Durability of COVID-19 humoral immunity post infection and different SARS-COV-2 vaccines.

BACKGROUND: The global challenge posed by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been a major concern for the healthcare sector in recent years. Healthcare workers have a relatively high risk of encountering COVID-19 patients, making protective immunity against SARS-CoV-2 is a priority for them. This study aims to evaluate the longitudinal measurement of SARS-CoV-2 IgG spike protein antibodies in healthcare workers (HCWs) after COVID-19 infection and after receiving the first and second doses of SARS-CoV-2 vaccines, including Pfizer-BioNTech (BNT162b2) and Oxford-AstraZeneca (AZD1222). METHODS: This longitudinal cohort study involved 311 healthcare workers working in two tertiary hospitals in Saudi Arabia. All participants were followed between July 2020 and July 2022 after completing the study questionnaire. A total of 3 ml of the blood samples were collected at four intervals: before/after vaccination. RESULTS: HCWs post-infection had lower mean SARS-CoV-2 IgG levels three months post-infection than post-vaccination. 92.2% had positive IgG levels two weeks after the first dose and reached 100% after the second dose. Over 98% had positive antibodies nine months after the second dose, regardless of vaccine type. The number of neutralizing antibodies decreased and was around 50% at nine months after the second dose. CONCLUSION: The results show different antibody patterns between infected and vaccinated HCWs. A high proportion of participants had positive antibodies after vaccination, with high levels persisting nine months after the second dose. Neutralizing antibodies decreased over time, with only about 50% of participants having positive antibodies nine months after the second dose. These results contribute to our understanding of immunity in healthcare workers and highlight the need for the continuous monitoring and possible booster strategies.

Novel Presentation of Major Histocompatibility Complex Class II Deficiency with Hemophagocytic Lymphohistiocytosis.

PURPOSE: Major histocompatibility complex (MHC) class II deficiency is one of the combined immune deficiency disorders caused by defects in the MHC class II regulatory genes leading to abnormal T cells development and function. Therefore, patients mainly present with increased susceptibility to infections, diarrhea, and failure to thrive. In this report, we present one MHC class II deficient patient with a novel presentation with Hemophagocytic Lymphohistiocytosis (HLH). METHODS: Immunophenotyping of lymphocyte subpopulations and HLA-DR expression was assess by flow cytometry. Gene mutational analysis was performed by whole exome and Sanger sequencing. RESULTS: We reported a 7-year-old girl, who was diagnosed at age of 2 years with MHC class II deficiency by genetic testing and flow cytometry. Two years later, she developed disseminated BCGitis which was treated with proper antimicrobial agents. At the age of 7 years, she presented with clinical features fulfilling 6 diagnostic criteria of HLH including evidence of hemophagocytic activity in bone marrow aspiration. Accordingly, the diagnosis of HLH was established and the patient was started on IV Dexamethasone, Anakinra and IVIG. Eventually, patient started to improve and was discharged in good condition. Few months later, the patient was readmitted with severe pneumonia and sepsis leading to death. CONCLUSION: Patients with MHC class II deficiency might present with disseminated BCGitis especially if the patient has severe T cell lymphopenia. Additionally, this immune defect might be added to the list of inborn errors of immunity that can be complicated with HLH.

Repurposing the oncolytic virus VSV∆51M as a COVID-19 vaccine.

The coronavirus disease 2019 (COVID-19) pandemic imposes an urgent and continued need for the development of safe and cost-effective vaccines to induce preventive responses for limiting major outbreaks around the world. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we repurposed the VSV∆51M oncolytic virus platform to express the spike receptor-binding domain (RBD) antigen. In this study, we report the development and characterization of the VSV∆51M-RBD vaccine. Our findings demonstrate successful expression of the RBD gene by the VSV∆51M-RBD virus, inducing anti-RBD responses without attenuating the virus. Moreover, the VSV∆51M-RBD vaccine exhibited safety, immunogenicity, and the potential to serve as a safe and effective alternative or complementary platform to current COVID-19 vaccines.

Quantitative proteomics analysis of COVID-19 patients: Fetuin-A and tetranectin as potential modulators of innate immune responses.

