NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins.

Increased recruitment of transitional and non-classical monocytes in the lung during SARS-CoV-2 infection is associated with COVID-19 severity. However, whether specific innate sensors mediate the activation or differentiation of monocytes in response to different SARS-CoV-2 proteins remain poorly characterized. Here, we show that SARS-CoV-2 Spike 1 but not nucleoprotein induce differentiation of monocytes into transitional or non-classical subsets from both peripheral blood and COVID-19 bronchoalveolar lavage samples in a NFκB-dependent manner, but this process does not require inflammasome activation. However, NLRP3 and NLRC4 differentially regulated CD86 expression in monocytes in response to Spike 1 and Nucleoprotein, respectively. Moreover, monocytes exposed to Spike 1 induce significantly higher proportions of Th1 and Th17 CD4 + T cells. In contrast, monocytes exposed to Nucleoprotein reduce the degranulation of CD8 + T cells from severe COVID-19 patients. Our study provides insights in the differential impact of innate sensors in regulating monocytes in response to different SARS-CoV-2 proteins, which might be useful to better understand COVID-19 immunopathology and identify therapeutic targets.

Beneficial effect of temporary methotrexate interruption on B and T cell responses upon SARS-CoV-2 vaccination in patients with rheumatoid arthritis or psoriatic arthritis.

B and T cell responses were evaluated in patients with rheumatoid arthritis (RA) or psoriatic arthritis (PsA) after 1 or 2 weeks of methotrexate (MTX) withdrawal following each COVID-19 vaccine dose and compared with those who maintained MTX. Adult RA and PsA patients treated with MTX were recruited and randomly assigned to 3 groups: MTX-maintenance (n = 72), MTX-withdrawal for 1 week (n = 71) or MTX-withdrawal for 2 weeks (n = 73). Specific antibodies to several SARS-CoV-2 antigens and interferon (IFN)-γ and interleukin (IL)-21 responses were assessed. MTX withdrawal in patients without previous COVID-19 was associated with higher levels of anti-RBD IgG and neutralising antibodies, especially in the 2-week withdrawal group and with higher IFN-γ secretion upon stimulation with pools of SARS-CoV-2 S peptides. No increment of RA/PsA relapses was detected across groups. Our data indicate that two-week MTX interruption following COVID-19 vaccination in patients with RA or PsA improves humoral and cellular immune responses.

Analysis of humoral and cellular immunity after SARS-CoV-2 vaccination in patients with multiple sclerosis treated with immunomodulatory drugs.

We analyzed immune response to SARS-CoV-2 vaccination by measuring specific IgG titers and T-cell reactivity to different SARS-CoV-2 peptides in multiple sclerosis patients taking different disease-modifying treatments. Of the 88 patients included, 72 developed any kind of immune response after vaccination. Although DMTs such as fingolimod and anti-CD20+ treatments prevented patients from developing a robust humoral response to the vaccine, most of them were still able to develop a cellular response, which could be crucial for long-term immunity. It is probably advisable that all MS patients take additional/booster doses to increase their humoral and/or cellular immune response to SARS-CoV-2.

T regulatory lymphocytes specific for SARS-CoV-2 display increased functional plasticity.

The study of phenotypic and functional characteristics of immune cells involved in host response to SARS-CoV-2 is relevant for understanding COVID-19 pathogenesis and individual differences in disease progression. We have analyzed chemokine receptor expression in SARS-CoV-2-specific CD4+ T lymphocytes from vaccinated donors, and have found an increase of CCR9+ and CCR6+ cells. CCR9+ specific CD4+ cells are enriched in T regulatory (Treg) lymphocytes. These cells specifically show heterogeneous regulatory activity, associated with different profiles of CCR9/CCR6 expression, individual differences in IL-10 and IL-17 production, and variable FoxP3 and Notch4 expression. A higher heterogeneity in FoxP3 is selectively observed in convalescent individuals within vaccinated population. Accordingly, SARS-CoV-2-specific CD4+ lymphocytes from COVID-19 patients are also enriched in CCR9+ and CCR6+ cells. CCR6+ specific Treg lymphocytes are mainly increased in critically ill individuals, indicating a preferential role for these cells in lung injury pathogenesis. We provide experimental evidence for a SARS-CoV-2-specific Treg population with increased plasticity, which may contribute to the differential pathogenic response against SARS-CoV-2 among individuals, and underlie the development of autoimmune conditions following SARS-CoV-2 infection.

