target recognition. In contrast, CD107a and cytokine expression did not differ between expansion conditions. However, IL-2/IL-15-expanded Vγ9Vδ2 T cells showed higher levels of transcription factor T-bet expression, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Granule-associated killing molecules released from cytotoxic T lymphocytes participate as a crucial step in immunity against tuberculosis (TB), but the role of coordinated production remains controversial. Coordinated release of effector molecules in vitro after stimulating peripheral blood mononuclear cells (PBMCs) of active TB or HIV/TB coinfection patients with PPD, purified protein derivative of tuberculin and avirulent Mtb, H37Ra, an attenuated strain were investigated in association with clinical outcomes. Perforin, granzyme-B, granulysin and IFN-&gamma, were measured using ELISA. Before anti-TB treatment, PBMCs of TB stimulated with PPD or H37Ra released higher perforin, granzyme-B, and granulysin levels than in HIV/TB and released significantly higher IFN-&gamma, (p = 0.045, p = 0.022). Granulysin positively correlated with perforin in TB (p = 0.042, r = 0.385), HIV/TB coinfection (p = 0.003, r = 0.941) after PPD stimulation, and after H37Ra stimulation in TB (p = 0.005, r = 0.549), but negatively correlated with granzyme B in TB (p = 0.042, r = &minus, 0.386), HIV/TB coinfection (p = 0.042, r = 0.754) were noted. After anti-TB treatment, increased levels of perforin, granulysin and IFN-&gamma, in TB or HIV/TB upon PPD or H37Ra stimulation, and decreased granzyme-B levels after PPD (p = 0.003) or H37Ra (p = 0.028) stimulation in TB were observed. These results suggest that granulysin may act synergistic with perforin and IFN-&gamma, in TB, indicating its crucial function in host immunity to tuberculosis. Future studies with larger numbers of patients ought to be conducted in the future.

Conventional T cells exhibit a delayed response to the initial priming of peptide antigens presented by major histocompatibility complex (MHC) proteins. Unlike conventional T cells, mucosal-associated invariant T (MAIT) cells quickly respond to non-peptidic metabolite antigens presented by MHC-related protein 1 (MR1). To elucidate the MR1-dependent activation program of MAIT cells in response to mycobacterial infections, we determined the surface markers, transcriptomic profiles, and effector responses of activated human MAIT cells. Results revealed that mycobacterial-incubated antigen-presenting cells stimulated abundant human CD8+ MAIT cells to upregulate the co-expression of CD69 and CD26, as a combinatorial activation marker. Further transcriptomic analyses demonstrated that CD69+CD26++ CD8+MAIT cells highly expressed numerous genes for mediating anti-mycobacterial immune responses, including pro-inflammatory cytokines, cytolytic molecules, NK cell receptors, and transcription factors, in contrast to inactivated counterparts CD69+/−CD26+/− CD8+MAIT cells. Gene co-expression, enrichment, and pathway analyses yielded high statistical significance to strongly support that activated CD8+ MAIT cells shared gene expression and numerous pathways with NK and CD8+ T cells in activation, cytokine production, cytokine signaling, and effector functions. Flow cytometry detected that activated CD8+MAIT cells produced TNFα, IFNγ, and granulysin to inhibit mycobacterial growth and fight mycobacterial infection. Together, results strongly support that the combinatorial activation marker CD69+CD26++ labels the activated CD8+MAIT cells that develop an innate-like activation program in anti-mycobacterial immune responses. We speculate that the rapid production of anti-mycobacterial effector molecules facilitates MAIT cells to fight early mycobacterial infection in humans.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli., Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans that recognize bacterial metabolites. This study shows that MAIT cells exert potent antimicrobial activity against both cell-associated and extracellular forms of Escherichia coli, including strains that are resistant to the last resort antibiotics carbapenems.

