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The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16- NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

Allogeneic CD8+ cytotoxic T cells play an essential role in rejecting transplanted allografts, but how their effector function is regulated on a transcriptional level remains unclear. Herein, we investigate the role of interferon regulatory factor 4 (IRF4) in controlling CD8+ T-cell function in response to transplant. B6.Rag1-/- mice were adoptively transferred with CD8+ T cells isolated from either Irf4fl/fl Cd4-Cre (T-cell-specific Irf4-deficient) or Irf4fl/fl control mice, followed by BALB/c skin transplantation. Recipients that received Irf4-deficient CD8+ T cells permanently accepted the skin allografts, whereas recipients that received control CD8+ T cells acutely rejected the transplanted skins. Mechanistically, compared with the transferred control CD8+ T cells in B6.Rag1-/- recipients, the transferred Irf4-deficient CD8+ T cells lost the capacity to differentiate into CD127- KLRG1+ terminal effector cells, barely produced effector cytokines and cytotoxic molecules (e.g. IL-2, IFN-γ, TNF-α, granzyme A and granzyme B), and displayed defect in proliferative capacity, evident by their decreased Ki67 expression and lower frequencies. Moreover, the transferred Irf4-deficient CD8+ T cells displayed low expression of transcription factors ID2 and T-bet that govern the terminal effector T-cell programmes, and high expression of transcription factor TCF1 that maintains the naive-memory T-cell programmes. Hence, IRF4 deficiency in CD8+ T cells abrogates their terminal effector differentiation and promotes transplant acceptance. These findings suggest that targeting IRF4 expression represents an attractive and promising therapeutic approach for inducing transplant acceptance.

To determine the levels of granzyme A in amniotic fluid in pregnancies complicated by preterm prelabor rupture of membranes (PPROM), based on the presence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI).A total of 166 women with singleton pregnancies complicated by PPROM were included. Amniocentesis was performed at the time of admission and assessments of MIAC (using both cultivation and non-cultivation techniques) and IAI (interleukin-6 in amniotic fluid) were performed on all subjects. Based on the presence/absence of MIAC and IAI, the women were further divided into the following subgroups: intra-amniotic infection, sterile IAI, colonization, and absence of both MIAC and IAI. Amniotic fluid granzyme A levels were assessed using ELISA.Women with MIAC had lower levels of granzyme A in the amniotic fluid than women without this condition (with MIAC: median 15.9 pg/mL vs. without MIAC: median 19.9 pg/mL,The presence of sterile IAI was associated with elevated levels of granzyme A in amniotic fluid.

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.

NK cells play an important role in immune regulation and defense of infection, but their characteristics in patients with decompensated cirrhosis and their relationship with liver function remain unclear. We studied the functional properties of NK cells (including CD56dim NK and CD56bright NK cells) in patients with HBV-related decompensated liver cirrhosis (HBV-DLC) and analyzed their relationship with decompensation of liver function. Thirty patients with HBV-DLC and 25 patients with HBV-related compensated liver cirrhosis (HBV-CLC) were recruited in this study. Twenty five age- and sex-matched healthy individuals were recruited as healthy controls (HCs). The phenotypical and functional characteristics of NK cell subsets were detected by flow cytometry, and the correlation between NK cells and decompensation of liver function was analyzed. The frequency of circulating CD56bright NK cells was significantly increased while circulating CD56dim NK cells was significantly decreased in HBV-DLC patients as compared with HCs and HBV-CLC patients. Peripheral activated-CD56bright NK cells from HBV-DLC patients might express lower levels of inhibitory receptor CD158b1/2 and higher levels of activating receptor NKG2D and their expression of perforin and granzyme A/B also increased significantly compared with HCs, suggesting a high immune activation status of peripheral CD56bright NK cells in HBV-DLC patients. In HBV-DLC patients, the expression of CD107a and perforin in circulating CD56dim NK cells was positively correlated with cytolytic capacity while CD107a and perforin expression in circulating CD56dim NK cells were significantly decreased, suggesting an impaired cytolytic capacity of circulating CD56dim NK cells. Besides, we found that the perforin expression of circulating CD56dim NK cells correlated negatively with child-pugh classification in HBV-DLC patients. The functional properties of circulating NK cell subsets in HBV-DLC patients have changed significantly, especially of CD56dim NK cells which closely related to decompensation of liver function. These findings may help provide new perspectives and theoretical basis for the treatment of patients with HBV-DLC.

Objective: The present study aimed to determine whether granzymes are implicated in the pathogenesis of infection-associated acute encephalopathy (AE). Methods: We investigated granzyme and cytokine levels in the cerebrospinal fluid of patients with acute encephalopathy or complex febrile seizures (cFS). A total of 24 acute encephalopathy patients and 22 complex febrile seizures patients were included in the present study. Levels of granzymes A and B were measured using enzyme-linked immunosorbent assay, and levels of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin 1β (IL-1β), IL-1 receptor antagonist (IL-1RA), IL-4, IL-6, IL-8, and IL-10 were assessed using the Bio-Plex suspension array system. Results: Cerebrospinal fluid levels of granzyme A were significantly higher, and those of TNF-α and IL-1RA were significantly lower in the AE group than in the cFS group; however, no significant differences in the levels of granzyme B, IFN-γ, IL-1β, IL-4, IL-6, IL-8, and IL-10 were observed between the 2 groups. In addition, no significant differences in granzyme A, granzyme B, or cytokine levels were observed between acute encephalopathy patients with and those without neurologic sequelae. Conclusions: Our findings indicate the involvement of granzyme A in the pathogenesis of acute encephalopathy.