Tertiary lymphoid structures and B lymphocytes: a promising therapeutic strategy to fight cancer.

Tertiary lymphoid structures (TLSs) are clusters of lymphoid cells with an organization that resembles that of secondary lymphoid organs. Both structures share common developmental characteristics, although TLSs usually appear in chronically inflamed non-lymphoid tissues, such as tumors. TLSs contain diverse types of immune cells, with varying degrees of spatial organization that represent different stages of maturation. These structures support both humoral and cellular immune responses, thus the correlation between the existence of TLS and clinical outcomes in cancer patients has been extensively studied. The finding that TLSs are associated with better prognosis in some types of cancer has led to the design of therapeutic strategies based on promoting the formation of these structures. Agents such as chemokines, cytokines, antibodies and cancer vaccines have been used in combination with traditional antitumor treatments to enhance TLS generation, with good results. The induction of TLS formation therefore represents a novel and promising avenue for the treatment of a number of tumor types.

A Comprehensive Analysis of Immune Response in Patients with Non-Muscle-Invasive Bladder Cancer.

BACKGROUND: Bladder carcinoma has elevated morbimortality due to its high recurrence and progression in localized disease. A better understanding of the role of the tumor microenvironment in carcinogenesis and response to treatment is needed. METHODS: Peripheral blood and samples of urothelial bladder cancer and adjacent healthy urothelial tissue were collected from 41 patients and stratified in low- and high-grade urothelial bladder cancer, excluding muscular infiltration or carcinoma in situ. Mononuclear cells were isolated and labeled for flow cytometry analysis with antibodies aimed at identifying specific subpopulations within T lymphocytes, myeloid cells and NK cells. RESULTS: In peripheral blood and tumor samples, we detected different percentages of CD4+ and CD8+ lymphocytes, monocyte and myeloid-derived suppressor cells, as well as differential expression of activation- and exhaustion-related markers. Conversely, only a significant increase in bladder total monocytes was found when comparing bladder and tumor samples. Interestingly, we identified specific markers differentially expressed in the peripheral blood of patients with different outcomes. CONCLUSION: The analysis of host immune response in patients with NMIBC may help to identify specific markers that allow optimizing therapy and patient follow-up. Further investigation is needed to establish a strong predictive model.

Development of an Effective Immune Response in Adults With Down Syndrome After Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccination.

BACKGROUND: Immune dysregulation in individuals with Down syndrome (DS) leads to an increased risk for hospitalization and death due to coronavirus disease 2019 (COVID-19) and may impair the generation of protective immunity after vaccine administration. METHODS: The cellular and humoral responses of 55 individuals with DS who received a complete SARS-CoV-2 vaccination regime at 1 to 3 (visit [V 1]) and 6 (V2) months were characterized. RESULTS: SARS-CoV-2-reactive CD4+ and CD8+ T lymphocytes with a predominant Th1 phenotype were observed at V1 and increased at V2. Likewise, an increase in SARS-CoV-2-specific circulating Tfh (cTfh) cells and CD8+ CXCR5+ PD-1hi lymphocytes was already observed at V1 after vaccine administration. Specific immunoglobulin G (IgG) antibodies against SARS-CoV-2 S protein were detected in 96% and 98% of subjects at V1 and V2, respectively, although IgG titers decreased significantly between both time points. CONCLUSIONS: Our findings show that DS individuals develop an effective immune response to usual regimes of SARS-CoV-2 vaccination.