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that gd T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites.Here, we demonstrate that Plasmodium falciparum infection depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

Objectives Immunoglobulin A vasculitis/Henoch–Schönlein purpura (IgAV/HSP) is a major cause of vasculitis in children. It is often accompanied by nephritis (HSPN) and could progress to chronic kidney disease. Galactose‐deficient IgA1 was recently reported to be involved in the pathogenesis of HSPN, for which immunosuppressive drugs are considered key treatment. However, the involvement of immune cells in the development of HSPN remains unclear. Methods We compared gene expressions of peripheral blood mononuclear cells (PBMCs) among healthy controls (n = 10), IgAV/HSP patients (n = 21) and HSPN patients (n = 8), which required nephritis development within 3 months of IgAV/HSP onset. Immunohistochemistry analysis and flow cytometry were performed to assess renal biopsy specimens and PBMCs, respectively. Serum CX3CL1 levels were measured by ELISA. Results GNLY and GZMB expressions increased in HSPN patients, consistent with increased number of glomerular granulysin‐ and/or granzyme B‐positive cells demonstrated by immunohistochemistry analysis. Additionally, circulating cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells were activated with the up‐regulated surface expressions of human leucocyte antigen DR (HLA‐DR) and CX3CR1 in HSPN patients with severe proteinuria. Renal biopsies demonstrated increased number of CD8+ cells and/or CD56+ cells and up‐regulated expression of glomerular CX3CL1, a specific ligand for CX3CR1, along with increased serum CX3CL1 level. Conclusion Activated CTLs and NK cells play roles in the development of nephritis in IgAV/HSP patients and can be used as novel biomarkers for HSPN., In this study, we found that activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells play roles in the development of Henoch–Schönlein purpura nephritis (HSPN). We also suggest that activation of circulating CTLs and NK cells can be used as a novel biomarker for HSPN with severe proteinuria.

Summary: Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing. : The mechanisms of cytolytic granules deployment and the events leading to selective use of perforin, and not granulysin, in NK-cell-mediated killing of Cryptococcus are unknown. Ogbomo et al. demonstrate that Eg5-kinesin and dynein control these events. Eg5-kinesin activity is required to turn on dynein activity for directed cytotoxicity. Keywords: Eg5-kinesin, dynein, NK cell cytotoxicity, granule congregation, granule convergence, microtubule organizing center polarization, perforin, granulysin

1,25-dihydroxyvitaminD3 [1,25(OH)2D3] modulates both the innate and adaptive immunity in tuberculosis. We explored the effect of 1,25(OH)2D3 on cytolytic molecules like perforin, granulysin, and granzyme-B in T-cells and natural killer cells during M. tuberculosis (Mtb) infection. Peripheral blood mononuclear cells (PBMCs) from 45 healthy controls (HCs) and 45 pulmonary tuberculosis (PTB) patients were cultured with Mtb in the absence or presence of 1,25(OH)2D3 for 72 h. The percentage of perforin, granulysin, and granzyme-B positive cells were estimated by flow cytometry. 1,25(OH)2D3 significantly decreased the percentage of cytolytic molecules in total, CD4+, CD8+ and CD56+ cells in HCs and PTB patients (p < 0.05). Moreover, 1,25(OH)2D3 downregulates IFN-γ levels while upregulate the anti-inflammatory cytokine IL-10. Correlation revealed that the total percentage of cytolytic molecules were positively correlated with IFN-γ level, whereas negatively correlated with IL-10 level in both the study subjects (p < 0.05). This results suggests that 1,25(OH)2D3 downregulate the expression of cytolytic molecues and act as anti-inflammatory in adaptive immune response, which might help to reduce inflammation and tissue damage during the active stage of the disease.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax–infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax–infected reticulocytes in a human leukocyte antigen–dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax–infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax–infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy. T cells kill blood-stage Plasmodium vivax, providing a rationale for the development of a T cell vaccine against this parasite.