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.

Introduction Whether the efficacy of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors declines with senescence remains controversial for lung adenocarcinoma (LUAD). Responsiveness to anti–PD-1/PD-L1 therapy is thought to rely on neoantigen exposure and immune elements in the tumor microenvironment. In this study, we explored the features of the tumor immune microenvironment in elderly patients with treatment-naive LUAD. Methods Transcriptome profiles and clinical characteristics of patients with LUAD were retrieved from The Cancer Genome Atlas as a discovery cohort. Immune cell infiltration (quantified by a single-sample gene set enrichment analysis), immunoregulatory molecule expression, and mutational patterns (from The Cancer Immunome Atlas) were compared between young and elderly patients. Immune cell infiltration was verified by immunohistochemistry using a validation cohort including 105 treatment-naive patients with LUAD. A tissue microarray consisting of 120 LUAD patients was used in the immunohistochemistry validation. Results Activated CD8+ T cell numbers increased slightly with age, but cytolytic molecules in T cells (granzyme B [GZMB], perforin 1 [PRF1], granzyme A [GZMA], granzyme M [GZMM], and granulysin [GNLY]) gradually declined. PD-L1 expression was not associated with age; however, a number of immunosuppressive elements beyond PD-L1 were upregulated in aging patients, including regulatory T cells and co-inhibitory molecules, for example, TIM-3, TIGIT, and HHLA2. Finally, senescence was accompanied by a loss of clonal neoantigens, which is believed to be correlated with responsiveness to immune checkpoint inhibitors. Conclusions Elderly patients were characterized by increased numbers of CD8+ T cells and impaired cytolytic molecule expression. The observed immune signature was also associated with a loss of clonal neoantigens and the accumulation of immunosuppressive elements. These findings show a unique immune microenvironment in senescence and support biomarker-guided candidate identification for anti–PD-1/PD-L1 therapeutic strategies for elderly patients with LUAD.

Mycobacterium tuberculosis(Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γ9δ2T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion ofGZMAfrom γ9δ2T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA+ ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm+ cells skewed toward a GzmA- CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1.

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.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Cavitation is a major pathological feature of pulmonary tuberculosis (TB). The study is aimed at investigating the mechanism of natural killer (NK) cells participating the cavity formation during Mycobacterium tuberculosis (MTB) infection. Human peripheral blood samples were donated by pulmonary TB patients with cavity or not. Real-time quantitative PCR and enzyme-linked immunosorbent assay were performed to analyze the expression of cytokines secreted by NK cells. And the cytotoxicity of NK cells was compared between two groups. Our data showed that NK cells were more abundant in cohorts of cavity. Increased abundance of granzyme A and granzyme B was observed in culture supernatants of NK cells isolated from cavitary TB patients, which also resulted in a higher level of nonviable MTB-infected monocytes. Our data firstly demonstrates that NK cells participate in cavity formation in pulmonary TB patients. The elevated level and increased cytotoxicity of NK cells accelerate the cavitary formulation.

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmA’s physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmA’s bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

Aims: Recent in vitro findings suggest that the serine protease Granzyme K (GzmK) may act as a proinflammatory mediator. However, its role in sepsis is unknown. Here we aim to understand the role of GzmK in a mouse model of bacterial sepsis and compare it to the biological relevance of Granzyme A (GzmA). Methods: Sepsis was induced in WT, GzmA-/- and GzmK-/- mice by an intraperitoneal injection of 2x108 CFU from E. coli. Mouse survival was monitored during 5 days. Levels of IL-1α, IL-1β, TNFα and IL-6 in plasma were measured and bacterial load in blood, liver and spleen was analyzed. Finally, profile of cellular expression of GzmA and GzmK was analyzed by FACS. Results: GzmA and GzmK are not involved in the control of bacterial infection. However, GzmA and GzmK deficient mice showed a lower sepsis score in comparison with WT mice, although only GzmA deficient mice exhibited increased survival. GzmA deficient mice also showed reduced expression of some proinflammatory cytokines like IL1-α, IL-β and IL-6. A similar result was found when extracellular GzmA was therapeutically inhibited in WT mice using serpinb6b, which improved survival and reduced IL-6 expression. Mechanistically, active extracellular GzmA induces the production of IL-6 in macrophages by a mechanism dependent on TLR4 and MyD88. Conclusions: These results suggest that although both proteases contribute to the clinical signs of E. coli-induced sepsis, inhibition of GzmA is sufficient to reduce inflammation and improve survival irrespectively of the presence of other inflammatory granzymes, like GzmK.

3 figuras.-- Póster presentado en el 42º Congreso SEI, Congreso de la Sociedad Española de Inmunología, formato virtual, 24-26 marzo de 2021, Madrid., Peritonitis is one of the most common causes of sepsis. Evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as proinflammatory mediator and could play an important role in the pathogenesis of sepsis. Here, we aim to analyze the role and therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis.

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell–based cancer immunotherapy.

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.

Theileria annulata is a tick‐transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumours that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria‐induced leukocyte transformation. Dataset comparisons highlighted a small set of genes associated with Theileria‐transformed leukocyte dissemination. The roles of Granzyme A (GZMA) and RAS guanyl‐releasing protein 1 (RASGRP1) were verified by CRISPR/Cas9‐mediated knockdown. Knocking down expression of GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in Rag2/γC mice confirming their role as dissemination suppressors in vivo. We further evaluated the roles of GZMA and RASGRP1 in human B lymphomas by comparing the transcriptome of 934 human cancer cell lines to that of Theileria‐transformed bovine host cells. We confirmed dampened dissemination potential of human B lymphomas that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and RASGRP1 have a novel tumour suppressor function in both T. annulata‐infected bovine host leukocytes and in human B lymphomas., Summarising the main findings of this study that identified new players in dissemination and oxidative stress regulation of Theileria‐transformed leukocytes and provided evidence for similar roles for GZMA and RASGRP1 in transcriptionally matched human B lymphoma cell lines.

Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8+ T cell subsets. PD-1-expressing CD8+ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development.

SARS-CoV-2 infection induces a T cell response that most likely contributes to virus control in COVID-19 patients, but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients.Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B, as well as perforin within different effector CD8+ T cell subsets. PD-1 expressing CD8+ T cells also produced cytotoxic molecules during acute infection indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2.Our data provides valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development.ImportanceCytotoxic T cells are responsible for the elimination of infected cells and are key players for the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group in comparison to younger patients.

Purpose of review New insights into IgG4-related disease (IgG4-RD) have recently been obtained. A better understanding of the mechanisms underlying this disease is important for identification of therapeutic targets, which will lead to the development of specific strategies for treatment. Recent findings Infiltration of activated T follicular helper (Tfh) cells is observed in affected tissues of IgG4-RD. Such Tfh cells have a greater capacity than tonsillar Tfh cells to help B cells produce IgG4. Circulating PD-1hiCXCR5- peripheral T helper (Tph)-like cells are also increased in patients with IgG4-RD. Because Tph-like cells express high levels of chemokine receptors and granzyme A, they have the capacity to infiltrate affected tissues and exert a cytotoxic function. Tph-like cells can also produce CXCL13, and CXCR5+ Tfh cells and B cells are therefore preferentially recruited to form ectopic lymphoid structures in the sites. Tph cells may have a role to ignite inflammation and maintain persistent fibroinflammation in collaboration with Tfh cells in lesions of IgG4-RD. Summary Recent advances in understanding the pathogenesis of IgG4-RD are remarkable. In this review, we summarize and discuss the possible pathologic role of CD4+ T-cell subsets in IgG4-RD.

Clinical evaluation methods for therapeutic effectiveness based on scientific evidence are desired as tools for contributing to therapeutic strategies and advancements in cancer immunotherapy research. By quantitatively measuring multiple immune response factors responsible for T cell activity and cytotoxic activity, we can be expected to evaluate a patient's immune response. We used plasma samples of human peripheral blood and compared the T cell and antitumor immune response by measuring various immune related cytokines (TNFα, sCD137, IL-2, IL-5, INFɤ, Perforin, Granzyme A, Granzyme B, IL-10, IL-4). In this pilot study, we compared samples from healthy donors with those from cancer patients treated with multivalent dendritic cell (DC) vaccines and natural killer (NK) cell immunotherapy before and after treatment. We examined the usefulness of this method as a biomarker to assess the therapeutic effectiveness of immune cell therapy by statistical analysis and by plotting a cancer immunogram correlating patient data with clinical outcomes.

Dysregulated inflammation is implicated in the pathobiology of aging, yet platelet–leukocyte interactions and downstream cytokine synthesis in aging remains poorly understood. Platelets and monocytes were isolated from healthy younger (age

Simple SummaryCD8(+) T cells are prominent decidual cells in the third trimester of healthy human pregnancy. They have a cytotoxic capacity which may control invasion of extravillous trophoblast and therefore affect placentation and play the role in development of preeclampsia. In this study, we examined the expression of CD8(+) T cells in decidual tissue and peripheral blood of women with severe and mild preeclampsia in comparison to gestational age-matched healthy pregnancies. Additionally, the expression of cytotoxic proteins in CD8(+) T cells was examined in order to specify their subpopulations.In our study, we aimed to establish expression of cytotoxic CD8(+) T cells in the decidua basalis and the maternal peripheral blood (mPBL) of severe and mild preeclampsia (PE) and compare to healthy pregnancies. Decidual tissue and mPBL of 10 women with mild PE, 10 women with severe PE, and 20 age- matched healthy pregnancy controls were analyzed by double immunofluorescence and qPCR, respectively. By double immunofluorescence staining, we found a decreased total number of cells/mm(2) in decidua basalis of granulysin (GNLY)(+) (p < 0.0001), granzyme B (GzB)(+)(p < 0.0001), GzB(+)CD8(+)(p < 0.0001), perforin (PRF1) (+) (p < 0.0001), and PRF1(+)CD8(+) (p < 0.01) in the severe PE compared to control group. Additionally, we noticed the trend of lower mRNA expression for GNLY, granzyme A (GZMA), GzB, and PRF1 in CD8(+) T cells of mPBL in mild and severe PE, with the latter marker statistically decreased in severe PE (p < 0.001). Forkhead box P3 (FOXP3) mRNA in CD8(+) T cells mPBL was increased in mild PE (p < 0.001) compared to controls. In conclusion, severe PE is characterized by altered expression of cytotoxic CD8(+) T cells in decidua and mPBL, suggesting their role in pathophysiology of PE and fetal-maternal immune tolerance.

