Disease activity drives divergent epigenetic and transcriptomic reprogramming of monocyte subpopulations in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is characterised by systemic inflammation involving various immune cell types. Monocytes, pivotal in promoting and regulating inflammation in SLE, differentiate from classic monocytes into intermediate and non-classic monocytes, assuming diverse roles and changing their proportions in inflammation. In this study, we investigated the epigenetic and transcriptomic profiles of these and novel monocyte subsets in SLE in relation to activity and progression. METHODS: We obtained the DNA methylomes and transcriptomes of classic, intermediate, non-classic monocytes in patients with SLE (at first and follow-up visits) and healthy donors. We integrated these data with single-cell transcriptomics of SLE and healthy donors and interrogated their relationships with activity and progression. RESULTS: In addition to shared DNA methylation and transcriptomic alterations associated with a strong interferon signature, we identified monocyte subset-specific alterations, especially in DNA methylation, which reflect an impact of SLE on monocyte differentiation. SLE classic monocytes exhibited a proinflammatory profile and were primed for macrophage differentiation. SLE non-classic monocytes displayed a T cell differentiation-related phenotype, with Th17-regulating features. Changes in monocyte proportions, DNA methylation and expression occurred in relation to disease activity and involved the STAT pathway. Integration of bulk with single-cell RNA sequencing datasets revealed disease activity-dependent expansion of SLE-specific monocyte subsets, further supported the interferon signature for classic monocytes, and associated intermediate and non-classic populations with exacerbated complement activation. CONCLUSIONS: Disease activity in SLE drives a subversion of the epigenome and transcriptome programme in monocyte differentiation, impacting the function of different subsets and allowing to generate predictive methods for activity and progression.

An algorithm based on immunotherapy discontinuation and liver biopsy spares corticosteroids in two thirds of cases of severe checkpoint inhibitor-induced liver injury.

BACKGROUND: There are few data on corticosteroids (CS)-sparing strategies for checkpoint inhibitor (ICI)-induced liver injury (ChILI). AIM: We aimed to assess the performance of a 2-step algorithm for severe ChILI, based on ICI temporary discontinuation (step-1) and, if lack of biochemical improvement, CS based on the degree of necroinflammation at biopsy (step-2). METHODS: Prospective study that included all subjects with grade 3/4 ChILI. Peripheral extended immunophenotyping was performed. Indication for CS: severe necroinflammation; mild or moderate necroinflammation with later biochemical worsening. RESULTS: From 111 subjects with increased transaminases (January 2020 to August 2023), 44 were diagnosed with grade 3 (N = 35) or grade 4 (N = 9) ChILI. Main reason for exclusion was alternative diagnosis. Lung cancer (13) and melanoma (12) were the most common malignancies. ICI: 23(52.3%) anti-PD1, 8(18.2%) anti-PD-L1, 3(6.8%) anti-CTLA-4, 10(22.7%) combined ICI. Liver injury pattern: hepatocellular (23,52.3%) mixed (12,27.3%) and cholestatic (9,20.5%). 14(32%) presented bilirubin >1.2 mg/dL. Overall, 30(68.2%) patients did not require CS: 22(50.0%) due to ICI discontinuation (step-1) and 8/22 (36.4%) based on the degree of necroinflammation (step-2). Biopsy mainly impacted on grade 3 ChILI, sparing CS in 8 out of 15 (53.3%) non-improvement patients after ICI discontinuation. CD8+ HLA-DR expression (p = 0.028), central memory (p = 0.046) were lower in CS-free managed subjects, but effector-memory cells (p = 0.002) were higher. Time to transaminases normalisation was shorter in those CS-free managed (overall: p < 0.001, grade 3: p < 0.001). Considering our results, a strategy based on ICI discontinuation and biopsy for grade 3 ChILI is proposed. CONCLUSIONS: An algorithm based on temporary immunotherapy discontinuation and biopsy allows CS avoidance in two thirds of cases of severe ChILI.

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

Kinetics of cellular and humoral immunogenicity and effectiveness of SARS-CoV-2 booster vaccination in hematologic neoplasms.

