Porcine γδ T cells express cytotoxic cell-associated markers and display killing activity but are not selectively cytotoxic against PRRSV- or swIAV-infected macrophages.

Background: Gamma-delta (γδ) T cells are a major immune cell subset in pigs. Approximately 50% of circulating T cells are γδ T cells in young pigs and up to 30% in adult sows. Despite this abundance, the functions of porcine γδ T cells are mostly unidentified. In humans and mice, activated γδ T cells exhibit broad innate cytotoxic activity against a wide variety of stressed, infected, and cancerous cells through death receptor/ligand-dependent and perforin/granzyme-dependent pathways. However, so far, it is unknown whether porcine γδ T cells have the ability to perform cytotoxic functions. Methods: In this study, we conducted a comprehensive phenotypic characterization of porcine γδ T cells isolated from blood, lung, and nasal mucosa. To further analyze the cytolytic potential of γδ T cells, in vitro cytotoxicity assays were performed using purified γδ T cells as effector cells and virus-exposed or mock-treated primary porcine alveolar macrophages as target cells. Results: Our results show that only CD2+ γδ T cells express cytotoxic markers (CD16, NKp46, perforin) with higher perforin and NKp46 expression in γδ T cells isolated from lung and nasal mucosa. Moreover, we found that γδ T cells can exhibit cytotoxic functions in a cell-cell contact and degranulation-dependent manner. However, porcine γδ T cells did not seem to specifically target Porcine Reproductive and Respiratory Syndrome Virus or swine Influenza A Virus-infected macrophages, which may be due to viral escape mechanisms. Conclusion: Porcine γδ T cells express cytotoxic markers and can exhibit cytotoxic activity in vitro. The specific mechanisms by which porcine γδ T cells recognize target cells are not fully understood but may involve the detection of cellular stress signals.

Littoporins: Novel actinoporin-like proteins in caenogastropod genus Littorina.

In the course of searching for genes controlling the immune system in caenogastropod mollusks, we characterized and phylogenetically placed five new actinoporin-like cytolysins expressed in periwinkles of the genus Littorina. These newly discovered proteins, named littoporins (LitP), contain a central cytolysin/lectin domain and exhibit a predicted protein fold that is almost identical to the three-dimensional structures of actinoporins. Two of these proteins, LitP-1 and LitP-2, were found to be upregulated in L. littorea kidney tissues and immune cells in response to natural and experimental infection with the trematode Himasthla elongata, suggesting their potential role as perforins in the systemic anti-trematode immune response. The primary sequence divergence of littoporins is hypothesized to be attributed to the taxonomic range of cell membranes they can recognize and permeabilize.

The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis.

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies.

Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.

Multifunctional T cell response in active pulmonary tuberculosis patients.

BACKGROUND: Tuberculosis still threatens human health. We aimed to investigate the T cell immune status and the role of multifunctional T cells in pulmonary tuberculosis patients. METHODS: Thirty active pulmonary tuberculosis (APTB) patients, 30 latent tuberculosis infection (LTBI) patients, 25 cured pulmonary tuberculosis (CPTB) patients and 25 healthy controls (HCs) enrolled in this study. Flow cytometer for detecting T cell phenotype and function. CBA Flex Set was used to measure chemokine. RESULTS: Compared with HCs and LTBI patients, APTB patients had fewer CD4+ T and CD8+ T cells, but the expression of granzyme A, granzyme B and perforin on CD8+ T cells increased. Compared to LTBI and CPTB patients, Mycobacterium tuberculosis-specific CD8+ T cells in APTB patients appeared to be more differentiated CD45RA-CCR7- cells, and there were more multifunctional CD4+ T and CD8+ T cells. Importantly, the frequency of multifunctional CD4+ T cells in the pleural fluid of APTB patients was higher than that of peripheral blood. And the proportion of multifunctional CD4+ T cells expressing the migration receptor CXCR3 in the peripheral blood of APTB patients decreased, while the concentrations of its ligands, chemokine MIG, IP-10 and I-TAC increased significantly in plasma, especially in pleural fluid. CONCLUSIONS: Decreased T lymphocytes in APTB patients may cause compensatory activation of CD8+ T cells. Multifunctional CD4+ T cells in peripheral blood could migrate to the lungs under the action of CXCR3 and associated chemokine. Multifunctional CD4+ T cells and Multifunctional CD8+ T cells were of great significance in monitoring disease treatment.

A Profound Membrane Reorganization Defines Susceptibility of Plasmodium falciparum Infected Red Blood Cells to Lysis by Granulysin and Perforin.

