Clinical and Experimental Immunology

In order to explore the effect of IFN{beta}-1b treatment on B cell phenotype and function in RRMS patients, blood was drawn from RRMS patients before treatment and again 2 and 6 months after every other day injections of IFN{beta}-1b. Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and stained with panels of antibodies against B cell surface antigens and the intracellular cytokines, IL-10 and IL-6. At baseline, PBMCs from RRMS patients have increased frequencies of B1 cells and a decreased frequencies of memory B cells when compared with PBMCs from age- and gender-matched healthy controls. CpG-stimulated PBMCs from patients treated with IFN{beta}-1b show an increase in IL-10 production and a decrease in IL-6 production by naive, memory and B1 cells compared with healthy controls. In addition, this treatment alters the composition of circulating B cell subsets, leading to an increase after six months in circulating naive B cells and a decrease in both memory and B1 cells, both cell types of which are potentially pathogenic in RRMS. Patients were divided into two groups based on disease activity, no/low or moderate/high. A significantly higher frequency of B1 cells and higher expression of CD27 on these cells was seen at baseline in the moderate/high disease activity group to IFN{beta}-1b compared with patients in the no/low disease activity group. Although the number of subjects in this study was limited, these findings suggest that alterations in the B cell compartment may be a mechanism by which IFN{beta}-1b reduces disease activity.

ObjectiveSystemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune dysregulation and widespread inflammation. Natural killer (NK) cells, essential for immune surveillance, exhibit profound dysfunction in SLE, including impaired cytotoxicity and cytokine production. However, the mechanisms underlying these abnormalities remain poorly understood. This study investigates how the accumulation of dysfunctional mitochondria due to defective mitophagy contributes to NK cell impairment in SLE and explores strategies to restore their function. MethodsMitochondrial structure and function in NK cells from SLE patients (n=104) and healthy controls (n=104) were assessed using flow cytometry, transmission electron microscopy, and proteomics. Mitophagy-related gene expression was quantified by RT-qPCR. The effects of Urolithin A, a mitophagy activator, and hydroxychloroquine (HCQ) on mitochondrial recycling and NK cell function were evaluated in vitro. ResultsSLE NK cells exhibited accumulation of enlarged, dysfunctional mitochondria, impaired lysosomal acidification, and increased cytosolic mitochondrial DNA leakage, consistent with defective mitophagy. Proteomic and transcriptional analyses revealed downregulation of key mitophagy-related genes. These abnormalities were associated with diminished NK cell effector functions, including reduced degranulation and cytokine production. In vitro, treatment with Urolithin A enhanced mitophagy, improved mitochondrial and lysosomal function, and restored NK cell effector responses. HCQ was also associated with partial recovery of mitochondrial recycling and NK cell function. ConclusionThese findings identify mitochondrial dysfunction and impaired mitophagy as major contributors to NK cell abnormalities in SLE. By uncovering a novel immunometabolic mechanism, this offers new insight into SLE pathogenesis and highlights potential therapeutic strategies targeting mitochondrial quality control.

IntroductionGlobal WHO data indicate that Sub-Saharan African (SSA) countries, such as Kenya, experienced reduced coronavirus disease 2019 (COVID-19) severe-morbidity and mortality burdens relative to their more affluent counterparts in Europe, Asia, and North America. MethodsWe analysed peripheral blood mononuclear cells (PBMC) samples collected from Kenya and Sweden before and during COVID-19. Pre-COVID-19 samples were available for 80 adults and 10 infants from Kenya, and 20 adults from Sweden. COVID-19 samples were available for 39 Kenyan adults. The samples were analysed for ex vivo IFN-{gamma} secretion using an Enzyme-Linked Immunosorbent (ELISpot) assay following in vitro stimulations with overlapping SARS-CoV-2 spike-protein peptides. T-cells expressing IFN-{gamma}, IL-2, TNF-, CD154, and CD107a were assessed following similar stimulations, using intracellular cytokine staining (ICS) and multiparameter flow cytometry. Results55.7% of the Kenyan pre-COVID-19 adults were classified as responders by ELISPOT responses to spike-protein peptides, compared with 28% of Swedish pre-COVID-19 adults (p = 0.04). The frequencies for SARS-CoV-2 spike-specific TNF- CD4+, TNF- CD8+ and IFN-{gamma} CD8+ T-cell responses, tended to be higher in the Kenyan adults although these differences did not reach statistical significance. ConclusionPre-COVID-19 T-cell responses could contribute to lower morbidity and mortality associated with SARS-CoV-2 infections in SSA relative to Europe, Asia, and North America. HighlightsO_LIHigher frequencies of cross-reactive IFN-{gamma}-secreting cells among Kenyan pre-COVID-19 adults compared to Sweden. C_LIO_LITrends of higher TNF- and IFN-{gamma} T-cell responses among Kenyan adults. C_LIO_LIPresence of pre-existing T-cell immunity in Sub-Saharan Africa. C_LI