Data on the effect of booster SARS-CoV-2 vaccination are mainly focused on humoral immunogenicity, while the kinetics of vaccine-induced cellular response and its correlation with effectiveness in hematologic patients are less explored. Our aim was to evaluate the longitudinal cellular and humoral immunogenicity induced by two and three doses of the mRNA-1273 SARS-CoV-2 vaccine in 270 patients with hematologic malignancies, and its relationship with the severity of breakthrough SARS-CoV-2 infection. Results indicate that at 23 weeks after the second dose, the seroconversion rate declined from 68.5% to 59.3%, with a reduction in median anti-S titers from 1577 to 456 BAU/mL, mainly in patients over 65 years of age or chronic lymphocytic leukemia (CLL) patients undergoing active therapy. Cellular immunogenicity, however, remained positive in 84.4% of cases. A third vaccine dose seroconverted 42.7% (41/96) and triggered cellular response in 36.7% (11/30) of previously negative patients. Notably, only 7.2% (15/209) of patients failed to develop both humoral and cellular response. Active therapy, anti-CD20 antibodies, lymphopenia, hypogammaglobulinemia, and low CD19+ cell count were associated with poor humoral response, while active disease, GvHD immunosuppressive therapy, lymphopenia, and low CD3+ , CD4+ , CD56+ cell count determined an impaired cellular response. After 13.8 months of follow-up, the incidence of SARS-CoV-2 infection was 24.8% (67/270), including 6 (9%) severe/critical cases associated with a weaker cellular (median interferon gamma (IFN-γ) 0.19 vs. 0.35 IU/mL) and humoral response (median anti-S titer <4.81 vs. 788 BAU/mL) than asymptomatic/mild cases. In conclusion, SARS-CoV-2 booster vaccination improves humoral response and COVID-19 severity is associated with impaired vaccine-induced immunogenicity.

Thyroid cells from normal and autoimmune thyroid glands suppress T lymphocytes proliferation upon contact revealing a new regulatory inhibitory type of interaction independent of PD1/PDL1.

Immune Checkpoint Receptors include a number of inhibitory receptors that limit tissue damage during immune responses; blocking PD-1/PD-L1 checkpoint receptor axis led to a paradigm shift in cancer immunotherapy but also to autoimmune adverse effects, prominently thyroid autoimmunity. Although PD-L1 is known to be expressed on thyroid follicular cells (TFCs) of autoimmune glands the role on PD-1/PD-L1 in the interaction between T cells and thyroid cells in the tissue has not been investigated. Here we report that autologous primary TFCs, but not transformed TFCs, inhibit CD4 and CD8 T cell proliferation but no cytokine production. This effect is not, however, mediated by PD-1/PD-L1 nor locally produced cytokines. Beta galactosidase analysis excluded culture-induced senescence as an explanation. High resolution flow cytometry demonstrated that autologous TFC/T cells co-culture induced the expansion of several clusters of double negative (DN) T cells characterized by high expression of activation markers and negative immune checkpoints. Single cell transcriptomic profiling demonstrated that dissociated TFC express numerous candidate molecules for mediating this suppressive activity, including CD40, E-Cadherin and TIGIT ligands. These ligands directly or through the generation of a suppressor population of DN T cells, and not the PD-1/PD-L1 axis, are most likely the responsible of TFC immunosuppressive activity. These results contribute to reveal the complex network of inhibitory mechanism that operate at the tissue level to restrain autoimmunity but also point to pathways, other that PD-1/PD-L1, that can contribute to tumor evasion.