Treatment of severe cases of coronavirus disease 2019 (COVID-19) is extremely important to minimize death and end-organ damage. Here we performed a proteomic analysis of plasma samples from mild, moderate and severe COVID-19 patients. Analysis revealed differentially expressed proteins and different therapeutic potential targets related to innate immune responses such as fetuin-A, tetranectin (TN) and paraoxonase-1 (PON1). Furthermore, protein changes in plasma showed dysregulation of complement and coagulation cascades in COVID-19 patients compared to healthy controls. In conclusion, our proteomics data suggested fetuin-A and TN as potential targets that might be used for diagnosis as well as signatures for a better understanding of the pathogenesis of COVID-19 disease.

SARS-CoV-2 RBD protein enhances the oncolytic activity of the vesicular stomatitis virus.

Despite recent advances in the research on oncolytic viruses (OVs), a better understanding of how to enhance their replication is key to improving their therapeutic index. Understanding viral replication is important to improve treatment outcomes based on enhanced viral spreading within the tumor milieu. The VSV-Δ51 oncolytic virus has been widely used as an anticancer agent with a high selectivity profile. In this study, we examined the role of the SARS-CoV-2 spike protein receptor-binding domain (RBD) in enhancing VSV-Δ51 viral production and oncolytic activity. To test this hypothesis, we first generated a novel VSV-Δ51 mutant that encoded the SARS-COV-2 RBD and compared viral spreading and viral yield between VSV-Δ51-RBD and VSV-Δ51 in vitro. Using the viral plaque assay, we demonstrated that the presence of the SARS-CoV-2 RBD in the VSV-Δ51 genome is associated with a significantly larger viral plaque surface area and significantly higher virus titers. Subsequently, using an ATP release-based assay, we demonstrated that the SARS-CoV-2 RBD could enhance VSV-Δ51 oncolytic activity in vitro. This observation was further supported using the B16F10 tumor model. These findings highlighted a novel use of the SARS-CoV-2 RBD as an anticancer agent.

The Middle East and North Africa Diagnosis and Management Guidelines for Inborn Errors of Immunity.

Human inborn errors of immunity (IEI) are a group of 485 distinct genetic disorders affecting children and adults. Signs and symptoms of IEI are heterogeneous, and accurate diagnosis can be challenging and depends on the available human expertise and laboratory resources. The Middle East and North Africa (MENA) region has an increased prevalence of IEI because of the high rate of consanguinity with a predominance of autosomal recessive disorders. This area also exhibits more severe disease phenotypes compared with other regions, probably due to the delay in diagnosis. The MENA-IEI registry network has designed protocols and guidelines for the diagnosis and treatment of IEI, taking into consideration the variable regional expertise and resources. These guidelines are primarily meant to improve the care of patients within the region, but can also be followed in other regions with similar patient populations.

Angiotensin II Exaggerates SARS-CoV-2 Specific T-Cell Response in Convalescent Individuals following COVID-19.

Dysregulation of renin-angiotensin systems during coronavirus disease 2019 (COVID-19) infection worsens the symptoms and contributes to COVID-19 severity and mortality. This study sought to investigate the effect of exogenous angiotensin II (Ang-II) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cells response in recovered COVID-19 patients. Human peripheral blood mononuclear cells (PBMCs) were treated with Ang II and then stimulated with a SARS-CoV-2 peptide pool. T-cell responses were measured using flow cytometry, while enzyme-linked immunosorbent assay (ELISA) and intracellular cytokine staining (ICS) assays determined functional capability and polarization. Additionally, the relative level of protein phosphorylation was measured using a phosphokinase array. Our results showed that Ang II treatment significantly increased the magnitude of SARS-CoV-2-specific T-cell response in stimulated PBMCs with a SARS-CoV-2 peptide pool. Moreover, the phosphorylation levels of numerous proteins implicated in cardiovascular diseases, inflammation, and viral infection showed significant increases in the presence of Ang II. The mitogenic stimulation of PBMCs after Ang II and SARS-CoV-2 peptide pool stimulation showed functional polarization of T-cells toward Th1/Th17 and Th17 phenotypes, respectively. Meanwhile, ELISA showed increased productions of IL-1ß and IL-6 in Ang II-stimulated PBMCs without affecting the IL-10 level. To our knowledge, this study is the first to demonstrate that Ang II exaggerates SARS-CoV-2-specific T-cells response. Therefore, during COVID-19 infection, Ang II may aggravate the inflammatory response and change the immune response toward a more inflammatory profile against SARS-CoV-2 infection.