Background The role of CD4 + T cells in the immunopathogenesis of pulmonary sarcoidosis is well-established, while less is known about the phenotype and function of CD8 + cytolytic T cells (CTLs). Methods CD8 + CTLs were explored in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from up to 25 patients with sarcoidosis and 25 healthy controls. The proportion of CTLs was assessed by the expression of cytolytic effector molecules perforin, granzyme B and granulysin in CD8 + T cells, using flow cytometry. Cytolytic function in blood lymphocytes was assessed using a standard 51 Cr-release assay. Patients with Lofgren´s syndrome (LS) and an acute disease onset, were compared to non-LS patients with an insidious onset. Results Higher proportions of peripheral CD8 + CTLs expressing perforin and granzyme B were observed in sarcoidosis compared to healthy controls. Blood CTLs from non-LS patients had significantly higher expression of perforin, granzyme B and granulysin compared to matched BAL, while LS patients maintained lower levels of effector molecules in both compartments. Mitogen-stimulated peripheral lymphocytes from sarcoidosis patients, particularly from the non-LS group, showed a higher target cell lysis compared to controls. Conclusion These results demonstrated enhanced peripheral CD8 + CTL responses in sarcoidosis, especially in non-LS patients who have an increased risk of chronic disease. Further comprehensive clinical studies are warranted to increase our understanding of CD8 + CTL responses in sarcoidosis.

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

The presence of memory T cells in COVID-19 patients has been acknowledged, however the functional potency of memory responses is critical for protection. In this study, naïve, effector, effector memory, and central memory CD4+ and CD8+ T cells obtained from the COVID-19 survivors were re-exposed to autologous monocyte-derived DCs that were loaded with SARS-CoV-2 spike glycoprotein S1. Proliferation capacity, CD25, 4-1BB, and PD-1 expression, and IFN-γ, IL-6, granzyme, granulysin, and FasL secretion were enhanced in CD4+ and CD8+ effector memory and central memory T cells. Albeit being at heterogeneous levels, the memory T cells from the individuals with COVID-19 history possess functional capacities to reinvigorate anti-viral immunity against SARS-CoV-2.

Chemokines and chemokine receptors have rapidly diversified in teleost fish but their immune functions remain unclear. We report in this study that CCL19, a chemokine known to control lymphocyte migration and compartmentalization of lymphoid tissues in mammals, diversified in salmonids leading to the presence of six CCL19-like genes named CK10a, CK10b, CK12a, CK12b, CK13a, and CK13b. Salmonid CCL19-like genes all contain the DCCL-conserved motif but share low amino acid sequence identity. CK12 (but not CK10 or CK13) is constitutively expressed at high levels in all four trout MALT. Nasal vaccination with a live attenuated virus results in sustained upregulation of CK12 (but not CK10 or CK13) expression in trout nasopharynx-associated lymphoid tissue. Recombinant His-tagged trout CK12a (rCK12a) is not chemotactic in vitro but it increases the width of the nasal lamina propria when delivered intranasally. rCK12a delivered intranasally or i.p. stimulates the expression of CD8α, granulysin, and IFN-γ in mucosal and systemic compartments and increases nasal CD8α+ cell numbers. rCK12a is able to stimulate proliferation of head kidney leukocytes from Ag-experienced trout but not naive controls, yet it does not confer protection against viral challenge. These results show that local nasal production of CK12a contributes to antiviral immune protection both locally and systemically via stimulation of CD8 cellular immune responses and highlight a conserved role for CK12 in the orchestration of mucosal and systemic immune responses against viral pathogens in vertebrates.