Detection and evolutionary dynamics of somatic FAS variants in autoimmune lymphoproliferative syndrome: Diagnostic implications.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by impaired apoptotic homeostasis. The clinical characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), and an increased risk of lymphoma. A distinctive biological feature is accumulation (>2.5%) of an abnormal cell subset composed of TCRαß+ CD4-CD8- T cells (DNTs). The most common genetic causes of ALPS are monoallelic pathogenic variants in the FAS gene followed by somatic FAS variants, mainly restricted to DNTs. Identification of somatic FAS variants has been typically addressed by Sanger sequencing in isolated DNTs. However, this approach can be costly and technically challenging, and may not be successful in patients with normal DNT counts receiving immunosuppressive treatment. In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) by Sanger sequencing on purified CD3+ cells. We then followed the evolutionary dynamics of the variant along time with an NGS-based approach involving deep amplicon sequencing (DAS) at high coverage (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs<2%) periods. DAS enabled detection of the somatic variant in all samples, even the one obtained after five years of immunosuppressive treatment (DNTs: 0.89%). The variant allele frequency (VAF) range was 4%-5% in pre-treatment samples and <1.5% in treatment samples, and there was a strong positive correlation between DNT counts and VAF (Pearson's R: 0.98, p=0.0003). We then explored whether the same approach could be used in a discovery setting. In the last follow-up sample (DNT: 0.89%) we performed somatic variant calling on the FAS exon 9 DAS data from whole blood and purified CD3+ cells using VarScan 2. The c.718_719insGTCG variant was identified in both samples and showed the highest VAF (0.67% blood, 1.58% CD3+ cells) among >400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts.

Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines.

BACKGROUND: COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical. Severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes. However, little is known about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients. METHODS: In this study, we obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14 + CD15- monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types. RESULTS: We observed DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes. These changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified. We also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines. A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production. CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells. CONCLUSION: Our findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.

Activation of the Monocyte/Macrophage System and Abnormal Blood Levels of Lymphocyte Subpopulations in Individuals with Autism Spectrum Disorder: A Systematic Review and Meta-Analysis.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with a so far unknown etiology. Increasing evidence suggests that a state of systemic low-grade inflammation may be involved in the pathophysiology of this condition. However, studies investigating peripheral blood levels of immune cells, and/or of immune cell activation markers such as neopterin are lacking and have provided mixed findings. We performed a systematic review and meta-analysis of studies comparing total and differential white blood cell (WBC) counts, blood levels of lymphocyte subpopulations and of neopterin between individuals with ASD and typically developing (TD) controls (PROSPERO registration number: CRD CRD42019146472). Online searches covered publications from 1 January 1994 until 1 March 2022. Out of 1170 publication records identified, 25 studies were finally included. Random-effects meta-analyses were carried out, and sensitivity analyses were performed to control for potential moderators. Results: Individuals with ASD showed a significantly higher WBC count (k = 10, g = 0.29, p = 0.001, I2 = 34%), significantly higher levels of neutrophils (k = 6, g = 0.29, p = 0.005, I2 = 31%), monocytes (k = 11, g = 0.35, p < 0.001, I2 = 54%), NK cells (k = 7, g = 0.36, p = 0.037, I2 = 67%), Tc cells (k = 4, g = 0.73, p = 0.021, I2 = 82%), and a significantly lower Th/Tc cells ratio (k = 3, g = −0.42, p = 0.008, I2 = 0%), compared to TD controls. Subjects with ASD were also characterized by a significantly higher neutrophil-to-lymphocyte ratio (NLR) (k = 4, g = 0.69, p = 0.040, I2 = 90%), and significantly higher neopterin levels (k = 3, g = 1.16, p = 0.001, I2 = 97%) compared to TD controls. No significant differences were found with respect to the levels of lymphocytes, B cells, Th cells, Treg cells, and Th17 cells. Sensitivity analysis suggested that the findings for monocyte and neutrophil levels were robust, and independent of other factors, such as medication status, diagnostic criteria applied, and/or the difference in age or sex between subjects with ASD and TD controls. Taken together, our findings suggest the existence of a chronically (and systemically) activated inflammatory response system in, at least, a subgroup of individuals with ASD. This might have not only diagnostic, but also, therapeutic implications. However, larger longitudinal studies including more homogeneous samples and laboratory assessment methods and recording potential confounding factors such as body mass index, or the presence of comorbid psychiatric and/or medical conditions are urgently needed to confirm the findings.

Case report: Cytokine hemoadsorption in a case of hemophagocytic lymphohistiocytosis secondary to extranodal NK/T-cell lymphoma.