Excess weight and anti-PD-1 immune checkpoint inhibitor’s outcomes in non-small cell lung cancer

Immune checkpoint inhibitors are one of the most effective treatments available in advanced non-small cell lung cancer. However, at present, there are no clinical or analytical biomarkers that define which patients benefit with certainty from these treatments. In our study, we evaluated whether excess weight could be a good predictive biomarker of benefit from these drugs.MethodsWe studied a population of 79 patients, divided into a study group with 39 patients diagnosed with non-small cell lung cancer treated with immunotherapy and 40 patients in a control group, diagnosed with different advanced cancers, treated with non-immunotherapy treatment. We analyzed according to the presence of excess weight or not, the treatment’s outcome in the study group and in the control group (objective response, and progression-free and overall survival).ResultsIn our study, we detected a better response rate to immunotherapy in patients with excess weight (62.50 vs 26.08%, OR 4.72, p = 0.02), and a better median progression-free survival (14.19 vs 5.03 months, HR 0.50, p = 0.058) and median overall survival (33.84 months vs 20.76 months, HR 0.43, p = 0.01) in the study group. These findings were specific to the immunotherapy group since in the control group, with patients who did not receive immune checkpoint inhibitors, these findings were not found.ConclusionOur study suggests that patients with excess weight who receive anti-PD-1 immune checkpoint inhibitors diagnosed with non-small cell lung cancer have a better outcome. This effect is specific to patients receiving immunotherapy. (AU)

Excess weight and anti-PD-1 immune checkpoint inhibitor's outcomes in non-small cell lung cancer.

PURPOSE: Immune checkpoint inhibitors are one of the most effective treatments available in advanced non-small cell lung cancer. However, at present, there are no clinical or analytical biomarkers that define which patients benefit with certainty from these treatments. In our study, we evaluated whether excess weight could be a good predictive biomarker of benefit from these drugs. METHODS: We studied a population of 79 patients, divided into a study group with 39 patients diagnosed with non-small cell lung cancer treated with immunotherapy and 40 patients in a control group, diagnosed with different advanced cancers, treated with non-immunotherapy treatment. We analyzed according to the presence of excess weight or not, the treatment's outcome in the study group and in the control group (objective response, and progression-free and overall survival). RESULTS: In our study, we detected a better response rate to immunotherapy in patients with excess weight (62.50 vs 26.08%, OR 4.72, p = 0.02), and a better median progression-free survival (14.19 vs 5.03 months, HR 0.50, p = 0.058) and median overall survival (33.84 months vs 20.76 months, HR 0.43, p = 0.01) in the study group. These findings were specific to the immunotherapy group since in the control group, with patients who did not receive immune checkpoint inhibitors, these findings were not found. CONCLUSION: Our study suggests that patients with excess weight who receive anti-PD-1 immune checkpoint inhibitors diagnosed with non-small cell lung cancer have a better outcome. This effect is specific to patients receiving immunotherapy.

Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV.

BACKGROUND: Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. METHODS: We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. FINDINGS: HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. INTERPRETATION: Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines. FUNDING: NIH (R21AI140930), MINECO/FEDER RETOS (RTI2018-097485-A-I00) and CIBERINF grants.

Peripheral Blood Mononuclear Cells Predict Therapeutic Efficacy of Immunotherapy in NSCLC.

In lung cancer immunotherapy, biomarkers to guide clinical decisions are limited. We now explore whether the detailed immunophenotyping of circulating peripheral blood mononuclear cells (PBMCs) can predict the efficacy of anti-PD-1 immunotherapy in patients with advanced non-small-cell lung cancer (NSCLC). We determined 107 PBMCs subpopulations in a prospective cohort of NSCLC patients before starting single-agent anti-PD-1 immunotherapy (study group), analyzed by flow cytometry. As a control group, we studied patients with advanced malignancies before initiating non-immunotherapy treatment. The frequency of PBMCs was correlated with treatment outcome. Patients were categorized as having either high or low expression for each biomarker, defined as those above the 55th or below the 45th percentile of the overall marker expression within the cohort. In the study group, three subpopulations were associated with significant differences in outcome: high pretreatment levels of circulating CD4+CCR9+, CD4+CCR10+, or CD8+CXCR4+ T cells correlated with poorer overall survival (15.7 vs. 35.9 months, HR 0.16, p = 0.003; 22.0 vs. NR months, HR 0.10, p = 0.003, and 22.0 vs. NR months, HR 0.29, p = 0.02). These differences were specific to immunotherapy-treated patients. High baseline levels of circulating T cell subpopulations related to tissue lymphocyte recruitment are associated with poorer outcomes of immunotherapy-treated advanced NSCLC patients.

Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

RGB-Marking to Identify Patterns of Selection and Neutral Evolution in Human Osteosarcoma Models.

Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models.