Background and Design: Psoriasis is a chronic papulosquamous disease where histologically epidermal hyperproliferation and infiltration involving natural killer cells and cytotoxic T-cells are observed. These cells have been shown to carry cytolytic molecules containing high amount of perforin, granzyme B and granulysin (GNLY). The roles of these molecules in the pathogenesis of psoriasis are still disputed, with serum GNLY and cathepsin-l (CL) levels thought to be associated with cellular immunity. In this study, we investigated the relationship between the severity and duration of psoriasis and the levels of CL and GNLY. Materials and Methods: Prospective and randomized study of 40 patients (23 males, 17 females) with psoriasis who admitted to hospital between December 2014 and August 2015, and 40 age and sex-matched healthy controls (23 males, 17 females) were investigated. CL and GNLY serum levels were measured by ELISA method. Results: There was no significant differences in GNLY and CL levels between psoriasis patients and the control group (p=0.243 and p=0.606). There was also no statistically significant difference between psoriasis patients with low Psoriasis Area Severity Index (PASI) (≤10) and those with high PASI (>10) (p=0.86 and p=0.61) score. Conclusion: There are studies that have shown GNLY and CL in the psoriazis are important markers for disease pathogenesis. However, according to the results of this study, CL and GNLY levels are not sufficient markers to indicate the level of cellular immunity and disease severity in psoriasis. Future studies are needed on this subject with a wider range of patients.

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-γ-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-γ-induced transcription of IRF-1, MHC-I and β2-microglobulin (β2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-γ- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-γ KO mice remained parasitemic and all died. β2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-γ promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY., Author summary CD8+ cytotoxic T lymphocytes (CTLs) are important for immune defense against intracellular pathogens, such as viruses, bacteria and parasites, and tumor surveillance. CTLs, which recognize peptide epitopes presented by MHC-I molecules expressed in nucleated cells, become activated and kill infected target cells by releasing the contents of cytotoxic granules into the immunological synapse. Since most Plasmodium spp. infect erythrocytes that are enucleated and do not express MHC-I, the role of CD8+ T cells in the blood-stage of malaria has been neglected. We recently showed that P. vivax-infected reticulocytes express MHC-I and are killed in a manner dependent on granulysin (GNLY), a cytotoxic granule effector protein. However, the protective role of CD8+ T cells is controversial and the role of GNLY in vivo remains to be demonstrated. Here, we show that CTLs and GNLY mediate mouse resistance to blood-stage infection with P. yoelii, a rodent malaria parasite that preferably infects reticulocytes.

The viral reservoir is the major hurdle in developing and establishing an HIV cure. Understanding factors affecting the size and decay of this reservoir is crucial for the development of therapeutic strategies. Recent work highlighted that CD8+ T cells are involved in the control of viral replication in ART-treated HIV-1-infected individuals, but how CD8+ T cells sense and restrict the HIV reservoir are not fully understood. Here, we demonstrate that a population of unconventional CD45RA+, PanKIR+, and/or NKG2A+ virtual memory CD8+ T cells (TVM cells), which confer rapid and robust protective immunity against pathogens, plays an important role in restraining the HIV DNA reservoir in HIV-1-infected patients with effective ART. In patients undergoing ART, TVM cells negatively correlate with HIV DNA and positively correlate with circulating IFN-α2 and IL-15. Moreover, TVM cells constitutively express high levels of cytotoxic granule components, including granzyme B, perforin and granulysin, and demonstrate the capability to control HIV replication through both cytolytic and noncytolytic mechanisms. Furthermore, by using an ex vivo system, we showed that HIV reactivation is effectively suppressed by TVM cells through KIR-mediated recognition. This study suggests that TVM cells are a promising target to predict posttreatment virological control and to design immune-based interventions to reduce the reservoir size in ART-treated HIV-1-infected individuals.