Summary Objective Natural killer (NK) cells have been involved in the pathology of different inflammatory and autoimmune disorders. Inflammation is an important regulator of osteoarthritis (OA), but the molecular and cellular mechanisms regulating this process are not well defined. Design To understand the role of NK cells in OA, we have compared the phenotype (CD56 subsets and perforin and granzyme expression) and cytotoxic function of NK cells in peripheral blood and synovial fluid from patients with OA undergoing total knee arthroplasty. Results In contrast to peripheral blood lymphocytes (PBLs), the majority of NK cells from the synovial fluid were CD56 bright CD16(−) cells. As expected the expression of the cytolytic mediators perforin and granzyme B in CD56 bright CD16(−) cells was low and correlated with a poor cytotoxic potential against K562 sensitive target cells. Surprisingly, this low cytotoxic NK cell subset expressed high levels of granzyme A (a protease recently characterized as a key modulator of inflammation in mouse models) in synovial fluid but not in peripheral blood. The presence of the CD56(+) bright CD16(−) cells expressing granzyme A correlated with increased levels of pro-inflammatory cytokines in synovial fluid from OA patients. Conclusion Our results indicate that NK cells from the synovium of patients with OA, which present an immunoregulatory non-cytotoxic phenotype, show different phenotype comparing with NK cells from peripheral blood, especially expressing granzyme A, a pro-inflammatory molecule which may contribute to the establishment of chronic articular inflammation in this type of patients.

B7-H4, another member of costimulatory molecule, has been shown to be overexpressed in multiple types of tumors, including hepatocellular carcinoma (HCC). However, the specific biological role of B7-H4 in HCC still needs to be further explored. In this study, we observed that B7-H4 was highly overexpressed in HCC tissues and cells, and its overexpression strongly correlated with patient's TNM stage, overall survival and early recurrence. Downregulation of B7-H4 significantly suppressed cell growth, invasion, and stemness of HCC by inducing apoptosis in the in vitro experiment. In addition, depletion of B7-H4 could help restore CD8+ T anti-tumor immunity by elevating the expression and secretion levels of CD107a, granzyme A, granzyme B, perforin and IFN-γ. In a xenografted mouse model of HCC, stable depletion of B7-H4 resulted in significantly smaller mean tumor volume and less mean tumor weight after 30 days of growth, compared to the control group. Together, our results provide insights into the diverse functions of B7-H4 involved in the pathogenesis, recurrence and anti-tumor immunity of HCC, indicating B7-H4 as a novel and effective approach for future treatment strategies that benefits anticancer therapy.

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A–deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.

Cytotoxic T lymphocytes and natural killer cells eliminate infected cells from the organism by triggering programmed cell death (apoptosis). The contents of the lytic granules of killer cells, including pore-forming proteins perforins and proteolytic enzymes granzymes, are released with the following penetration of the released proteins into the target cells. Granzyme B initiates mitochondria-dependent apoptosis via (i) proapoptotic Bid protein, (ii) Mcl-1 and Bim proteins, or (iii) p53 protein. As a result, cytochrome c is released from the mitochondria into the cytoplasm, causing formation of apoptosomes that initiate the proteolytic cascade of caspase activation. Granzymes M, H, and F cause cell death accompanied by the cytochrome c release from the mitochondria. Granzyme A induces generation of reactive oxygen species (ROS), which promotes translocation of the endoplasmic reticulum-associated SET complex to the nucleus where it is cleaved by granzyme A, leading to the activation of nucleases that catalyze single-strand DNA breaks. Granzymes A and B penetrate into the mitochondria and cleave subunits of the respiratory chain complex I. One of the complex I subunits is also a target for caspase-3. Granzyme-dependent damage to complex I leads to the ROS generation and cell death.

Summary Tumor necrosis factor (TNF)-α is largely regarded as a proinflammatory cytokine, but several recent researches have demonstrated that TNF-α could possess immunoregulatory roles with potential to suppress anti-tumor immunity. Chronic hepatitis B virus (HBV) infection is a major risk factor of hepatocellular carcinoma (HCC), and HBV-specific CD8 T cells could exert anti-tumor roles in HCC patients. Here, we found that HBV-specific CD8 T cells, both in the peripheral blood and in the tumor microenvironment, were more enriched with TNF-α-expressing cells than interferon (IFN)-γ-expressing cells. Compared to IFN-γ-expressing HBV-specific CD8 T cells, TNF-α-expressing HBV-specific CD8 T cells presented lower expression of inhibitory checkpoint molecules, including programmed cell death (PD)-1, T cell immunoglobulin mucin-3 (TIM-3) and cytotoxic T lymphocyte antigen (CTLA)-4. HBV-specific CD8 T cells could mediate the lysis of autologous primary tumor cells, and the inhibition of TNF-α could further elevate their cytotoxic capacity. Subsequently, we demonstrated that TNF-α inhibition in HBV-specific CD8 T cells could significantly increase granzyme B (GZMB) and perforin 1 (PRF1) expression while having no effect towards granzyme A (GZMA) expression. The addition of exogenous TNF-α at low levels had no consistent effect on the expression of GZMA, GZMB and PRF1, but at higher levels, exogenous TNF-α significantly reduced GZMA, GZMB and PRF1 expression. Overall, these results suggested that TNF-α-expressing cells probably presented a deleterious role in HCC but were enriched in HBV-specific CD8 T cells.

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4+ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD., A subset of granzyme A–producing CD4+ T cells accumulate in the intestines of mice and mediate the development of graft-versus-host disease after hematopoietic cell transplantation.