We discuss a single case of Hemophagocytic lymphohistiocytosis (HLH) due to NK-type non-Hodgkin lymphoma and Epstein-Barr virus reactivation with multiorgan dysfunction and distributive shock in which we performed cytokine hemoadsorption with Cytosorb ®. A full microbiological panel was carried out, including screening for imported disease, standard serologies and cultures for bacterial and fungal infection. A liver biopsy and bone marrow aspirate were performed, confirming the diagnosis. The patients fulfilled the HLH-2004 diagnostic criteria, and according to the 2018 Consensus Statements by the HLH Steering Committee of the Histiocyte Society, dexamethasone and etoposide were started. There was an associated hypercytokinemia and, due to refractory distributive shock, rescue therapy with cytokine hemoadsorption was performed during 24 h (within day 2 and 3 from ICU admission). After starting this procedure, rapid hemodynamic control was achieved with a significant reduction in vasopressor support requirements. This case report highlights that cytokine hemoadsorption can be an effective since rapid decrease in IL-10 levels and a significant hemodynamic improvement was achieved.

Commercialized kits to assess T-cell responses against SARS-CoV-2 S peptides. A pilot study in health care workers.

Background: It is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses. Methods: Twenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined. Results: IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (R > 0.8) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between specific antibody levels and the IGRA assay in the ability to detect immune response to SARS-CoV-2, there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one, with Ig(S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA-test. Conclusion: Whole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers.

Introducción: Es fundamental evaluar los niveles de protección inmune en infectados o tras la vacunación frente a SARS-CoV-2. La cuantificación de la respuesta inmune celular T puede complementar la determinación de anticuerpos. Evaluamos la viabilidad de un ensayo comercial validado de respuesta celular T específica frente a SARS-CoV-2. Métodos: Se incluyeron veinte trabajadores sanitarios (TS). Medimos anticuerpos contra las proteínas N y S de SARS-CoV-2 y realizamos el ensayo de liberación de interferón-gamma (IFNγ) en sangre completa (IGRA) frente a péptidos de la proteína S. IFNγ se determinó mediante dos métodos de detección: CLIA y ELISA. Resultados: IGRA detectó respuesta celular T en TS tanto infectados como vacunados. La correlación de los dos métodos de detección de IFNγ fue muy alta (R >0,8) y la sensibilidad y la especificidad variaron entre 100 y 86% y 100-73% respectivamente. Hubo una concordancia muy alta entre los niveles de anticuerpos específicos y el ensayo IGRA aunque la correlación cuantitativa fue relativamente baja. En el grupo de infectados, una dosis de vacuna fue suficiente para alcanzar el «plateau¼ de respuesta inmune. IGRA fue claramente positivo en un profesional vacunado inmunosuprimido que presentaba anticuerpos contra la proteína S negativos. Conclusiones: IGRA frente a péptidos de la proteína-S es susceptible de automatización y constituye una herramienta prometedora para medir la respuesta inmune celular frente a SARS-CoV-2; es aplicable a un gran número de muestras y puede servir para valorar la protección, particularmente en los grupos vulnerables en riesgo de volver a exponerse a la infección, como los TS.

Commercialized kits to assess T-cell responses against SARS-CoV-2 S peptides. A pilot study in health care workers

BackgroundIt is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses.MethodsTwenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined.ResultsIGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (R>0.8) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between specific antibody levels and the IGRA assay in the ability to detect immune response to SARS-CoV-2, there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one, with Ig(S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA-test.ConclusionWhole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers. (AU)

IntroducciónEs fundamental evaluar los niveles de protección inmune en infectados o tras la vacunación frente a SARS-CoV-2. La cuantificación de la respuesta inmune celular T puede complementar la determinación de anticuerpos. Evaluamos la viabilidad de un ensayo comercial validado de respuesta celular T específica frente a SARS-CoV-2.MétodosSe incluyeron veinte trabajadores sanitarios (TS). Medimos anticuerpos contra las proteínas N y S de SARS-CoV-2 y realizamos el ensayo de liberación de interferón-gamma (IFNγ) en sangre completa (IGRA) frente a péptidos de la proteína S. IFNγ se determinó mediante dos métodos de detección: CLIA y ELISA.ResultadosIGRA detectó respuesta celular T en TS tanto infectados como vacunados. La correlación de los dos métodos de detección de IFNγ fue muy alta (R>0,8) y la sensibilidad y la especificidad variaron entre 100 y 86% y 100-73% respectivamente. Hubo una concordancia muy alta entre los niveles de anticuerpos específicos y el ensayo IGRA aunque la correlación cuantitativa fue relativamente baja. En el grupo de infectados, una dosis de vacuna fue suficiente para alcanzar el «plateau» de respuesta inmune. IGRA fue claramente positivo en un profesional vacunado inmunosuprimido que presentaba anticuerpos contra la proteína S negativos.ConclusionesIGRA frente a péptidos de la proteína-S es susceptible de automatización y constituye una herramienta prometedora para medir la respuesta inmune celular frente a SARS-CoV-2; es aplicable a un gran número de muestras y puede servir para valorar la protección, particularmente en los grupos vulnerables en riesgo de volver a exponerse a la infección, como los TS. (AU)