NK cell functions are tightly regulated by the balance between the inhibitory and stimulatory surface receptors. We investigated the surface expression of galectin-9 (Gal-9) and its function in NK cells from HIV-infected individuals on antiretroviral therapy, long-term nonprogressors, and progressors compared with healthy controls. We also measured the expression of TIGIT and TIM-3 on different NK cell subpopulations and compared their functionality to Gal-9+ NK cells. Our data demonstrated significant upregulation of Gal-9 on NK cells in HIV-infected groups versus healthy controls. Gal-9 expression was associated with impaired expression of cytotoxic effector molecules granzyme B, perforin, and granulysin. In contrast, Gal-9 expression significantly enhanced IFN-γ expression in NK cells of HIV-1–infected individuals. We also found an expansion of TIGIT+ NK cells in HIV-infected individuals; however, dichotomous to Gal-9+ NK cells, TIGIT+ NK cells expressed significantly higher amounts of cytotoxic molecules but lower IFN-γ. Moreover, lower expression of cytotoxic effector molecules in Gal-9+ NK cells was associated with higher CD107a expression, which suggests indiscriminate degranulation. Importantly, a positive correlation between the plasma viral load and Gal-9+ NK cells was observed in progressors. Finally, we found that a cytokine mixture (IL-12, IL-15, and IL-18) can improve effector functions of Gal-9+ NK cells in HIV-infected individuals, although, such an effect was observed for Gal-9− NK cells, as well. Overall, our data highlight the important role of Gal-9 in dysfunctional NK cells and, more importantly, a dichotomy for the role of Gal-9 versus TIGIT and suggest a potential new avenue for the development of therapeutic approaches.

Background: Human natural killer (NK) cells are key functional players in kidney transplant rejection. However, the respective contributions of the two functionally distinct human NK cell subsets (CD56bright cytokine-producing vs. CD56dim cytotoxic effector) in episodes of allograft rejection remain uncertain, with current immunohistochemical methods unable to differentiate these discrete populations. We report the outcomes of an innovative multi-color flow cytometric-based approach to unequivocally define and evaluate NK cell subsets in human kidney allograft rejection. Methods: We extracted renal lymphocytes from human kidney transplant biopsies. NK cell subsets were identified, enumerated, and phenotyped by multi-color flow cytometry. Dissociation supernatants were harvested and levels of soluble proteins were determined using a multiplex bead-based assay. Results were correlated with the histopathological patterns in biopsies—no rejection, borderline cellular rejection, T cell-mediated rejection (TCMR), and antibody-mediated rejection (AMR). Results: Absolute numbers of only CD56bright NK cells were significantly elevated in TCMR biopsies. In contrast, both CD56bright and CD56dim NK cell numbers were significantly increased in biopsies with histopathological evidence of AMR. Notably, expression of the activation marker CD69 was only significantly elevated on CD56dim NK cells in AMR biopsies compared with no rejection biopsies, indicative of a pathogenic phenotype for this cytotoxic NK cell subset. In line with this, we detected significantly elevated levels of cytotoxic effector molecules (perforin, granzyme A, and granulysin) in the dissociation supernatants of biopsies with a histopathological pattern of AMR. Conclusions: Our results indicate that human NK cell subsets are differentially recruited and activated during distinct types of rejection, suggestive of specialized functional roles.

