Quantitative or qualitative differences in immunity may drive and predict clinical severity in COVID-19. We therefore measured modules of serum pro-inflammatory, anti-inflammatory and anti-viral cytokines in combination with the anti-SARS-CoV-2 antibody response in COVID-19 patients admitted to tertiary care. Using machine learning and employing unsupervised hierarchical clustering, agnostic to severity, we identified three distinct immunotypes that were shown post-clustering to predict very different clinical courses such as clinical improvement or clinical deterioration. Immunotypes did not associate chronologically with disease duration but rather reflect variations in the nature and kinetics of individual patient's immune response. Here we demonstrate that immunophenotyping can stratify patients to high and low risk clinical subtypes, with distinct cytokine and antibody profiles, that can predict severity progression and guide personalized therapy.

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BackgroundIn a previous in-depth phenotypic analysis of PBLs in JIA, we detected differences in cell subpopulations dependent on the age and also some cellular changes that could be often associated with an exhausted phenotype.ObjectivesTo analyze the expression of co-inhibitory and co-stimulatory molecules in different cellular subpopulations in Oligoarticular JIA patients stratified by age and treatment.MethodsExploratory cross-sectional study. 53 patients fulfilling Oligoarticular JIA (ILAR) criteria and 22 controls were included. All of them were children or young adults (2 to 35 years of age). JIA patients were either not treated (n=14) or treated with MTX (n=14), anti-TNF (n=8), or a combination of both (n=16). JIA disease activity was clinically and biologically assessed. Four flow cytometry panels were designed for the analysis of co-stimulatory/co-inhibitory markers, some typical of “exhausted” cells (PD1, TIM 3, TIGIT, CD226, CD137, HVEM, LIGHT, BTLA), assessed in memory, effector and naïve CD4+ and CD8+ T lymphocytes, and also in B lymphocytes and NK cells. Statistical analysis was performed, and FDR correction was applied for p values.ResultsNo correlation was found among the different exhaustion/activation markers and age neither in JIA nor in controls. Interestingly, age-related differential trends were observed in the CD8+ compartment in controls, while in JIA patients were detected in the B cell compartment. We didn’t observe, after correcting for treatment, any significant difference in lymphocyte subpopulations defined by the above co-inhibitory and co-stimulatory markers in JIA patients in relation to indexes of disease activity. However, there was a tendency towards a higher expression of CD137 in several CD8+ subpopulations of active patients as compared to inactive.ConclusionNo clear differences were found in PBL subpopulations of oligoarticular-JIA patients regarding co-stimulatory/co-inhibitory molecules. This finding could be explained, at least in part, by the good therapeutic control achieved in this patient population, or by the particular characteristics of this group of patients, in which prevails a more localized expression of the disease. It could be of interest to carry out complementary analysis to further define the differential tendencies observed.AcknowledgementInstituto de Salud Carlos III (ISCIII), Sociedad Española Reumatología (SER)Disclosure of InterestsNone declared

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BackgroundThe potential of expanded flow cytometric analysis of PBLs has not been yet been fully exploited to study JIA heterogeneity neither to monitor JIA patients' on biological treatmentsObjectivesTo analyze in depth lymphocyte subpopulations in treated JIA patients stratified by age and gender.MethodsOne hundred one patients, 69 children and 32 adult fulfilling JIA (ILAR) criteria attending HUVH rheumatology clinic were included. All patients were treated with either MTX, anti-TNFα, or a combination of both. Controls were sex/age matched volunteers either healthy or attending the clinics of HUVH for non inflammatory conditions (59 controls, 29 paediatric and 30 adult). Extended immunophenotype following FITMaN protocol that discerns 57 peripheral blood lymphocyte (PBL) subpopulations was applied. Bonferrroni's correction indicated that the cut off for significance was p<0.00041667.ResultsChanges common to each group of patients irrespective of treatment are as follows: paediatric patients showed lower percentage of CD8 effector cells (CD45RA+CCR7-) when compared to controls (10.53±5.71 vs 16.92±5.71). Percentage of total CD4 cells was higher on JIA children than in controls (42.21±7.40 vs 36.04±5.22). No differences were found on percentages nor in absolute number of Th1 (CXCR3+CCR6-), Th2 (CXCR3-CCR6-), or Th17 (CXCR3-CCR6-) CD4 cells. Interestingly all Th subpopulations showed decreased expression of HLA-DR in JIA children when compared with controls (6.06±3.08 vs 10.49±5.02; 0.81±0.51 vs 1.48±0.64; 8.57±3.81 vs 12.6±4.95; respectively). JIA adults presented no significant differences in T cells subpopulations compared to adult controls, but showed higher percentage of CD21- switch-memory B cells (IgD-IgM-CD27-) than controls (10.82±5.33 vs 4.29±4.33).In a second analysis of the JIA patients few differences were observed according to the administered therapy. Paediatric patients treated with MTX (n=27) showed a lower proportion of the effector memory (CD45RA-CCR7-) cells both CD4+ and CD8+, when compared to controls, anti-TNF, and anti-TNF plus MTX groups (20.01±7.76 vs 27.64±9.27; 20.01±7.76 vs 27.84±4.27; 20.01±7.76 vs 27.07±10.05; for CD4s and 26.42±7.23 vs 37.10±12.38; 26.42±7.23 vs 42.08±11.59; 26.42±7.23 vs 37.27±12.54 for CD8s, respectively). Percentage of CD25+ lymphocytes was higher in the anti-TNF plus MTX group. The Th1–17 (CCR6+CXCR3+) CD4 cell subpopulation was significantly increased in anti-TNF and anti-TNFα plus MTX groups compared with controls and MTX (10.45±3.14 vs 6.38±3.89; 10.45±3.14 vs 4.78±2.56 and 9.43±4.63 vs 6.38±3.89; 9.43±4.63 vs 4.78±2.56, respectively). Adult patients treated with anti-TNFα+MTX and MTX presented higher levels of CD21- naïve B cells compared to HC and to anti-TNF groups (4.86±3.24 vs 2.32±1.69; 4.86±3.24 vs 2.95±1.94 and 5.38±1.54 vs 2.32±1.69; 5.38±1.54 vs 2.95±1.94).ConclusionsIn depth phenotypic analysis of PBL in JIA showed alterations in cell subpopulations that seem dependent on age group. Our data also suggests that detailed in depth phenotypic analysis ...