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 γδ T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites. Here, we follow up on the molecular mechanisms of parasite growth inhibition by human pore-forming proteins. We confirm that Plasmodium falciparum infection efficiently 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, by revealing the molecular events we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.

T cells at work: How post-transcriptional mechanisms control T cell homeostasis and activation.

T cells are central players of the adaptive immune system by protecting us from recurring infections and by killing malignant cells. Protective T cell responses rely on the concerted production of effector molecules such as cytolytic mediators, granzymes, and perforins, as well as pro-inflammatory cytokines and chemokines. Once activated, T cells drastically change their gene expression and rapidly respond to insults by producing ample amounts of effector molecules. In the absence of antigen, T cells remain in a quiescent state and survey our body for possible pathogenic insults. Resting T cells are, however, not inert, but continuously regulate their protein production to survive and to be prepared for possible re-infections. Here, we review our current knowledge on the regulation of gene expression in activated and quiescent T cells. We specifically focus on post-transcriptional mechanisms that define the protein output and that allow dormant cells to undergo active signaling and selective translation, keeping them poised for activation. Finally, we discuss which signals drive T cell survival and their preparedness to respond to insults and which mechanisms are involved in these processes.

CXCL12-CXCR4-Mediated Chemotaxis Supports Accumulation of Mucosal-Associated Invariant T Cells Into the Liver of Patients With PBC.

Objectives: To explore the potential role of CD3+CD8+CD161high TCRVα7.2+ mucosal-associated invariant T (MAIT) cells in the pathogenesis of primary biliary cholangitis (PBC). Methods: We enrolled 55 patients with PBC, 69 healthy controls (HCs), and 8 patients with hepatic hemangioma. Circulating MAIT cells and their chemokine receptor profiles and cytokine production were quantified using flow cytometry. Liver-resident MAIT cells were examined by immunofluorescence staining. CXCL12-mediated chemotaxis of MAIT cells was measured using a transwell migration assay. Plasma interleukin (IL)-18 was measured using ELISA, and cytokine production in IL-18-stimulated MAIT cells was detected using flow cytometry. Result: Peripheral MAIT cells were found to be significantly lower in patients with PBC (3.0 ± 3.2% vs. 9.4 ± 8.0%, p < 0.01) and negatively correlated with alkaline phosphatase (ALP) levels (r = -0.3209, p < 0.05). Liver immunofluorescence staining suggested that MAIT cells might accumulate in PBC liver. MAIT cells from patients with PBC expressed higher levels of CXCR4 (84.8 ± 18.0% vs. 58.7 ± 11.4%, p < 0.01), and the expression of CXCL12 was higher in PBC liver. CXCL12 promoted MAIT cell chemotaxis (70.4 ± 6.8% vs. 52.2 ± 3.5%, p < 0.01), which was attenuated by CXCR4 antagonist. MAIT cells from PBC produced significantly more interferon-γ (IFN-γ) (88.3 ± 4.2% vs. 64.2 ± 10.1%, p < 0.01), tumor necrosis factor-α (TNF-α) (93.0 ± 1.1% vs. 80.1 ± 5.3%, p < 0.01), Granzyme B (89.3 ± 3.3% vs. 72.1 ± 7.0%, p < 0.01), and perforin (46.8 ± 6.6% vs. 34.8 ± 7.7%, p < 0.05). MAIT cells from PBC expressed higher levels of IL18-Rα (83.8 ± 10.2% vs. 58.3 ± 8.7%, p < 0.01). Plasma IL-18 was more abundant in patients with PBC (286.8 ± 75.7 pg/ml vs. 132.9 ± 78.1 pg/ml, p < 0.01). IL-18 promoted IFN-γ production in MAIT cells (74.9 ± 6.6% vs. 54.7 ± 6.7%, p < 0.01), which was partially attenuated by blocking IL-18R (68.6 ± 8.3% vs. 43.5 ± 4.2%, p < 0.01). Conclusion: Mucosal-associated invariant T cells from patients with PBC accumulated in the liver via CXCL12-CXCR4-mediated chemotaxis, produced pro-inflammatory cytokines, and contributed to portal inflammation, which was potentially mediated by elevated IL-18. Targeting MAIT cells might be a therapeutic approach for PBC.

Distinct genomic features across cytolytic subgroups in skin melanoma.