CD4+ T Cell Immune Specificity Changes After Vaccination in Healthy And COVID-19 Convalescent Subjects.

The immune response promoted by SARS-CoV-2 vaccination is relevant to develop novel vaccines and optimized prevention strategies. We analyzed the adaptive immunity in healthy donors (HD) and convalescent individuals (CD), before and after administering BNT162b2 vaccine. Our results revealed specific changes in CD4+ T cell reactivity profile in vaccinated HD and CD, with an increase in S1 and S2 positive individuals, proportionally higher for S2. On the contrary, NCAP reactivity observed in HD and CD patients was no longer detectable after vaccination. Despite the substantial antibody response in CD, MPro-derived peptides did not elicit CD4+ lymphocyte activation in our assay in either condition. HD presented an increment in anti-S and anti-RBD IgG after first dose vaccination, which increased after the second vaccination. Conversely, anti-S and anti-RBD IgG and IgA titers increased in already positive CD after first dose administration, remaining stable after second dose inoculation. Interestingly, we found a strong significant correlation between S1-induced CD4+ response and anti-S IgA pre-vaccination, which was lost after vaccine administration.

A Differential Signature of Circulating miRNAs and Cytokines Between COVID-19 and Community-Acquired Pneumonia Uncovers Novel Physiopathological Mechanisms of COVID-19.

Coronavirus Disease 2019 (COVID-19) pneumonia is a life-threatening infectious disease, especially for elderly patients with multiple comorbidities. Despite enormous efforts to understand its underlying etiopathogenic mechanisms, most of them remain elusive. In this study, we compared differential plasma miRNAs and cytokines profiles between COVID-19 and other community-acquired pneumonias (CAP). A first screening and subsequent validation assays in an independent cohort of patients revealed a signature of 15 dysregulated miRNAs between COVID-19 and CAP patients. Additionally, multivariate analysis displayed a combination of 4 miRNAs (miR-106b-5p, miR-221-3p, miR-25-3p and miR-30a-5p) that significantly discriminated between both pathologies. Search for targets of these miRNAs, combined with plasma protein measurements, identified a differential cytokine signature between COVID-19 and CAP that included EGFR, CXCL12 and IL-10. Significant differences were also detected in plasma levels of CXCL12, IL-17, TIMP-2 and IL-21R between mild and severe COVID-19 patients. These findings provide new insights into the etiopathological mechanisms underlying COVID-19.

IL-6 serum levels predict severity and response to tocilizumab in COVID-19: An observational study.

BACKGROUND: Patients with coronavirus disaese 2019 (COVID-19) can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome requiring invasive mechanical ventilation (IMV). Because IL-6 is a relevant cytokine in acute respiratory distress syndrome, the blockade of its receptor with tocilizumab (TCZ) could reduce mortality and/or morbidity in severe COVID-19. OBJECTIVE: We sought to determine whether baseline IL-6 serum levels can predict the need for IMV and the response to TCZ. METHODS: A retrospective observational study was performed in hospitalized patients diagnosed with COVID-19. Clinical information and laboratory findings, including IL-6 levels, were collected approximately 3 and 9 days after admission to be matched with preadministration and postadministration of TCZ. Multivariable logistic and linear regressions and survival analysis were performed depending on outcomes: need for IMV, evolution of arterial oxygen tension/fraction of inspired oxygen ratio, or mortality. RESULTS: One hundred forty-six patients were studied, predominantly males (66%); median age was 63 years. Forty-four patients (30%) required IMV, and 58 patients (40%) received treatment with TCZ. IL-6 levels greater than 30 pg/mL was the best predictor for IMV (odds ratio, 7.1; P < .001). Early administration of TCZ was associated with improvement in oxygenation (arterial oxygen tension/fraction of inspired oxygen ratio) in patients with high IL-6 (P = .048). Patients with high IL-6 not treated with TCZ showed high mortality (hazard ratio, 4.6; P = .003), as well as those with low IL-6 treated with TCZ (hazard ratio, 3.6; P = .016). No relevant serious adverse events were observed in TCZ-treated patients. CONCLUSIONS: Baseline IL-6 greater than 30 pg/mL predicts IMV requirement in patients with COVID-19 and contributes to establish an adequate indication for TCZ administration.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients.

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.