Recent evidence has shown that B cells may play a key role in the pathogenesis of relapsing-remitting multiple sclerosis. Studies in our laboratory have shown a difference in the production of IL-6 and IL-10 by B cells isolated from RRMS patients compared with healthy controls. In order to further characterize the nature of the B cells in RRMS patients, we analyzed samples from patients on no disease modifying treatment for B cell expression of multiple phenotypic and regulatory markers. We observed an increased frequency in the number of circulating B cells, an increase in B1 B cells and a decrease in memory B cells in RRMS patients. These B1 cells showed a significantly higher frequency of CD5 expression and the memory B cells a significant increase in the class-switched IgD-phenotype. We also examined death receptors involved in apoptotic pathways. CD95 frequency was significantly lower in RRMS patients tan healthy controls in all B cell subsets. Conversely, frequency of PD-1 was elevated in both the naive and memory B cell subsets, and PD-L1 was elevated in B1 cells from RRMS patients. Finally, we examined a series of immunoreceptor tyrosine-based inhibition motif (ITIM)-containing inhibitory receptors, including members of the SIGLEC family. Significantly higher levels of CD22, CD305 and CD307d were seen in RRMS patients, while significantly lower levels of SIGLEC-10 were observed. Taken together, these results indicate a potential for differential regulation of B cells in RRMS patients that may provide an avenue for B cell directed therapies for the disease.

Immune responses in people with multiple sclerosis (pwMS) on disease-modifying therapies (DMTs) have been of significant interest throughout the COVID-19 pandemic. Lymphocyte-targeting immunotherapies including anti-CD20 treatments and sphingosine-1-phosphate receptor (S1PR) modulators attenuate antibody responses after vaccination. Evaluation of cellular responses after vaccination is therefore of particular importance in these populations. In this study, we analysed CD4 and CD8 T cell functional responses to SARS-CoV-2 spike peptides in healthy controls and pwMS on five different DMTs by flow cytometry. Although pwMS on anti-CD20 and S1PR therapies had low antibody responses after both 2 and 3 vaccine doses, T cell responses in pwMS on anti-CD20 therapies were preserved after a third vaccination, even when additional anti-CD20 treatment was administered between vaccine doses 2 and 3. PwMS taking S1PR modulators had low detectable T cell responses in peripheral blood. CD4 and CD8 T cell responses to SARS-CoV-2 variants of concern Delta and Omicron were lower than to the ancestral Wuhan-Hu-1 variant. Our results indicate the importance of assessing both cellular and humoral responses after vaccination and suggest that even in the absence of robust antibody responses vaccination can generate immune responses in pwMS.