BACKGROUND: Skin melanoma is a highly immunogenic cancer. The intratumoral immune cytolytic activity (CYT) reflects the ability of cytotoxic T and NK cells to eliminate cancer cells, and is associated with improved patient survival. Despite the enthusiastic clinical results seen in advanced-stage metastatic melanoma patients treated with immune checkpoint inhibitors, a subgroup of them will later relapse and develop acquired resistance. We questioned whether CYT associates with different genomic profiles and thus, patient outcome, in skin melanoma. METHODS: We explored the TCGA-SKCM dataset and stratified patients to distinct subgroups of cytolytic activity. The tumor immune contexture, somatic mutations and recurrent copy number aberrations were calculated using quanTIseq, MutSigCV and GISTIC2. Chromothriptic events were explored using CTLPScanner and cancer neoepitopes were predicted with antigen garnish. Each tumor's immunophenoscore was calculated using Immunophenogram. Mutational signatures and kataegis were explored using SigProfiler and compared to the known single or doublet base substitution signatures from COSMIC. RESULTS: Metastatic skin melanomas had significantly higher CYT levels compared to primary tumors. We assessed enrichment for immune-related gene sets within CYT-high tumors, whereas, CYT-low tumors were enriched for non-immune related gene sets. In addition, distinct mutational and neoantigen loads, primarily composed of C > T transitions, along with specific types of copy number aberrations, characterized each cytolytic subgroup. We found a broader pattern of chromothripsis across CYT-low tumors, where chromosomal regions harboring chromothriptic events, contained a higher number of cancer genes. SBS7a/b, SBS5 and SBS1 were the most prevalent mutational signatures across both cytolytic subgroups, but SBS1 differed significantly between them. SBS7a/b was mutually exclusive with SBS5 and SBS1 in both CYT subgroups. CYT-high patients had markedly higher immunophenoscore, suggesting that they should display a clinical benefit upon treatment with immune checkpoint inhibition therapy, compared to CYT-low patients. CONCLUSIONS: Overall, our data highlight the existence of distinct genomic features across cytolytic subgroups in skin melanoma, which might affect the patients' relapse rate or their acquisition of resistance to immune checkpoint inhibition therapies.

Bone marrow-derived mesenchymal stem cells inhibit CD8+ T cell immune responses via PD-1/PD-L1 pathway in multiple myeloma.

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8+ T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD-1 expression on CD8+ T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD-1 expression of CD4+ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8+ T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+ T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells to promote the immune escape of MM.

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process, which is partially reversed by benznidazole treatment.

BACKGROUND: Signs of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4+ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4+ T cells. METHODOLOGY/PRINCIPAL FINDINGS: The functional capacity of CD4+ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4+ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4+ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4+ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24-48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9-12 months after treatment, an increase in the CD4+ T cell subset coproducing three molecules, which were mainly granzyme B+, perforin+ and IFN-γ+ (1.4% versus 4.5%). CONCLUSIONS/SIGNIFICANCE: A CD4+ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9-12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4+ T cells.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

Granzyme B in Inflammatory Diseases: Apoptosis, Inflammation, Extracellular Matrix Remodeling, Epithelial-to-Mesenchymal Transition and Fibrosis.

Inflammation is strictly interconnected to anti-inflammatory mechanisms to maintain tissue homeostasis. The disruption of immune homeostasis can lead to acute and chronic inflammatory diseases, as cardiovascular, pulmonary, metabolic diseases and cancer. The knowledge of the mechanisms involved in the development and progression of these pathological conditions is important to find effective therapies. Granzyme B (GrB) is a serine protease produced by a variety of immune, non-immune and tumor cells. Apoptotic intracellular and multiple extracellular functions of GrB have been recently identified. Its capability of cleaving extracellular matrix (ECM) components, cytokines, cell receptors and clotting proteins, revealed GrB as a potential multifunctional pro-inflammatory molecule with the capability of contributing to the pathogenesis of different inflammatory conditions, including inflammaging, acute and chronic inflammatory diseases and cancer. Here we give an overview of recent data concerning GrB activity on multiple targets, potentially allowing this enzyme to regulate a wide range of crucial biological processes that play a role in the development, progression and/or severity of inflammatory diseases. We focus our attention on the promotion by GrB of perforin-dependent and perforin-independent (anoikis) apoptosis, inflammation derived by the activation of some cytokines belonging to the IL-1 cytokine family, ECM remodeling, epithelial-to-mesenchymal transition (EMT) and fibrosis. A greater comprehension of the pathophysiological consequences of GrB-mediated multiple activities may favor the design of new therapies aim to inhibit different inflammatory pathological conditions such as inflammaging and age-related diseases, EMT and organ fibrosis.

NK and T Cell Differentiation at the Maternal-Fetal Interface in Sows During Late Gestation.