Forest therapy is a fast-growing treatment approach, as it has the potential to alleviate stressful life events and to improve psychological well-being and physical health. Bamboo forests are widespread in southwestern China. Nevertheless, a knowledge gap on the specific health benefits of bamboo forest (BF) therapy still exists. To explore the psycho-physiologic responses of participants to the effects of BF therapy, 60 male adults aged between 19 and 24, with similar healthy conditions, were selected to participate in this study. A one-group pretest&ndash, posttest design was used for the BF sites and the city site (CS) to compare the difference in the psycho-physiologic responses of participants before and after the test. Participants at the BF sites participated in a three-day bamboo forest therapy session, and those at the CS participated in a three-day urban program. Blood pressure, heart rate, and peripheral oxygen saturation were measured as the physical signs, and the profile of mood state (POMS) questionnaire was completed by the participants for the psychological evaluation. Blood was sampled, and natural killer (NK) activity, the number of NK cells, and the levels of corticosterone, granulysin, perforin, and granzyme A/B in peripheral blood lymphocytes (PBLs) were measured. All the measurements mentioned above were performed at 08:00 on the first and fourth days within the test. Results indicated that the three-day BF therapy was capable of enhancing positive mood states and also reducing negative mood states in the male participants. The blood pressure and heart rates of the male participants decreased, while the peripheral oxygen saturation increased after the three-day BF therapy session. Furthermore, BF therapy significantly increased NK activity and the number of NK cells and perforin-, granulysin-, and granzyme A/B-expressing cells and significantly decreased the corticosterone level in PBLs in the male participants. The three-day BF therapy session improved the psychological and physiological well-being and enhanced the immune functions of the male college students.

8 figures, 1 table.-- Electronic supplementary information available, If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC., This work was supported in part by FEDER/Gobierno de Aragón (group B29), Ministerio de Economia y Competitividad (SAF2014-54763-C2-1 and SAF2017-83120-C2-1-R to J.P. and SAF2014-54763-C2-2-R to E.M.G.), and Instituto de Salud Carlos III (PI13/00864 to L.M.-L.). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (to L.S. and M.A.A.), Gobierno de Aragon (to I.U.-M., L.C., L.S., and P.J.-S.), and FPU/Ministerio de Educación, Cultura y Deportes (to P.M.L.). I.U.-M. was supported by Fondo Garantía Empleo Juvenil/INAEM. M.A.A. has a Juan de la Cierva contract (Ministerio de Ciencia, Innovación y Universidades). J.P. was supported by Fundación Aragon I+D (ARAID).

Oral presentation made at the Serpins2019 Conference, held in Sevilla (Spain), 19th – 22nd September 2019.

Infectious laryngotracheitis (ILT) is an acute respiratory disease of poultry caused by infectious laryngotracheitis virus (ILTV). Control of the disease with live attenuated vaccines administered via eye drop build upon immune responses generated by the eye-associated lymphoid tissues. The aim of this study was to assess cytokine and lymphocyte changes in the conjunctiva-associated lymphoid tissues (CALT) and Harderian gland (HG) stimulated by the ocular inoculation of the ILTV chicken embryo origin (CEO) vaccine strain and virulent strain 63140. This study offers strong evidence to support the roles that the CALT and HG play in the development of protective ILTV immune responses. It supports the premise that ILTV-mediated immunomodulation favors the B cell response over those of T cells. Further, it provides evidence that expansions of CD8&alpha, + cells, with the concomitant expression of the Granzyme A gene, are key to reducing viral genomes in the CALT and halting ILTV cytolytic replication in the conjunctiva. Ultimately, this study revealed that the early upregulation of interleukin (IL)-12p40 and Interferon (IFN)-&gamma, cytokine genes, which shape the antigen-specific cell-mediated immune responses, retarded the decline of virus replication, and enhanced the development of lesions in the conjunctiva epithelium.

Mucosal leishmaniasis (ML) is characterized by high production of inflammatory cytokines. Administration of pentoxifylline (PTX), an inhibitor of TNF-alpha, with pentavalent antimony (Sb(v)), has been successfully used as alternative treatment for refractory ML. Our study aims to investigate the in situ cellular response underlying the effectiveness of this therapy, by evaluating the intensity of the inflammatory infiltrate, cellular composition, and expression of cytokines and granzyme A in lesions from ML before and after treatment with Sb(v) alone or in combination with PTX. Our data showed no differences in the intensity of inflammatory infiltrate comparing before and after treatment, and comparing between different treatments. However, although the number and frequency of CD4(+) and CD8(+) cells were not different before and after treatments or comparing different treatments, frequency of CD68(+) cells decreased after treatment with Sb(v) + PTX, but not with Sb(v). This was due to a reduction in CD68(+) TNF-alpha(+) and not in CD68(+) IL-10(+) cells. The frequency of TNF-alpha(+) cells was correlated with the intensity of the inflammatory infiltrate before treatment, but this correlation was lost after treatment with Sb(v) + PTX. Although the total expression of granzyme A did not significantly change after treatments, a clear trend of decrease was observed after treatment with Sb(v) + PTX. Interestingly, patients who took longer to heal, regardless of the treatment, displayed a higher frequency of granzyme A(+) cells. Our data suggest that treatment with Sb(v) + PTX acts in CD68(+) cells reducing the expression of TNF-alpha but not IL-10, resulting in more efficient modulation of the inflammatory response, accelerating the healing process.