SARS-CoV-2 Coronavirus Spike Protein-Induced Apoptosis, Inflammatory, and Oxidative Stress Responses in THP-1-Like-Macrophages: Potential Role of Angiotensin-Converting Enzyme Inhibitor (Perindopril).

A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process. Graphical: Suggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).

Seroprevalence of SARS-CoV-2 among high-risk healthcare workers in a MERS-CoV endemic area.

INTRODUCTION: Healthcare workers (HCWs) in Saudi Arabia are a unique population who have had exposures to the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It follows that HCWs from this country could have pre-existingMERS-CoV antibodies that may either protect from coronavirus disease 2019 (COVID-19) infection or cause false SARS-CoV-2 seropositive results. In this article, we report the seroprevalence of MERS-CoV and SARS-CoV-2 among high-risk healthcare workers in Riyadh city, Saudi Arabia. METHODS: This is a cross-sectional study enrolling 420 high-risk HCWs who are physically in contact with COVID-19 patients in three tertiary hospitals in Riyadh city. The participants were recruited between the 1st of July to the end of December 2020. A 3 ml of the venous blood samples were collected and tested for the presence of IgG antibodies against the spike proteins of SARS-CoV-2 and MERS-CoV using enzyme-linked immunosorbent assay (ELISA). RESULTS: The overall prevalence of SARS-CoV-2 in high-risk HCWs was 14.8% based on SARS-CoV-2 IgG testing while only 7.4% were positive by Polymerase Chain Reaction (PCR) for viral RNA. Most of the SARS-CoV-2 seropositive HCWs had symptoms and the most frequent symptoms were body aches, fever, cough, loss of smell and taste, and headache. The seroprevalence of MERS-CoV IgG was 1% (4 participants) and only one participant had dual seropositivity against MERS-CoV and SARS-CoV-2. Three MERS-CoV positive samples (75%) turned to be negative after using in-house ELISA and none of the MERS-CoV seropositive samples had detectable neutralization activity. CONCLUSION: Our SARS-CoV-2 seroprevalence results were higher than reported regional seroprevalence studies. This finding was expected and similar to other international findings that targeted high-risk HCWs. Our results provide evidence that the SARS-CoV-2- seropositivity in Saudi Arabia similar to other countries was due to exposure to SARS-CoV-2 rather than MERS-CoV antibody.

Nationwide Seroprevalence of SARS-CoV-2 in Saudi Arabia.

BACKGROUND: Estimated seroprevalence of Coronavirus Infectious Disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is a critical evidence for a better evaluation of the virus spread and monitoring the progress of COVID-19 pandemic in a population. In the Kingdom of Saudi Arabia (KSA), SARS-CoV-2 seroprevalence has been reported in specific regions, but an extensive nationwide study has not been reported. Here, we report a nationwide study to determine the prevalence of SARS-CoV-2 in the population of KSA during the pandemic, using serum samples from healthy blood donors, non-COVID patients and healthcare workers (HCWs) in six different regions of the kingdom, with addition samples from COVID-19 patients. METHODS: A total of 11,703 serum samples were collected from different regions of the KSA including; 5395 samples from residual healthy blood donors (D); 5877 samples from non-COVID patients collected through residual sera at clinical biochemistry labs from non-COVID patients (P); and 400 samples from consented HCWs. To determine the seroprevalence of SARS-CoV-2, all serum samples, in addition to positive control sera from RT-PCR confirmed COVID-19 patients, were subjected to in-house ELISA with a sample pooling strategy, which was further validated by testing individual samples that make up some of the pools, with a statistical estimation method to report seroprevalence estimates. RESULTS: Overall (combining D and P groups) seroprevalence estimate was around 11% in Saudi Arabia; and was 5.1% (Riyadh), 1.5% (Jazan), 18.4% (Qassim), 20.8% (Hail), 14.7% (ER; Alahsa), and 18.8% in Makkah. Makkah samples were only D group and had a rate of 24.4% and 12.8% in the cities of Makkah and Jeddah, respectively. The seroprevalence in Saudi Arabia across the sampled areas would be 12 times the reported COVID-19 infection rate. Among HCWs, 7.5% (4.95-10.16 CI 95%) had reactive antibodies to SARS-CoV-2 without reporting any previously confirmed infection. This was higher in HCWs with hypertension. The study also presents the demographics and prevalence of co-morbidities in HCWs and subset of non-COVID-19 population. INTERPRETATION: Our study estimates the overall national serological prevalence of COVID-19 in Saudi Arabia to be 11%, with an apparent disparity between regions. This indicates the prevalence of asymptomatic or mild unreported COVID-19 cases.

Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Classical and Counter-Regulatory Renin-Angiotensin System: Potential Key Roles in COVID-19 Pathophysiology.

In the current COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 uses angiotensin-converting enzyme-2 (ACE-2) receptors for cell entry, leading to ACE-2 dysfunction and downregulation, which disturb the balance between the classical and counter-regulatory renin-angiotensin system (RAS) in favor of the classical RAS. RAS dysregulation is one of the major characteristics of several cardiovascular diseases; thus, adjustment of this system is the main therapeutic target. RAS inhibitors-particularly angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 receptor blockers (ARBs)-are commonly used for treatment of hypertension and cardiovascular disease. Patients with cardiovascular diseases are the group most commonly seen among those with COVID-19 comorbidity. At the beginning of this pandemic, a dilemma occurred regarding the use of ACEIs and ARBs, potentially aggravating cardiovascular and pulmonary dysfunction in COVID-19 patients. Urgent clinical trials from different countries and hospitals reported that there is no association between RAS inhibitor treatment and COVID-19 infection or comorbidity complication. Nevertheless, the disturbance of the RAS that is associated with COVID-19 infection and the potential treatment targeting this area have yet to be resolved. In this review, the link between the dysregulation of classical RAS and counter-regulatory RAS activities in COVID-19 patients with cardiovascular metabolic diseases is investigated. In addition, the latest findings based on ACEI and ARB administration and ACE-2 availability in relation to COVID-19, which may provide a better understanding of the RAS contribution to COVID-19 pathology, are discussed, as they are of the utmost importance amid the current pandemic.

Dans l'actuelle pandémie de la COVID-19, le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) utilise les récepteurs de l'enzyme de conversion de l'angiotensine 2 (ECA-2) pour entrer dans les cellules, s'ensuit le dysfonctionnement et la régulation à la baisse de l'ECA-2, qui perturbent l'équilibre entre le système rénine-angiotensine (SRA) traditionnel et le SRA contre-régulateur en faveur du SRA traditionnel. La dysrégulation du SRA est l'une des caractéristiques principales des maladies cardiovasculaires. Par conséquent, l'ajustement de ce système est l'objectif thérapeutique principal. Les inhibiteurs du SRA, particulièrement les inhibiteurs de l'ECA (IECA) et les antagonistes des récepteurs de type 1 de l'angiotensine II (ARA), sont communément utilisés pour traiter l'hypertension et les maladies cardiovasculaires. Les patients atteints de maladies cardiovasculaires représentent le groupe le plus fréquemment observé parmi les patients atteints de comorbidités associées à la COVID-19. Au début de la pandémie, un dilemme à propos de l'utilisation des IECA et des ARA s'est posé, puisqu'ils aggravaient potentiellement la dysfonction cardiovasculaire et pulmonaire chez les patients atteints de la COVID-19. Des essais cliniques urgents issus de différents pays et hôpitaux ont montré qu'il n'y avait pas d'association entre le traitement par inhibiteurs du SRA et les complications liées à l'infection par la COVID-19 ou aux comorbidités. Néanmoins, la perturbation du SRA qui est associée à l'infection par la COVID-19 et le traitement potentiel dans ce champ restent à résoudre. Dans la présente revue, le lien entre la dysrégulation du SRA traditionnel et les activités contre-régulatrices du SRA chez les patients atteints de la COVID-19 qui ont des maladies cardiovasculaires métaboliques est étudié. De plus, nous nous penchons sur les plus récentes conclusions fondées sur l'administration des IECA et des ARA et la disponibilité de l'ECA2 en relation avec la COVID-19 pour offrir une meilleure compréhension de la contribution du SRA à la pathologie de la COVID-19, puisqu'ils sont de la plus haute importance dans le contexte de l'actuelle pandémie.

NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.

Homozygous truncating NEK10 mutation, associated with primary ciliary dyskinesia: a case report.

