Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients.

Introduction: Common Variable Immunodeficiency (CVID) patients are characterized by hypogammaglobulinemia and poor response to vaccination due to deficient generation of memory and antibody-secreting B cells. B lymphocytes are essential for the development of humoral immune responses, and mitochondrial function, hreactive oxygen species (ROS) production and autophagy are crucial for determining B-cell fate. However, the role of those basic cell functions in the differentiation of human B cells remains poorly investigated. Methods: We used flow cytometry to evaluate mitochondrial function, ROS production and autophagy processes in human naïve and memory B-cell subpopulations in unstimulated and stimulated PBMCs cultures. We aimed to determine whether any alterations in these processes could impact B-cell fate and contribute to the lack of B-cell differentiation observed in CVID patients. Results: We described that naïve CD19+CD27- and memory CD19+CD27+ B cells subpopulations from healthy controls differ in terms of their dependence on these processes for their homeostasis, and demonstrated that different stimuli exert a preferential cell type dependent effect. The evaluation of mitochondrial function, ROS production and autophagy in naïve and memory B cells from CVID patients disclosed subpopulation specific alterations. Dysfunctional mitochondria and autophagy were more prominent in unstimulated CVID CD19+CD27- and CD19+CD27+ B cells than in their healthy counterparts. Although naïve CD19+CD27- B cells from CVID patients had higher basal ROS levels than controls, their ROS increase after stimulation was lower, suggesting a disruption in ROS homeostasis. On the other hand, memory CD19+CD27+ B cells from CVID patients had both lower ROS basal levels and a diminished ROS production after stimulation with anti-B cell receptor (BCR) and IL-21. Conclusion: The failure in ROS cell signalling could impair CVID naïve B cell activation and differentiation to memory B cells. Decreased levels of ROS in CVID memory CD19+CD27+ B cells, which negatively correlate with their in vitro cell death and autophagy, could be detrimental and lead to their previously demonstrated premature death. The final consequence would be the failure to generate a functional B cell compartment in CVID patients.

Elevated complement C3 and increased CD8 and type 1 helper lymphocyte T populations in patients with post-COVID-19 condition.

BACKGROUND: Biological markers associated to post-COVID-19 condition (PCC) have not been clearly identified. METHODS: Eighty-two patients attending our post-COVID-19 outpatient clinic were recruited and classified as fully recovered (40.2%) or presenting with PCC (59.8%). Clinical and radiological data, laboratory markers, cytokines, and lymphocyte populations were analyzed. RESULTS: Median number of days after hospitalization was 78.5 [p25-p75: 60-93] days. PCC was significantly more frequent in women, in patients with a previously critical COVID-19, and in those with two or more comorbidities. No differences were found in lymphocyte counts, ferritin, C-reactive protein, D-dimer or sCD25, IL-1ß, IL-1Ra, IL-6, CXCL8, IL-17A, IL-18, IL-22, IFN-γ, TNF-α, and IL-10 cytokines levels. PCC patients showed significantly higher levels of complement factor C3 than fully recovered patients: median C3 128 mg/dL [p25-p75:107-135] vs 111 mg/dL [p25-p75: 100-125] (p =.005), respectively. In the flow cytometry assessment of peripheral blood lymphocyte subpopulations, PCC patients showed significantly increased CD8 populations compared to fully recovered patients: median CD8: 529 [p25-p75: 384-683] vs 370/mm3 [p25-p75:280-523], p =.007. When type 1, 2, 17/22, and 17.1 helper and follicular T lymphocyte subpopulations were analyzed, the frequency of Th1 was significantly higher in PCC patients compared to fully recovered patients (30% vs 38.5%, p =.028). CONCLUSION: Patients with a post-COVID-19 condition showed significantly increased immunological parameters of inflammation (complement factor C3 and CD8 and Th1 T lymphocyte populations) compared to fully recovered patients. These parameters could be used as biological markers of this condition.

Hyperinflammatory State and Low T1 Adaptive Immune Response in Severe and Critical Acute COVID-19 Patients.