Background: In OA joint, synovial membrane contain immunocompetent cells (1-4), which produce predominantly pro-inflammatory cytokines (2,5), justifying the name “osteoarthritis” and directing the development of disease towords mild systemic inflammatory condition (6). Granulysin (GNLY) is mediator of cellular immunity expressed in T and NK cells in 15 kDa precursor and 9 kDa cytotoxic forms (2). It is regulated by interleukin 15. We investigated GNLY expression in perifperal blood lymphocytes and GNLY mediated apoptosis in vitro, serum concentration of IL-15 and the correlation of GNLY expression with intensity of the pain in the knee of OA patients and with 6-minute walk distance. Objectives: Women with knee OA (20), and healthy control (17) were medically examined, and their blood samples tested. All of them signed informed consent before medical sampling of peripheral blood (PB). Methods: Visual analogue scale (VAS) of pain and results of 6-minute walk test were noticed in all participants. Peripheral blood mononuclear cells were isolated by gradient density centrifugation and intracellular GNLY labeling in CD3-CD56+NK cells, CD3+CD56- T cells and CD3+CD56+ NKT cells was performed and analyzed by flow cytometry. NK cells’ apoptotic activity against NK sensitive K-562 cells was measured in 18-hour PKH-26 (red) cytotoxicity assay with evaluation of propidium iodide-/annexin V+ target cells by flow cytometry. In some samples anti-GNLY and/or anti-perforin antibodies were added. IL-15concentration was measured by ELISA. Nonparametric Kruskal-Wallis and Mann-Whitney U-test, as well as Spearman correlation test were used for statistical evaluation. Results: In lymphocytes of OA patients GNLY expression and NK cell-mediated apoptosis of K-562 cells did not differ significantly from the healthy control. In OA patients only, RC8 antibody against cytotoxic GNLY molecule significantly decreased apoptosis of K-562 cells. RF10 anti-15 kDa GNLY did not show such effect. Anti-perforin antibody completely abolished apoptosis in both groups tested and the effects of additionally added RC8 or RF10 anti-GNLY antibodies were not observed. Serum IL-15 concentration in healthy controls and OA patients was low and did not show statistically significant difference. GNLY expression in lymphocytes, and particularly in NK subset, positively correlated with VAS of pain and 6-minutes walking distance. Conclusion: In OA patients, GNLY mediated apoptosis is involved in apoptosis of NK sensitive K-562 cells in vitro and might be involved in the killing of damaged joint cells in vivo after direct contact. References [1] Lindblad S, Hedfors E. Arthritis Rheum. 1987;30(10):1081-8. [2] Wojdasiewicz P, et al. Mediators inflamm. 2014;561459. [3] Leheita O, et al. Egypt J Immunol2005;12:113-24. [4] Huss RS, et al.. Arthritis Rheum. 2010;62:3799-805. [5] Imamura M, et al. Int J inflam. 2015;2015:329792. [6] Maldonado M, Nam J. Biomed Res int. 2013;2013:284873. Acknowledgement: The research was financed by the University of Rijeka Science Support No. Uniri-biomed-18-110 -1248 to Gordana Laskarin and by hospital „Thalassotherapia-Opatija“. Disclosure of interests: Gordana Laskarin Speakers bureau: Yes, at the professional meetings, Tatjana Kehler Speakers bureau: at some congresses and meetings, Viktor Persic Speakers bureau: at some meetings and congresses, Merica aralica: None declared, Božena Curko-Cofek: None declared, Marija Rogoznica: None declared, Ivan Rosovic: None declared, Tamara Kauzlaric-Zivkovic: None declared, Sandra Rusac-Kukic: None declared, Daniel Rukavina: None declared

Primary T cell immunodeficiency and HIV-infected patients are plagued by non-viral infections caused by bacteria, fungi, and parasites, suggesting an important and underappreciated role for T lymphocytes in controlling microbes. Here, we review recent studies showing that killer lymphocytes use the antimicrobial cytotoxic granule pore-forming peptide granulysin, induced by microbial exposure, to permeabilize cholesterol-poor microbial membranes and deliver death-inducing granzymes into these pathogens. Granulysin and granzymes cause microptosis, programmed cell death in microbes, by inducing reactive oxygen species and destroying microbial antioxidant defenses and disrupting biosynthetic and central metabolism pathways required for their survival, including protein synthesis, glycolysis, and the Krebs cycle.

