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7 figures, 1 table., [Aims]: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis., [Methods]: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times., [Results]: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα., [Conclusions]: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology., This work was supported by grant SAF2017-83120-C2-1-R and SAF2014-54763-C2-2-R from the Ministry of Science, Innovation and Universities and FEDER (Group B29_17R, Aragon Government). MG and LS were supported by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM was supported by a PhD fellowship from Aragon Government, MA was supported by a post-doctoral fellowship “Juan de la Cierva-formación” from the Ministry of Science, Innovation and Universities. JP was supported by ARAID Foundation.

6 figures, 6 tables.-- Supplementary material available., Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis., [Methods]: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity., [Results]: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity., [Conclusions]: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death., The authors would like to thank the Biobank of the Aragon Health System integrated in the Spanish National Biobanks Network and the Servicios Científico Técnicos de Citometria de Flujo del CIBA for their collaboration. Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Health National Institute Carlos III (COV20-00308), Aragón Government (Fondo COVID-19), Fundación Santander-Universidad de Zaragoza (Programa COVID-19), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente, ASPANOA and Carrera de la Mujer de Monzón. EMG is funded by Agencia Estatal de Investigación (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). IUM and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from AECC, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. DDM is supported by a postdoctoral fellowship 'Sara Borrell', and MA is supported by a postdoctoral fellowship 'Juan de la Cierva-incorporacion' from the Ministry of Science, Innovation and Universities. EM and BGT are supported by Rio Hortega contract. JP is supported by the ARAID Foundation.

5 figures, 5 tables.-- Supplementary information available., (1) Despite the effectiveness of immune checkpoint inhibitors (ICIs) in lung cancer, there is a lack of knowledge about predictive biomarkers. The objective of our study is to analyze different subsets of T-lymphocytes and natural killer (NK) cells as predictive biomarkers in a cohort of patients with nonsmall cell lung cancer (NSCLC) treated with ICI. (2) This is an observational, prospective study with 55 NSCLC patients treated with ICI. A total of 43 T and NK cell subsets are analyzed in peripheral blood, including the main markers of exhaustion, differentiation, memory, activation, and inhibition. (3) Regarding the descriptive data, Granzyme B+CD4+ Treg lymphocytes stand out (median 17.4%), and within the NK populations, most patients presented cytotoxic NK cells (CD56+CD3−CD16+GranzymeB+; median 94.8%), and about half of them have highly differentiated adaptive-like NK cells (CD56+CD3−CD16+CD57+ (mean 59.8%). A statistically significant difference was observed between the expression of PD1 within the CD56bright NK cell subpopulation (CD56+CD3−CD16−PD-1+) (p = 0.047) and a better OS. (4) Circulating immune cell subpopulations are promising prognostic biomarkers for ICI. Pending on validation with a larger sample, here we provide an analysis of the major circulating T and NK cell subsets involved in cancer immunity, with promising results despite a small sample size., Work in the JP lab is funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_20R), Grant PID2020-113963RB-I00 by MICIN/AEI, CIBER—Consorcio Centro de Investigación Biomédica en Red—(CIBERINFEC, CB21/13/00087), Grant PTA2019-016739-I funded by MCIN/AEI/10.13039/501100011033 to PD, Instituto de Salud Carlos III, Aspanoa and Carrera de la mujer de Monzón. Grant PID2020-113963RB-I00 by MICIN/AEI to EMGB. Bristol Myers Squibb. Roche Farma S.A. Funders did not make any decisions about the study or the results to post.

1 figure, 1 table, Sepsis is a serious global health problem. In addition to a high incidence, this syndrome has a high mortality and is responsible for huge health expenditure. The pathophysiology of sepsis is very complex and it is not well-understood yet. However, it is widely accepted that the initial phase of sepsis is characterized by a hyperinflammatory response while the late phase is characterized by immunosuppression and immune anergy, increasing the risk of secondary infections. Granzymes (Gzms) are a family of serine proteases classified according to their cleavage specificity. Traditionally, it was assumed that all Gzms acted as cytotoxic proteases. However, recent evidence suggests that GzmB is the one with the greatest cytotoxic capacity, while the cytotoxicity of others such as GzmA and GzmK is not clear. Recent studies have found that GzmA, GzmB, GzmK, and GzmM act as pro-inflammatory mediators. Specially, solid evidences show that GzmA and GzmK function as extracellular proteases that regulate the inflammatory response irrespectively of its ability to induce cell death. Indeed, studies in animal models indicate that GzmA is involved in the cytokine release syndrome characteristic of sepsis. Moreover, the GZM family also could regulate other biological processes involved in sepsis pathophysiology like the coagulation cascade, platelet function, endothelial barrier permeability, and, in addition, could be involved in the immunosuppressive stage of sepsis. In this review, we provide a comprehensive overview on the contribution of these novel functions of Gzms to sepsis and the new therapeutic opportunities emerging from targeting these proteases for the treatment of this serious health problem., 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 (JP-P), SAF2014-54763-C2-2-R (EG)] and Instituto de Salud Carlos III (PI16-00526, LM-L; PI18/00527, JP-P). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (LS and MA), Ministerio de Ciencia, Innovación y Universidades (MG-T). MA has a Juan de la Cierva Contract (Ministerio de Ciencia, Innovación y Universidades) and JS-M a Rio Hortega Contract (Instituto de Salud Carlos III). JP was supported by Fundación Aragon I+D (ARAID).