BACKGROUND: Primary Ciliary Dyskinesia (PCD) is also known as immotile-cilia syndrome, an autosomal recessive disorder of ciliary function, leading to mucus retention in the respiratory system in childhood. Our knowledge in the pathophysiological aspect of this devastating disorder is increasing with the advancement of genetic and molecular testing. CASE PRESENTATION: Here in, we report two siblings with a classical clinical and radiological presentation of PCD. Using whole exome sequencing we identified a homozygous truncating variant (c.3402 T > A); p.(Tyr1134*) in the NEK10 gene. Western bolt analysis revealed a decrease in the expression of NEK10 protein in the patient cells. CONCLUSIONS: NEK10 plays a central role in the post-mitotic process of cilia assembly, regulating ciliary length and functions during physiological and pathological status. This study highlights the challenges of identifying disease-causing variants for a highly heterogeneous disorder and reports on the identification of a novel variant in NEK10 which recently associated with PCD.

Successful hematopoietic stem cell transplant in leukocyte adhesion deficiency type III presenting primarily as malignant infantile osteopetrosis.

Leukocyte adhesion deficiency type III (LAD-III) is caused by mutations in FERMT3 that encodes Kindlin-3 which regulates integrins activation. LAD-III predisposes to infections and bleeding. Osteopetrosis was reported in some cases. We report three patients who presented as malignant infantile osteopetrosis. One had recurrent infections and none had bleeding. Exome sequencing revealed a novel homozygous mutation in FERMT3 c.1555C > T (p.Gln519Ter). Two patients underwent successful hematopoietic stem cell transplant (HSCT) from matched siblings with resolution of osteopetrosis. The third patient died secondary to sepsis prior to HSCT. Our results support early HSCT in LAD-III prior to the occurrence of life-threatening complications.

Hypomorphic variants in AK2 reveal the contribution of mitochondrial function to B-cell activation.

BACKGROUND: The gene AK2 encodes the phosphotransferase adenylate kinase 2 (AK2). Human variants in AK2 cause reticular dysgenesis, a severe combined immunodeficiency with agranulocytosis, lymphopenia, and sensorineural deafness that requires hematopoietic stem cell transplantation for survival. OBJECTIVE: We investigated the mechanisms underlying recurrent sinopulmonary infections and hypogammaglobulinemia in 15 patients, ranging from 3 to 34 years of age, from 9 kindreds. Only 2 patients, both of whom had mildly impaired T-cell proliferation, each had a single clinically significant opportunistic infection. METHODS: Patient cells were studied with next-generation DNA sequencing, tandem mass spectrometry, and assays of lymphocyte and mitochondrial function. RESULTS: We identified 2 different homozygous variants in AK2. AK2G100S and AK2A182D permit residual protein expression, enzymatic activity, and normal numbers of neutrophils and lymphocytes. All but 1 patient had intact hearing. The patients' B cells had severely impaired proliferation and in vitro immunoglobulin secretion. With activation, the patients' B cells exhibited defective mitochondrial respiration and impaired regulation of mitochondrial membrane potential and quality. Although activated T cells from the patients with opportunistic infections demonstrated impaired mitochondrial function, the mitochondrial quality in T cells was preserved. Consistent with the capacity of activated T cells to utilize nonmitochondrial metabolism, these findings revealed a less strict cellular dependence of T-cell function on AK2 activity. Chemical inhibition of ATP synthesis in control T and B cells similarly demonstrated the greater dependency of B cells on mitochondrial function. CONCLUSIONS: Our patients demonstrate the in vivo sequelae of the cell-specific requirements for the functions of AK2 and mitochondria, particularly in B-cell activation and antibody production.

Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation.