Background: A better understanding of COVID-19 immunopathology is needed to identify the most vulnerable patients and improve treatment options. Objective: We aimed to identify immune system cell populations, cytokines, and inflammatory markers related to severity in COVID-19. Methods: 139 hospitalized patients with COVID-19-58 mild/moderate and 81 severe/critical-and 74 recovered patients were included in a prospective longitudinal study. Clinical data and blood samples were obtained on admission for laboratory markers, cytokines, and lymphocyte subsets study. In the recovered patients, lymphocyte subsets were analyzed 8-12 weeks after discharge. Results: A National Early Warning Score 2 >2 (OR:41.4; CI:10.38-167.0), ferritin >583 pg/mL (OR:16.3; CI: 3.88-69.9), neutrophil/lymphocyte ratio >3 (OR: 3.5; CI: 1.08-12.0), sIL-2rα (sCD25) >512 pg/mL (OR: 3.3; CI: 1.48-7.9), IL-1Ra >94 pg/mL (OR: 3.2; IC: 1.4-7.3), and IL-18 >125 pg/mL (OR: 2.4; CI: 1.1-5.0) were associated with severe/critical COVID-19 in the multivariate models used. Lower absolute values of CD3, CD4, CD8, and CD19 lymphocytes together with higher frequencies of NK cells, a CD4 and CD8 activated (CD38+HLA-DR+) memory T cell and effector memory CD45RA+ (EMRA) phenotype, and lower T regulatory cell frequencies were found in severe/critical patients relative to mild/moderate and recovered COVID-19 patients. A significant reduction in Th1, Tfh1, and Tc1 with higher Th2, Tfh2, Tc2, and plasma cell frequencies was found in the most severe cases. Conclusion: A characteristic hyperinflammatory state with significantly elevated neutrophil/lymphocyte ratio and ferritin, IL-1Ra, sIL-2rα, and IL-18 levels together with a "low T1 lymphocyte signature" was found in severe/critical COVID-19 patients.

Paper biosensors for detecting elevated IL-6 levels in blood and respiratory samples from COVID-19 patients.

Decentralizing COVID-19 care reduces contagions and affords a better use of hospital resources. We introduce biosensors aimed at detecting severe cases of COVID-19 in decentralized healthcare settings. They consist of a paper immunosensor interfaced with a smartphone. The immunosensors have been designed to generate intense colorimetric signals when the sample contains ultralow concentrations of IL-6, which has been proposed as a prognosis biomarker of COVID-19. This is achieved by combining a paper-based signal amplification mechanism with polymer-filled reservoirs for dispensing antibody-decorated nanoparticles and a bespoken app for color quantification. With this design we achieved a low limit of detection (LOD) of 10-3 pg mL-1 and semi-quantitative measurements in a wide dynamic range between 10-3 and 102 pg mL-1 in PBS. The assay time is under 10 min. The low LOD allowed us to dilute blood samples and detect IL-6 with an LOD of 1.3 pg mL-1 and a dynamic range up to 102 pg mL-1. Following this protocol, we were able to stratify COVID-19 patients according to different blood levels of IL-6. We also report on the detection of IL-6 in respiratory samples (bronchial aspirate, BAS) from COVID-19 patients. The test could be easily adapted to detect other cytokines such as TNF-α and IL-8 by changing the antibodies decorating the nanoparticles accordingly. The ability of detecting cytokines in blood and respiratory samples paves the way for monitoring local inflammation in the lungs as well as systemic inflammation levels in the body.

IL-21 and anti-CD40 restore Bcl-2 family protein imbalance in vitro in low-survival CD27+ B cells from CVID patients.