2 tables. Immune control inhibitor drugs (anti-PD1/PD-L1/CTLA-4) (ICIs) are showing efficacy in the treatment of lung cancer. Currently the only biomarker with clinical utility for ICIs, such as the expression of PDL1, does not appear to be perfect or effective. Our working group is conducting a pilot study in lung cancer patients receiving ICIs with the aim of analyze the factors that affect the sensitivity of the immunotherapy in lung Cancer. Tumor Mutational Burden (TMB) and the sequencing of the T Cell Receptor (TCR) repertoire in peripheral blood have been postulated as predictive biomarkers of efficacy of immunotherapy. The review focusses on the predictive value of TMB for clinical responses to ICIs and discusses its clinical need after a discussion of the limitations. TCR CDR3 beta analysis and parameters such as clonality and TCR convergence may be good alternatives. For the moment, the combination of biomarkers may be the optimal tool. Despite therapeutic advances, lung cancer (LC) is one of the leading causes of cancer morbidity and mortality worldwide. Recently, the treatment of advanced LC has experienced important changes in survival benefit due to immune checkpoint inhibitors (ICIs). However, overall response rates (ORR) remain low in unselected patients and a large proportion of patients undergo disease progression in the first weeks of treatment. Therefore, there is a need of biomarkers to identify patients who will benefit from ICIs. The programmed cell death ligand 1 (PD-L1) expression has been the first biomarker developed. However, its use as a robust predictive biomarker has been limited due to the variability of techniques used, with different antibodies and thresholds. In this context, tumor mutational burden (TMB) has emerged as an additional powerful biomarker based on the observation of successful response to ICIs in solid tumors with high TMB. TMB can be defined as the total number of nonsynonymous mutations per DNA megabases being a mechanism generating neoantigens conditioning the tumor immunogenicity and response to ICIs. However, the latest data provide conflicting results regarding its role as a biomarker. Moreover, considering the results of the recent data, the use of peripheral blood T cell receptor (TCR) repertoire could be a new predictive biomarker. This review summarises recent findings describing the clinical utility of TMB and TCRβ (TCRB) and concludes that immune, neontigen, and checkpoint targeted variables are required in combination for accurately identifying patients who most likely will benefit of ICIs.

2 tables., Immune control inhibitor drugs (anti-PD1/PD-L1/CTLA-4) (ICIs) are showing efficacy in the treatment of lung cancer. Currently the only biomarker with clinical utility for ICIs, such as the expression of PDL1, does not appear to be perfect or effective. Our working group is conducting a pilot study in lung cancer patients receiving ICIs with the aim of analyze the factors that affect the sensitivity of the immunotherapy in lung Cancer. Tumor Mutational Burden (TMB) and the sequencing of the T Cell Receptor (TCR) repertoire in peripheral blood have been postulated as predictive biomarkers of efficacy of immunotherapy. The review focusses on the predictive value of TMB for clinical responses to ICIs and discusses its clinical need after a discussion of the limitations. TCR CDR3 beta analysis and parameters such as clonality and TCR convergence may be good alternatives. For the moment, the combination of biomarkers may be the optimal tool., Despite therapeutic advances, lung cancer (LC) is one of the leading causes of cancer morbidity and mortality worldwide. Recently, the treatment of advanced LC has experienced important changes in survival benefit due to immune checkpoint inhibitors (ICIs). However, overall response rates (ORR) remain low in unselected patients and a large proportion of patients undergo disease progression in the first weeks of treatment. Therefore, there is a need of biomarkers to identify patients who will benefit from ICIs. The programmed cell death ligand 1 (PD-L1) expression has been the first biomarker developed. However, its use as a robust predictive biomarker has been limited due to the variability of techniques used, with different antibodies and thresholds. In this context, tumor mutational burden (TMB) has emerged as an additional powerful biomarker based on the observation of successful response to ICIs in solid tumors with high TMB. TMB can be defined as the total number of nonsynonymous mutations per DNA megabases being a mechanism generating neoantigens conditioning the tumor immunogenicity and response to ICIs. However, the latest data provide conflicting results regarding its role as a biomarker. Moreover, considering the results of the recent data, the use of peripheral blood T cell receptor (TCR) repertoire could be a new predictive biomarker. This review summarises recent findings describing the clinical utility of TMB and TCRβ (TCRB) and concludes that immune, neontigen, and checkpoint targeted variables are required in combination for accurately identifying patients who most likely will benefit of ICIs.