BACKGROUND: LPS-responsive beige-like anchor protein (LRBA) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) deficiencies give rise to overlapping phenotypes of immune dysregulation and autoimmunity, with dramatically increased frequencies of circulating follicular helper T (cTFH) cells. OBJECTIVE: We sought to determine the mechanisms of cTFH cell dysregulation in patients with LRBA deficiency and the utility of monitoring cTFH cells as a correlate of clinical response to CTLA4-Ig therapy. METHODS: cTFH cells and other lymphocyte subpopulations were characterized. Functional analyses included in vitro follicular helper T (TFH) cell differentiation and cTFH/naive B-cell cocultures. Serum soluble IL-2 receptor α chain levels and in vitro immunoglobulin production by cultured B cells were quantified by using ELISA. RESULTS: cTFH cell frequencies in patients with LRBA or CTLA4 deficiency sharply decreased with CTLA4-Ig therapy in parallel with other markers of immune dysregulation, including soluble IL-2 receptor α chain, CD45RO+CD4+ effector T cells, and autoantibodies, and this was predictive of favorable clinical responses. cTFH cells in patients with LRBA deficiency were biased toward a TH1-like cell phenotype, which was partially reversed by CTLA4-Ig therapy. LRBA-sufficient but not LRBA-deficient regulatory T cells suppressed in vitro TFH cell differentiation in a CTLA4-dependent manner. LRBA-deficient TFH cells supported in vitro antibody production by naive LRBA-sufficient B cells. CONCLUSIONS: cTFH cell dysregulation in patients with LRBA deficiency reflects impaired control of TFH cell differentiation because of profoundly decreased CTLA4 expression on regulatory T cells and probably contributes to autoimmunity in patients with this disease. Serial monitoring of cTFH cell frequencies is highly useful in gauging the clinical response of LRBA-deficient patients to CTLA4-Ig therapy.

DOCK8 Deficiency Presenting as an IPEX-Like Disorder.

PURPOSE: The dedicator of cytokinesis 8 (DOCK8) deficiency is an autosomal recessive-combined immunodeficiency whose clinical spectra include recurrent infections, autoimmunity, malignancies, elevated serum IgE, eczema, and food allergies. Here, we report on patients with loss of function DOCK8 mutations with profound immune dysregulation suggestive of an immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)-like disorder. METHODS: Immunophenotyping of lymphocyte subpopulations and analysis of DOCK8 protein expression were evaluated by flow cytometry. T regulatory (Treg) cells were isolated by cell sorting, and their suppressive activity was analyzed by flow cytometry. Gene mutational analysis was performed by whole-exome and Sanger sequencing. RESULTS: Patient 1 (P1) presented at 10 months of age with chronic severe diarrhea and active colitis in the absence of an infectious trigger, severe eczema with elevated serum IgE, and autoimmune hemolytic anemia, suggestive of an IPEX-related disorder. Whole-exome sequencing revealed a homozygous nonsense mutation in DOCK8 at the DOCK-homology region (DHR)-1 (c.1498C>T; p. R500X). Patient P2, a cousin of P1 who carries the same DOCK8 nonsense mutation, presented with eczema and recurrent ear infections in early infancy, and she developed persistent diarrhea by 3 years of age. Patient P3 presented with lymphoproliferation, severe eczema with allergic dysregulation, and chronic diarrhea with colitis. She harbored a homozygous loss of function DOCK8 mutation (c.2402 -1G→A). Treg cell function was severely compromised by both DOCK8 mutations. CONCLUSION: DOCK8 deficiency may present severe immune dysregulation with features that may overlap with those of IPEX and other IPEX-like disorders.

Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.

BACKGROUND: Gain-of-function (GOF) mutations in the human signal transducer and activator of transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity with impaired TH17 cell differentiation and exaggerated responsiveness to type I and II interferons. Allogeneic bone marrow transplantation has been attempted in severely affected patients, but outcomes have been poor. OBJECTIVE: We sought to define the effect of increased STAT1 activity on T helper cell polarization and to investigate the therapeutic potential of ruxolitinib in treating autoimmunity secondary to STAT1 GOF mutations. METHODS: We used in vitro polarization assays, as well as phenotypic and functional analysis of STAT1-mutated patient cells. RESULTS: We report a child with a novel mutation in the linker domain of STAT1 who had life-threatening autoimmune cytopenias and chronic mucocutaneous candidiasis. Naive lymphocytes from the affected patient displayed increased TH1 and follicular T helper cell and suppressed TH17 cell responses. The mutation augmented cytokine-induced STAT1 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reduced hyperresponsiveness to type I and II interferons, normalized TH1 and follicular T helper cell responses, improved TH17 differentiation, cured mucocutaneous candidiasis, and maintained remission of immune-mediated cytopenias. CONCLUSIONS: Autoimmunity and infection caused by STAT1 GOF mutations are the result of dysregulated T helper cell responses. Janus kinase inhibitor therapy could represent an effective targeted treatment for long-term disease control in severely affected patients for whom hematopoietic stem cell transplantation is not available.