Common variable immunodeficiency (CVID) is characterized by an abnormal B cell differentiation to memory and antibody-secreting B cells. The defective functionality of CVID patients' B cells could be the consequence of alterations in apoptosis regulation. We studied the balance of Bcl-2 family anti-/pro-apoptotic proteins to identify molecular mechanisms that could underlie B cell survival defects in CVID. We used flow cytometry to investigate Bcl-2, Bcl-XL, Bax, and Bim expression in B cells ex vivo and after anti-CD40 or anti-BCR activation with or without IL-21, besides to spontaneous and stimulation-induced Caspase-3 activation and viable/apoptotic B cell subpopulations. We found increased basal levels of Bax and Bim in CVID B cells that correlated with low viability and high Caspase-3 activation only in CD27+ B cells, particularly in a subgroup of apoptosis-prone CVID (AP-CVID) patients with low peripheral B cell counts and high autoimmunity prevalence (mostly cytopenias). We detected a broad B cell defect in CVID regarding Bcl-2 and Bcl-XL induction, irrespective of the stimulus used. Therefore, peripheral CVID memory B cells are prompted to die from apoptosis due to a constitutive Bcl-2 family protein imbalance and defective protection from activation-induced apoptosis. Interestingly, anti-CD40 and IL-21 induced normal and even higher levels of Bcl-XL, respectively, in CD27+ B cells from AP-CVID, which was accompanied by cell viability increase. Thus low-survival memory B cells from AP-CVID can overcome their cell death regulation defects through pro-survival signals provided by T cells. In conclusion, we identify apoptosis regulation defects as disease-contributing factors in CVID. B cell counts and case history of cytopenias might be useful to predict positive responses to therapeutic approaches targeting T-dependent signaling pathways.

Follicular T Cells from smB- Common Variable Immunodeficiency Patients Are Skewed Toward a Th1 Phenotype.

Germinal center follicular T helper (GCTfh) cells are essential players in the differentiation of B cells. Circulating follicular T helper (cTfh) cells share phenotypic and functional properties with GCTfh cells. Distinct subpopulations of cTfh with different helper capabilities toward B cells can be identified: cTfh1 (CXCR3+CCR6-), cTfh2 (CXCR3-CCR6-), and cTfh17 (CXCR3-CCR6+). Alterations in cTfh function and/or distribution have been associated with autoimmunity, infectious diseases, and more recently, with several monogenic immunodeficiencies. Common variable immunodeficiency (CVID) disease is the commonest symptomatic primary immunodeficiency with a genetic cause identified in only 2-10% of patients. Although a heterogeneous disease, most patients show a characteristic defective B cell differentiation into memory B cells or antibody-secreting cells. We investigated if alterations in CVID cTfh cells frequency or distribution into cTfh1, cTfh2, and cTfh17 subpopulations and regulatory follicular T (Tfr) cells could be related to defects in CVID B cells. We found increased percentages of cTfh exhibiting higher programmed death-1 expression and altered subpopulations distribution in smB- CVID patients. In contrast to smB+ patients and controls, cTfh from smB- CVID patients show increased cTfh1 and decreased cTfh17 subpopulation percentages and increased CXCR3+CCR6+ cTfh, a population analogous to the recently described pathogenic Th17.1. Moreover, Tfr cells are remarkably decreased only in smB- CVID patients. In conclusion, increased cTfh17.1 and cTfh1/cTfh17 ratio in CVID patients could influence B cell fate in smB- CVID patients, with a more compromised B cell compartment, and the decrease in Tfr cells may lead to high risk of autoimmune conditions in CVID patients.

Increased STAT3 phosphorylation on CD27(+) B-cells from common variable immunodeficiency disease patients.

Maturation and differentiation of B-cells are driven by T-cells' help through IL-21/STAT3 axis in GC centers or through extrafollicular pathways, in a T-independent manner. B-cell differentiation is defective in common variable immunodeficiency disease (CVID) patients. We investigated if IL-21/STAT3 axis alterations could influence B-cell fate. We activated purified CVID B-cells with surrogate T-dependent (anti-CD40), T-independent (TLR-9 ligand) stimuli or through B-cell receptor engagement (anti-IgM) with or without IL-21. IL-21 mediated STAT3 activation was greater on CD27(-) than CD27(+) B-cells depending on the stimulus. IL-21 alone induced STAT3 phosphorylation (pSTAT3) only on CD27(-) B-cells and IL-21 induced higher pSTAT3 levels on CD27(-) than CD27(+) B-cells after anti-IgM or anti-CD40 activation. CVID CD27(+) B-cells showed selective STAT3 hyperphosphorylation after activation with anti-IgM or anti-CD40 alone and anti-IgM, anti-CD40 or ODN combined with IL-21. Increased STAT3 activation during immune responses could result in B-cell differentiation defects in CVID.

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.