Identification and characterization of CD8+ and CD4+ T-cell epitopes elicited by HIV therapeutic vaccination is key for elucidating the nature of protective cellular responses and mechanism of the immune evasion of HIV. Here, we report the characterization of HIV-specific T-cell responses in cART (combination antiretroviral therapy) treated HIV-1 infected patients after vaccination with ex vivo-generated IFNα Dendritic Cells (DCs) loaded with LIPO-5 (HIV-1 Nef 66–97, Nef 116–145, Gag 17–35, Gag 253–284 and Pol 325–355 lipopeptides). Vaccination induced and/or expanded HIV-specific CD8+ T cells producing IFNγ, perforin, granzyme A and granzyme B, and also CD4+ T cells secreting IFNγ, IL-2 and IL-13. These responses were directed against dominant and subdominant epitopes representing all vaccine regions; Gag, Pol and Nef. Interestingly, IL-2 and IL-13 produced by CD4+ T cells were negatively correlated with the peak of viral replication following analytic treatment interruption (ATI). Epitope mapping confirmed that vaccination elicited responses against predicted T-cell epitopes, but also allowed to identify a set of 8 new HIV-1 HLA-DR-restricted CD4+ T-cell epitopes. These results may help to better design future DC therapeutic vaccines and underscore the role of vaccine-elicited CD4+ T-cell responses to achieve control of HIV replication., Author summary Improvement of therapeutic vaccine strategies in the perspective of HIV cure is warranted and one of the determinant factors for elucidating the nature of protective cellular responses is the identification and characterization of CD8+ and CD4+ T-cell epitopes elicited by the vaccine. However, fine characterization of vaccine response at the level of individual peptides contained in therapeutic vaccines is lacking. Here, we report in-depth characterization of HIV-specific T-cell responses observed in antiretroviral therapy treated HIV-1 infected patients after vaccination with autologous ex vivo-generated IFNα DCs loaded with 5 long HIV peptides coupled to a lipid tail. Our study shows that dominant and subdominant epitopes from Gag, Pol, and Nef vaccine regions are targeted by HIV-specific CD8+ T and CD4+ T cells and that vaccine-elicited HIV-specific CD4+ T cells producing IL-2 and IL-13 are significantly correlated with a better viral control following treatment interruption. These results underscore the role of vaccine-elicited CD4+ T-cell responses to achieve control of HIV replication and thus may inform the design of better therapeutic vaccines.

Aims CD8+ T cells are important in mediating protective responses to intracellular pathogens. However, an uncontrolled response may lead to pathology. The role of CD8+ T cells in different clinical manifestations of human leishmaniasis is controversial and poorly understood. We aim to study the response of CD8+ T cells to the first exposure to different strains of Leishmania, seeking to correlate these findings with clinical manifestations of disease. Methods and results We have evaluated the expression of granzyme A, inflammatory and anti-inflammatory cytokines, as well as CTLA-4 by human naive CD8+ T cells exposed to Leishmania braziliensis and two different strains of Leishmania infantum in vitro. We observed that while exposure to L braziliensis induced an inflammatory profile, as measured by the expression of granzyme A, IFN-gamma and IL-17, as well as a higher IFN/IL-10 ratio, exposure to L infantum led to a regulatory profile, as measured by lower IFN/IL-10 ratio and higher expression of CTLA-4. Conclusion These results may help explain why patients with the visceral clinical form present a weaker cellular response and, consequently, a worse outcome of the disease. The use of CTLA-4 checkpoint inhibitors may emerge as a potential immunotherapy to ameliorate the immune response in visceral leishmaniasis patients.

Background IgG 4 -related disease (IgG 4 -RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4 + T cells constitute the major inflammatory cell population in IgG 4 -RD lesions. Objective We used an unbiased approach to characterize CD4 + T-cell subsets in patients with IgG 4 -RD based on their clonal expansion and ability to infiltrate affected tissue sites. Methods We used flow cytometry to identify CD4 + effector/memory T cells in a cohort of 101 patients with IgG 4 -RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4 + SLAMF7 + cytotoxic T lymphocytes (CTLs) and CD4 + GATA3 + T H 2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging. Results CD4 + effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG 4 -RD. Next-generation sequencing revealed prominent clonal expansions of these CD4 + CTLs but not CD4 + GATA3 + memory T H 2 cells in patients with IgG 4 -RD. The dominant T cells infiltrating a range of inflamed IgG 4 -RD tissue sites were clonally expanded CD4 + CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4 + CTLs. Conclusions IgG 4 -RD is prominently linked to clonally expanded IL-1β– and TGF-β1–secreting CD4 + CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4 + T cells are now linked to a human disease characterized by chronic inflammation and fibrosis.

ObjectivesIgG4-related disease (IgG4-RD) is a chronic, systemic, inflammatory condition of unknown aetiology. We have recently described clonally expanded circulating CD4+ cytotoxic T lymphocytes (CTLs) in IgG4-RD that infiltrate affected tissues where they secrete interleukin (IL)-1β and transforming growth factor -β1 (TGF-β1). In this study, we sought to examine the role of CD4+ CTLs in the pathogenesis of IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) and to determine whether these cells secrete interferon-gamma (IFN-γ) at lesional sites.MethodsSalivary glands of 25 patients with IgG4-DS, 22 patients with Sjögren's syndrome (SS), 12 patients with chronic sialoadenitis (CS) and 12 healthy controls were analysed in this study. Gene expression analysis was performed on submandibular glands (SMGs) from five patients with IgG4-DS, three with CS and three healthy controls. Infiltrating CD4+ CTLs were examined by quantitative multicolour imaging in tissue samples from 20 patients with IgG4-DS, 22 patients with SS, 9 patients with CS and 9 healthy controls.ResultsIn IgG4-DS tissues, nine genes associated with CD4+ CTLs were overexpressed. The expression of granzyme A (GZMA) mRNA was significantly higher in samples from patients with IgG4-RD compared with corresponding tissues from SS and healthy controls. Quantitative imaging showed that infiltrating CD4+ GZMA+ CTLs were more abundant in patients with IgG4-DS than in the other groups. The ratio of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS correlated with serum IgG4 concentrations and the number of affected organs. A large fraction of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS secreted IFN-γ.ConclusionsThe pathogenesis of IgG4-DS is associated with tissue infiltration by CD4+GZMA+ CTLs that secrete IFN-γ.