Human milk has been previously found to contain various types of leukocytes however specific characteristics of these cells, such as whether they contain cytolytic antimicrobial proteins that may induce pathogen directed cell death, are unknown. This project aims to examine the presence and localization of immune proteins such as perforin, granulysin and granzymes in human milk cells at the protein and mRNA level. Genes encoding these proteins were confirmed in human milk cell samples, which were particularly enriched in early milk and in the case of maternal infection. Fluorescence activated cell sorting (FACS) was used to investigate the co-expression of these proteins with pan-immune cell marker CD45 and epithelial marker EPCAM. Co-expression of antimicrobial proteins was found predominantly in CD45 positive cells, also increasing in the case of maternal infection. Our study suggests that human milk contains cells that carry hallmarks of activated or memory T-cells which are enriched early in lactation and in the case of maternal infection. Presence and prevalence of these cells in human milk may indicate a role in the protection of the maternal breast or for delivery to the vulnerable infant.

BACKGROUND: Psoriasis is a systemic inflammatory disorder that involves complex pathogenic interactions between the innate and adaptive immune systems. The most accepted mechanism in the etiopathogenesis of psoriasis is the induction of inflammation with keratinocyte hyperproliferation. Granulysin (GNLY) is a cytolytic antimicrobial peptide (AMP) that is secreted together with granzyme and perforin from the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It has been immunohistochemically proven that the expression of granulysin is increased in lesions of psoriasis. OBJECTIVE: This study aimed to investigate the relationship between psoriasis disease and granulysin gene polymorphisms. METHODS: GNLY rs7908 and rs10180391 polymorphisms were studied by PCR-RFLP in 100 psoriasis patients under treatment in the Dermatology Polyclinic of Bulent Ecevit University. In addition, 100 healthy individuals with similar age and sex distribution were used as a control group. RESULTS: In the control group, GNLY rs7908 CC genotype was significantly higher than in psoriasis patients (P= 0.031; OR= 0.305; Cl= 0.305 (0.121 - 0.773). In our study, the genotype distributions in patients and control groups were GNLY rs7908 (SNP) GG (51%, 37%), GC (41%, 44%), CC (8%, 19%); GNLY rs10180391 (SNP) from the CC (41%, 44%), CT (42%, % 41), TT (17%, 15%). STUDY LIMITATIONS: The study only included Turkish patients. CONCLUSION: Our findings showed that GNLY rs7908 CC genotype and C allele had a protective effect against psoriasis and decreased the disease severity (according to PASI score), whereas rs10180391 SNP did not show any effective role in psoriasis pathogenesis.

The limited cytotoxicity of immune cells facilitates a successful establishment of pregnancy. However, the association between cytotoxic granules and unexplained repeated implantation failure (uRIF) remains unkown. Twenty-one fertile controls and 54 patients with uRIF were included in this study. The pregnancy outcomes were monitored at different gestational periods. The peripheral blood lymphocytes were detected using specific monoclonal antibodies by flow cytometry. The percentage of perforin+ (Pfr+), granzyme B+ (GrB+), or granulysin+ (Gnly+) lymphocytes was not significantly different among fertile controls, uRIF patients with successful pregnancy outcomes, and uRIF patients with pregnancy failure. The percentage of GrB+ γδ-T cells in lymphocytes was markedly higher in uRIF patients with implantation failure and clinical pregnancy failure than that in uRIF patients with a corresponding successful pregnancy outcome. A four-tier risk model showed that the risk of suffering clinical pregnancy failure in uRIF patients among high risk tier (83.3 %), normal risk tier (65.0 %) and low risk tier (39.1 %) was elevated by 2–4 fold compared with uRIF patients among lowest risk tier (20.0 %). In addition, the percentage of GrB+ NK cells in lymphocytes tended to decrease in uRIF patients with pregnancy failure. The AUC of the combined indicator with GrB+ γδ-T cells and GrB+ NK cells was increased than that of GrB+ γδ-T cells and GrB+ NK cells for predicting clinical pregnancy failure. In conclusion, the frequency of GrB-expressing γδ-T and NK cells in peripheral blood could serve as a predictor of clinical pregnancy failure in patients with uRIF.