The phenotype and function of immune cells that reside at the maternal-fetal interface in humans and mice have been, and still are, extensively studied with the aim to fully comprehend the complex immunology of pregnancy. In pigs, information regarding immune cell phenotypes is limited and mainly focused on early gestation whereas late gestation has not yet been investigated. We designed a unique methodology tailored to the porcine epitheliochorial placenta, which allowed us to address immune phenotypes separately in the maternal endometrium (ME) and fetal placenta (FP) by flow cytometry. In-depth phenotyping of NK cells, non-conventional and conventional T cells within maternal blood (mBld), ME, FP, and fetal spleen (fSpln) revealed major differences between these anatomic sites. In both maternal compartments, all NK cells were perforin+ and had NKp46-defined phenotypes indicative of late-stage differentiation. Likewise, T cells with a highly differentiated phenotype including CD2+CD8α+CD27dim/-perforin+ γδ T cells, CD27-perforin+ cytolytic T cells (CTLs), and T-bet+ CD4+CD8α+CD27- effector memory T (Tem) cells prevailed within these compartments. The presence of highly differentiated T cells was also reflected in the number of cells that had the capacity to produce IFN-γ. In the FP, we found NK cells and T cell populations with a naive phenotype including CD2+CD8α-CD27+perforin- γδ T cells, T-bet-CD4+CD8α-CD27+ T cells, and CD27+perforin- CTLs. However, also non-naive T cell phenotypes including CD2+CD8α+CD27+perforin- γδ T cells, T-bet+CD4+CD8α+CD27- Tem cells, and a substantial proportion of CD27-perforin+ CTLs resided within this anatomic site. Currently, the origin or the cues that steer the differentiation of these putative effector cells are unclear. In the fSpln, NKp46high NK cells and T cells with a naive phenotype prevailed. This study demonstrated that antigen-experienced immune cell phenotypes reside at the maternal-fetal interface, including the FP. Our methodology and our findings open avenues to study NK and T cell function over the course of gestation. In addition, this study lays a foundation to explore the interplay between immune cells and pathogens affecting swine reproduction.

Perforins Expression by Cutaneous Gamma Delta T Cells.

Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene MPEG1, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.

TIGIT presents earlier expression dynamic than PD-1 in activated CD8+ T cells and is upregulated in non-small cell lung cancer patients.

CD8+ T cells are considered a critical component of antitumor immunity. However, tumor-infiltrating CD8+ T cells may express more than one checkpoint molecules that have the potential to inhibit effector responses alone or cooperatively. Here, we focused on the expression dynamic of TIGIT and PD-1 in CD8+ T cells. TIGIT+ subset presented significantly higher PD-1 expression than TIGIT- subset in circulating CD8+ T cells. The expression dynamic of TIGIT and PD-1 was then tracked. In total CD8+ T cells, TIGIT mRNA increased more rapidly than PD-1 mRNA, and TIGIT+ CD8+ T cells upregulated PD-1 more rapidly than TIGIT- CD8+ T cells. Next, 24-h-stimulated CD8+ T cells were re-sorted into TIGIT+ and TIGIT- subsets, and the TIGIT+ cells that came from TIGIT- cells also presented significantly more rapid PD-1 induction than persistent TIGIT- CD8+ T cells. In non-small cell lung cancer (NSCLC) patients, the expression of PD-1 was more enriched in TIGIT+ cells than in TIGIT- cells in both circulating CD8+ T cells and tumor-infiltrating CD8+ T cells. Function analysis revealed that TIGIT+ CD8 T cells presented lower interferon-gamma, perforin 1, and granzyme B upregulation than TIGIT- CD8 T cells, especially in NSCLC patients. Overall, these data indicated that TIGIT presented earlier expression dynamic than PD-1 in activated CD8+ T cells and was upregulated in NSCLC patients.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1+  cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ T cells (TRM  cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM  cells by NK1.1+  cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM  cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

COVID-19 pneumonia: CD8+ T and NK cells are decreased in number but compensatory increased in cytotoxic potential.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is posing a huge threat to human health worldwide. We aim to investigate the immune status of CD8+ T and NK cells in COVID-19 patients. METHODS: The count and immune status of lymphocytes were detected by flow cytometry in 32 COVID-19 patients and 18 healthy individuals. RESULTS: As the disease progression in COVID-19 patients, CD8+ T and NK cells were significantly decreased in absolute number but highly activated. After patients' condition improved, the count and immune status of CD8+ T and NK cells restored to some extent. GrA+CD8+ T and perforin+ NK cells had good sensitivity and specificity for assisting diagnosis of COVID-19. CONCLUSIONS: As the disease progression, the declined lymphocytes in COVID-19 patients might lead to compensatory activation of CD8+ T and NK cells. GrA+CD8+ T and perforin+ NK cells might be used as meaningful indicators for assisting diagnosis of COVID-19.