Purpose: Malignant gliomas are the most common tumors in the central nervous system with a poor prognosis. Recently, CD4+ cytotoxic T cells (CTLs) are being increasingly recognized as possessing antitumor capacity. However, their presence, activity and regulation in glioma have not been investigated in detail. Methods: To examine this, 72 grade II and grade III Han Chinese glioma patients and 30 Han Chinese healthy controls were investigated. Results: We found that compared to healthy controls, glioma patients had significantly upregulated frequencies of circulating CD4+ CTLs, identified by the expression of granzyme A (GzmA), granzyme B (GzmB) and/or perforin. The stimulated CD4+ CTLs in grade II and grade III glioma patients also had less proliferative ability than those in healthy controls, a feature of suppression that progressed with tumor grade. The frequencies of GzmB-expressing circulating CD4+ CTLs were directly associated with prognosis. We hypothesized that the programed death 1 (PD-1)/PD-ligan...

Two major unanswered questions related to the pathogenesis of type 1 diabetes (T1D) prevent progress in our ability to intervene in disease development: What breaks immune tolerance to the pancreatic β-cells? And what kills the β-cells? By the surprising demonstration that knockout of the tryptase granzyme A (GrzmA), conventionally considered to be a key proapoptotic mediator of immune killer cell cytotoxicity, does not protect but markedly accelerates and increases incidence of T1D in the nonobese diabetic (NOD) mouse, Mollah et al. (1) add to answering both of these questions. On one hand, they show that GrzmA does not contribute to β-cell killing. On the other hand, their striking finding that diabetes resistance in the proinsulin II transgenic mouse is broken by GrzmA knockout provides evidence of an important role of GrzmA for maintenance of peripheral tolerance. By answering “No” to the question as to whether GrzmA is important in β-cell killing, this study adds to reports showing that granzyme B (GrzmB) (2), Fas (3,4), and perforin (5,6) are dispensable in β-cell killing in the NOD mouse. These observations challenge the view that β-cell destruction in T1D is caused by classic effector T-cell mechanisms and support the notion that these cells do ensure antigen-specific homing and reactivity but then interact with innate immune cells to orchestrate an intraislet inflammatory reaction leading to β-cell killing, the selectivity of which is in part determined by differentiation-dependent sensitivity of β-cells to cytotoxic inflammatory mediators (7). The “Yes” to the question as to whether GrzmA contributes to maintain peripheral tolerance is perhaps …

Simple Summary Cytotoxic T lymphocytes kill cancer or virally infected cells by exocytosis of lytic granules. This leads to perforin-mediated granzyme entry into the target cell, consequently killing the target cell. Granzymes and perforin are activated by cysteine cathepsins whose activity is regulated by the protein inhibitor cystatin F. Since cystatin F can be secreted by a range of cancer and immune cells in tumour microenvironments, we here investigated whether extracellular cystatin F can be taken up by and affect the function of cytotoxic T lymphocytes. We demonstrated cystatin F uptake into cytotoxic T lymphocytes, down-regulation of target peptidases, and reduced target cell killing. Overall, our results indicate that cystatin F is an important mediator that can impair the killing efficiency of cytotoxic T lymphocytes and thus suggest that it is a possible target for cancer immunotherapy. Abstract Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function.

While immune checkpoint inhibitor (CPI) therapies have revolutionized the treatment of metastatic melanoma and other malignancies, many patients fail to respond, necessitating to explore combination therapies to improve response rates. Histone deacetylases (HDACs) are epigenetic modifying proteins that remove acetyl groups from histones and non-histone proteins. HDAC inhibitors (HDACi) have shown success in the treatment of several hematologic malignancies. Beyond direct tumor cytotoxicity, HDACi have also demonstrated immunomodulatory effects. We previously published data showing the anti-tumor efficacy of the class I/IV HDACi mocetinostat in pre-clinical murine models through upregulation of tumor antigen presentation machinery and downregulation of Tregs. As a result of these and other data, a phase Iβ clinical trial (NCT03565406) was initiated to evaluate combination of mocetinostat with nivolumab and ipilimumab for the treatment of unresectable stage III/IV metastatic melanoma. A total of 10 patients were treated. Objective responses by RECIST 1.1 criteria were seen in 7/10 patients; 2 patients had complete responses, 5 had partial responses and 3 had progressive disease. To further understand the impact of mocetinostat on immune function, we evaluated serum and peripheral blood (PBMC) samples from patients prior to and on-treatment, in addition to in vitro treatment of treatment-naive patient PBMC samples with mocetinostat. Both in vitro treated and in vivo paired patient samples demonstrated a decrease in M2-like monocytes (CD80-CD86-CD163+CD206+) and an increase in M1-like monocytes (CD80+CD86+CD163-CD206-). We also observed a decrease in myeloid derived suppressor cells (MDSC, CD14+CD11b+HLA-DRlow) and expression of NOS2, an enzyme involved in MDSC mediated T-cell suppression, in this subset in both treated patients in vivo and in vitro. CD4+ and CD8+ T-cells showed increased levels of the proliferation marker Ki67 on-treatment relative to baseline in vivo in treated patients. Analysis of patient serum levels showed an increase in Granzyme A, Granzyme B, IFNγ and TNF. Similar upregulation of inflammatory and cytolytic proteins were seen in vitro. Assessment of histone 3 acetylation levels showed robust increases in all patients assessed on-treatment except 1 patient (progressive disease). Combining the results of in vitro assays with patient sample analyses, these data suggest that mocetinostat decreases immunosuppressive immune cell phenotypes while augmenting anti-tumor phenotypes, demonstrating potential of combination therapy with CPI. Citation Format: David M. Woods, Andressa S. Laino, Melinda Vassallo, Jeffrey Weber. The Class I/IV HDAC Inhibitor Mocetinostat Augments Anti-Tumor Immune Responses in Melanoma Patients [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-007.