Lichen planus is a chronic mucocutanous disorder histopathologically characterized with a keratinocytes apoptosis, subsequent basal cell layer liquefaction and accumulation of the inflammatory infiltrate in papillary dermis. A formation of apoptotic bodies in basal cell layer is due to a cytotoxic lymphocyte attack to the basal keratinocytes. It has been demonstrated that the cytotoxic molecules included in this attack are perforin and granzyme B. Both molecules are found upregulated in CD8+ lymphocytes that are in close contact to keratinocytes. However, their amount is lower in lichen planus than in other skin disease characterized by liquefaction and vacuolar degeneration of the basal epidermal layer. This could speculate about other cytotoxic molecule such as granulysin that could mediate keratinocyte apoptosis. Therefore, in this article we hypothesize about the crucial role of granulysin molecule in keratinocytes killing that could contribute to a lichen planus pathogenesis.

Cellular and antibody-mediated rejection processes and also interstitial fibrosis/tubular atrophy (IFTA) lead to allograft dysfunction and loss. The search for accurate, specific and non-invasive diagnostic tools is still ongoing and essential for successful treatment of renal transplanted patients. Molecular markers in blood cells and serum may serve as diagnostic tools but studies with high patient numbers and differential groups are rare. We validated the potential value of several markers on mRNA level in blood cells and serum protein level in 166 samples from kidney transplanted patients under standard immunosuppressive therapy (steroids ± mycophenolic acid ± calcineurin inhibitor) with stable graft function, urinary tract infection (UTI), IFTA, antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR) applying RT-PCR and ELISA. The mRNA expression of RANTES, granulysin, granzyme-B, IP-10, Mic-A and Interferon-γ in blood cells did not distinguish specifically between the different pathologies. We furthermore discovered that the mRNA expression of the chemokine IL-8 is significantly lower in samples from IFTA patients than in samples from patients with stable graft function (p

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species ( Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are a spectrum of T-cell mediated immune disorders. While the contributory mechanisms leading to the apoptosis of epidermal cells in SJS/TEN remain unproven, the keratinocyte apoptosis seen in SJS/TEN is thought to occur through the T-cell mediated Fas-Fas ligand (FasL), perforin/granzyme B, and other immune mediators. Most recently, emphasis has been placed on the granulysin pathway as being the primary mediator of apoptosis and widespread epidermal necrosis in SJS/TEN. This article aims to review the proposed mechanisms by which these pathways work and the immunomodulatory therapies that have been developed in an attempt to target them.

Background Granulysin (GNLY) is excreted from cytotoxic T lymphocytes and natural killer cells, and plays an important role in antitumor immunity. However, few studies have estimated serum GNLY concentrations in patients with nasopharyngeal carcinoma (NPC). We evaluated GNLY as a potential biomarker for NPC. Methods Serum GNLY concentrations were measured in blood samples taken from 98 NPC patients, 56 nasopharyngitis (NPT) patients, and 99 healthy subjects. The clinical relevance of GNLY in NPC was also investigated. We also assessed the association between serum GNLY and serum immunoglobulin A antibodies against the Epstein–Barr virus (EBV) viral capsid antigen (VCA-IgA) and EBV DNA. Results Serum GNLY levels were significantly lower in NPC patients and significantly higher in nasopharyngitis patients compared to healthy controls. Thus, serum GNLY performs well as a biomarker for distinguishing between NPC and NPT. The serum GNLY concentration is elevated with corresponding increases in clinical stage and shows a significant correlation with VCA-IgA and EBV DNA concentration. Conclusions Serum GNLY is closely associated with the clinical characteristics of NPC and may be a potential biomarker for NPC.

Protozoan infections are a serious global health problem1, 2. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents—granzyme (Gzm) proteases and the pore-forming perforin (PFN)—into the infected cell3. However, these cytotoxic molecules do not kill intracellular parasites. CD8+ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ4, 5, 6, 7, 8, 9, 10. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes11, 12, 13, 14, and GNLY, PFN and Gzms rapidly kill intracellular bacteria15. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell–mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.