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters; A Retrospective Cohort Analysis and Pilot Study.

Background: Two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. Objectives: To measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings: 1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. Conclusions: Laboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible.

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report.

Syndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient's CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient's complex clinical phenotype.

Precision medicine in sepsis and septic shock: From omics to clinical tools.

Sepsis is a heterogeneous disease with variable clinical course and several clinical phenotypes. As it is associated with an increased risk of death, patients with this condition are candidates for receipt of a very well-structured and protocolized treatment. All patients should receive the fundamental pillars of sepsis management, which are infection control, initial resuscitation, and multiorgan support. However, specific subgroups of patients may benefit from a personalized approach with interventions targeted towards specific pathophysiological mechanisms. Herein, we will review the framework for identifying subpopulations of patients with sepsis, septic shock, and multiorgan dysfunction who may benefit from specific therapies. Some of these approaches are still in the early stages of research, while others are already in routine use in clinical practice, but together will help in the effective generation and safe implementation of precision medicine in sepsis.

Is humoral and cellular response to SARS-CoV-2 vaccine modified by DMT in patients with multiple sclerosis and other autoimmune diseases?

BACKGROUND: The effect of disease-modifying therapies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine response is unclear. OBJECTIVES: We aim to determine the immunological responses to SARS-CoV-2 in multiple sclerosis (MS) and anti-CD20-treated patients with other autoimmune diseases (AID). METHODS: Humoral and cellular responses we determined before and 30-90 days after vaccination in patients with MS and anti-CD20-treated patients with other AID in two Catalan centers. RESULTS: 457 patients were enrolled. Findings showed that humoral response decreased under anti-CD20s or sphingosine 1-phosphate receptor modulators (S1PRM) and with longer treatment duration and increased after 4.5 months from the last anti-CD20 infusion. Cellular response decreased in S1PRM-treated. Patients on anti-CD20 can present cellular responses even in the absence of antibodies. CONCLUSION: Anti-CD20s and S1PRM modify the immunological responses to SARS-CoV-2 vaccines.

Case Report: X-Linked SASH3 Deficiency Presenting as a Common Variable Immunodeficiency.

SASH3 is a lymphoid-specific adaptor protein. In a recent study, SASH3 deficiency was described as a novel X-linked combined immunodeficiency with immune dysregulation, associated with impaired TCR signaling and thymocyte survival in humans. The small number of patients reported to date showed recurrent sinopulmonary, cutaneous and mucosal infections, and autoimmune cytopenia. Here we describe an adult patient previously diagnosed with common variable immunodeficiency (CVID) due to low IgG and IgM levels and recurrent upper tract infections. Two separate, severe viral infections drew our attention and pointed to an underlying T cell defect: severe varicella zoster virus (VZV) infection at the age of 4 years and bilateral pneumonia due type A influenza infection at the age of 38. Genetic testing using an NGS-based custom-targeted gene panel revealed a novel hemizygous loss-of-function variant in the SASH3 gene (c.505C>T/p.Gln169*). The patient's immunological phenotype included marked B cell lymphopenia with reduced pre-switch and switch memory B cells, decreased CD4+ and CD8+ naïve T cells, elevated CD4+ and CD8+ TEMRA cells, and abnormal T cell activation and proliferation. The patient showed a suboptimal response to Streptococcus pneumoniae (polysaccharide) vaccine, and a normal response to Haemophilus influenzae type B (conjugate) vaccine and SARS-CoV-2 (RNA) vaccine. In summary, our patient has a combined immunodeficiency, although he presented with a phenotype resembling CVID. Two severe episodes of viral infection alerted us to a possible T-cell defect, and genetic testing led to SASH3 deficiency. Our patient displays a milder phenotype than has been reported previously in these patients, thus expanding the clinical spectrum of this recently identified inborn error of immunity.

Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients.

BACKGROUND: SARS-CoV-2 infection portends a broad range of outcomes, from a majority of asymptomatic cases to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty, and co-morbidities such as obesity, diabetes, and cardiovascular disease. A precise knowledge of the molecular and biological mechanisms that may explain the association of severe disease with male sex is still lacking. Here, we analyzed the relationship of serum testosterone levels and the immune cell skewing with disease severity in male COVID-19 patients. METHODS: Biochemical and hematological parameters of admission samples in 497 hospitalized male and female COVID-19 patients, analyzed for associations with outcome and sex. Longitudinal (in-hospital course) analyses of a subcohort of 114 male patients were analyzed for associations with outcome. Longitudinal analyses of immune populations by flow cytometry in 24 male patients were studied for associations with outcome. RESULTS: We have found quantitative differences in biochemical predictors of disease outcome in male vs. female patients. Longitudinal analyses in a subcohort of male COVID-19 patients identified serum testosterone trajectories as the strongest predictor of survival (AUC of ROC = 92.8%, p < 0.0001) in these patients among all biochemical parameters studied, including single-point admission serum testosterone values. In lethal cases, longitudinal determinations of serum luteinizing hormone (LH) and androstenedione levels did not follow physiological feedback patterns. Failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and augmented circulating classical monocytes. CONCLUSIONS: Recovery or failure to reinstate testosterone levels is strongly associated with survival or death, respectively, from COVID-19 in male patients. Our data suggest an early inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by full recovery in survivors or a peripheral failure in lethal cases. These observations are suggestive of a significant role of testosterone status in the immune responses to COVID-19 and warrant future experimental explorations of mechanistic relationships between testosterone status and SARS-CoV-2 infection outcomes, with potential prophylactic or therapeutic implications.

Humoral and Cellular Responses to SARS-CoV-2 in Convalescent COVID-19 Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Information about humoral and cellular responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and antibody persistence in convalescent (COVID-19) patients with multiple sclerosis (PwMS) is scarce. The objectives of this study were to investigate factors influencing humoral and cellular responses to SARS-CoV-2 and its persistence in convalescent COVID-19 PwMS. METHODS: This is a retrospective study of confirmed COVID-19 convalescent PwMS identified between February 2020 and May 2021 by SARS-CoV-2 antibody testing. We examined relationships between demographics, MS characteristics, disease-modifying therapy (DMT), and humoral (immunoglobulin G against spike and nucleocapsid proteins) and cellular (interferon-gamma [IFN-γ]) responses to SARS-CoV-2. RESULTS: A total of 121 (83.45%) of 145 PwMS were seropositive, and 25/42 (59.5%) presented a cellular response up to 13.1 months after COVID-19. Anti-CD20-treated patients had lower antibody titers than those under other DMTs (p < 0.001), but severe COVID-19 and a longer time from last infusion increased the likelihood of producing a humoral response. IFN-γ levels did not differ among DMT. Five of 7 (71.4%) anti--CD20-treated seronegative patients had a cellular response. The humoral response persisted for more than 6 months in 41/56(81.13%) PwMS. In multivariate analysis, seropositivity decreased due to anti-CD20 therapy (OR 0.08 [95% CI 0.01-0.55]) and increased in males (OR 3.59 [1.02-12.68]), whereas the cellular response decreased in those with progressive disease (OR 0.04 [0.001-0.88]). No factors were associated with antibody persistence. DISCUSSION: Humoral and cellular responses to SARS-CoV-2 are present in COVID-19 convalescent PwMS up to 13.10 months after COVID-19. The humoral response decreases under anti-CD20 treatment, although the cellular response can be detected in anti-CD20-treated patients, even in the absence of antibodies.