Background Sepsis-related acute kidney injury (AKI) is associated with high morbidity and mortality among patients. Underlying pathomechanisms include capillary leakage and fluid loss into the interstitial tissue and constant exposure to pathogens results in activation of inflammatory cascades, organ dysfunction and subsequently organ damage. Methods To identify novel factors that trigger sepsis-related acute kidney injury, plasma levels of Granzyme A, as representative of a lymphocyte-derived protease, and heparin-binding protein as indicator for neutrophil-derived mediators, were investigated retrospectively in 60 sepsis patients. Results While no association was found between plasma levels of lymphocyte-derived Granzyme A and the incidence of sepsis-related AKI, sepsis patients with AKI had significantly higher plasma levels of heparin-binding protein compared to those without AKI. This applies both to heparin-binding protein peak values (43.30 ± 23.34 vs. 30.25 ± 15.63 pg/mL; p = 0.005) as well as mean values (27.93 ± 14.39 vs. 22.02 ± 7.65 pg/mL; p = 0.021). Furthermore, a heparin-binding protein cut-off value of 23.89 pg/mL was established for AKI diagnosis. Conclusion This study identifies the neutrophil-derived heparin-binding protein as a valuable new biomarker for AKI in sepsis. Beyond the diagnostic perspective, this offers prospect for further research on pathogenesis of AKI and novel therapeutic approaches.

Model selection for evaluation of immuno-oncology agents is often aided by both response to therapy and changes in immune cell subsets following treatment. To identify underlying factors that influence differences in therapeutic response, CT26 colorectal and 4T1-Luc (4T1) breast tumor models, which are immunologically warm and cold, respectively, were treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Flow cytometry and transcriptome analysis were used to describe treatment-related changes in immune cell infiltrate, TIL activation status, cytokine/granzyme B production, and overall changes in gene expression. Response to anti-CTLA-4 therapy was distinct between these models. Tumor growth inhibition was observed in CT26 (74%), but not 4T1. In CT26, CD8+ and NK T cell infiltration increased, while M2 macrophages decreased. In 4T1 tumors, only increased NKT cell infiltration was noted after treatment, and no demonstrable changes were observed in any other cell type or activation marker. Several pharmacodynamic changes in immune cell activation were observed in CT26. Notably, CT26-derived CD4+, CD8+, and NK T cell subsets had increased IFNγ production (70%, 25%, and 17%, respectively, versus control). An increase in polyfunctional T cells producing both IFNγ and TNFα was also noted for CT26 and was correlated with slower tumor growth. Granzyme B was reduced and CD107a was increased in CT26-derived NK T cells, suggesting functionally active T cells. This was also supported by upregulation of genes in the cytotoxicity, antigen presentation, and NFkappaB pathways. Following CT26 treatment with anti-CTLA-4, transcriptome analysis with the nCounter® Mouse PanCancer IO 360 Panel (NanoString) showed an 8-10 fold induction of several T cell-related genes as well as an 8-14 fold induction of several chemokines and their receptors. These data complement the flow cytometry data and support stimulation of T cell activation and recruitment to CT26 tumors following anti-CTLA-4 treatment. In contrast, analysis of 4T1 tumors indicated only a 2 to 3-fold induction of CCL4, CCL11, and Granzyme A after treatment. Interestingly, genes in the matrix remodeling pathway were upregulated in 4T1 tumors upon anti-CTLA-4 treatment. In summary, CT26 tumors were responsive to anti-CTLA-4 treatment, showing increased polyfunctional T cell recruitment, NK T cell activity, and reduced M2 macrophage infiltration, as well as marked upregulation of several genes involved in T cell recruitment and activation in tumor. In contrast, 4T1 tumors did not display any remarkable response to therapy, changes to TIL infiltrates, and only limited changes to gene expression with treatment. Collectively, these results suggest tumor profiles following treatment can provide insight into potential targets of interest and are important for preclinical model selection. Citation Format: Sheri R. Barnes, David Draper, Sarah E. Church, Maryland Rosenfeld Franklin. Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6679.

Previous studies by our lab have established that placental‐ischemia stimulated T‐helper 17 cells (TH17s) cause increased cytolytic natural killer (cNK) cell proliferation and activation during pregnancy; however, the exact mechanism is unknown. The objective of this study was to investigate the role of interlukin 17 (IL‐17) in inducing cNK cell activation in pregnancy. We infused 150 pg/day of recombinant IL‐17 into a subset of normal pregnant (NP) Sprague Dawley rats from gestation day (GD) 12–19 (NP+IL‐17). On GD 19, mean arterial pressure (MAP), fetal and placental weights, cytokines, cNK cell activation, cytotoxic enzymes, and vascular reactivity were assessed. MAP significantly increased from 99 ± 3 mmHg in NP to 120 ± 1 mmHg in NP+IL‐17 (P