BackgroundEpstein-Barr virus (EBV) infection is a known trigger and risk factor for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-COVID syndrome (PCS). In previous studies, we found enhanced IgG reactivity to EBV EBNA4 and EBNA6 arginine-rich sequences in postinfectious ME/CFS (piME/CFS). ObjectiveThis study aims to investigate IgG responses to arginine-rich (poly-R) EBNA4 and EBNA6 sequences and homologous human sequences in PCS and ME/CFS. MethodsThe IgG responses against poly-R EBNA4 and EBNA6 and corresponding homologous human 15-mer peptides and respective full-length proteins were analyzed using a cytometric bead array (CBA) and a multiplex dot-blot assay. Sera of 45 PCS patients diagnosed according to WHO criteria, with 26 patients fulfilling the Canadian Consensus criteria for ME/CFS (pcME/CFS), 36 patients with non-COVID post-infectious ME/CFS (piME/CFS), and 34 healthy controls (HC) were investigated. ResultsAutoantibodies to poly-R peptide sequences of the neuronal antigen SRRM3, the ion channel SLC24A3, TGF-{beta} signaling regulator TSPLY2, angiogenic regulator TSPYL5, as well as to full-length -adrenergic receptor (ADRA) proteins were more frequent in patients. Several autoantibodies were positively associated with key symptoms of autonomic dysfunction, fatigue, cognition, and pain. ConclusionCollectively, we identified autoantibodies with new antigen specificities with a potential role in PCS and ME/CFS. Clinical ImplicationThese finding should prompt further studies on the function of these autoantibodies, their exploitation for diagnostic use, and of drugs targeting autoantibodies. Capsule summaryOur study reveals elevated autoantibodies to EBV-related poly-R sequences and their human homologues in PCS and ME/CFS patients associated with symptom severity, suggesting a potential role in disease pathogenesis.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating but poorly-understood disease. ME/CFS symptoms can range from mild to severe, and include immune system effects alongside incapacitating fatigue and post-exertional disease exacerbation. In this study, we examined immunological profiles of people living with ME/CFS by flow cytometry, focusing on cytotoxic cells, to determine whether people with mild/moderate (n= 43) or severe ME/CFS (n=53) expressed different immunological markers. We found that people with mild/moderate ME/CFS had increased expression of cytotoxic effector molecules alongside enhanced proportions of early-immunosenescence cells, determined by the CD28-CD57- phenotype, indicative of persistent viral infection. In contrast, people with severe ME/CFS had higher proportions of activated circulating lymphocytes, determined by CD69+ and CD38+ expression, and expressed more pro-inflammatory cytokines, including IFN{gamma}, TNF and IL-17, following stimulation in vitro, indicative of prolonged non-specific inflammation. These changes were consistent across different cell types including CD8+ T cells, mucosal associated invariant T cells and Natural Killer cells, indicating generalised altered cytotoxic responses across the innate and adaptive immune system. These immunological differences likely reflect different disease pathogenesis mechanisms occurring in the two clinical groups, opening up opportunities for the development of prognostic markers and stratified treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/24319359v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1e3f2e5org.highwire.dtl.DTLVardef@5fd9fborg.highwire.dtl.DTLVardef@e015eforg.highwire.dtl.DTLVardef@1c03ae1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

B cell depletion using rituximab is widely used to treat autoimmune diseases, but patient response varies. The efficacy of rituximab is limited by the efficiency of depletion. Strategies to improve response include altering rituximab dosing, switching anti-CD20-mAb, alternative B cell targets, or non-B cell targeted therapies. Implementing an appropriate strategy requires understanding of the mechanism(s) of resistance to depletion and, if this varies between individuals, a means to test for it. Rituximab kills B cells via a variety of Fc{gamma} receptor (Fc{gamma}R)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), as well as non-Fc{gamma}R mechanisms. We conducted a longitudinal cohort study in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) using two national registries. Qualitative and quantitative FCGR functional variants were measured using multiplexed ligation-dependent probe amplification, supplemented by novel FCGR2C assays. We provide consistent evidence that FCGR3A, specifically increased number of copies of the FCGR3A-158V allele, was the major Fc{gamma}R gene associated with rituximab response, including clinical response in RA and SLE and depth of B cell depletion in the combined cohort. In SLE, we provide preliminary data suggesting increased FCGR2C ORF copies were also associated with improved clinical response. Furthermore, we demonstrated the impact of disease status and concomitant therapies on both natural killer cell Fc{gamma}RIIIa expression and rituximab-induced ADCC; demonstrating increased Fc{gamma}RIIIa expression and FCGR3A genotype were independently associated with clinical response and B cell depletion. Our findings highlight the importance of enhancing Fc{gamma}R-effector functions, may help stratify patients, and support ongoing development of next-generation CD20 depleting therapeutics. One Sentence SummaryThe high affinity Fc{gamma}RIIIa allotype on NK cells explains depth of B cell depletion and clinical response in rituximab therapy for autoimmune disease

Rheumatoid arthritis (RA) in an autoimmune disease that leads to inflammation of synovial joints and other organs. Many RA patients share a common peptide sequence within the HLA-DR (DR) molecule expressed on antigen-presenting cells (APC), suggesting that DRhi cells are important in RA. Here, we use DRhi to broadly define and immunophenotype RA APC, including potential APC not meeting standard definitions for lymphocytes, monocytes, dendritic cells (DC) from RA patients and healthy controls (HC). We measured mean fluorescence intensities (MFI) of molecules associated with DC (CD141, CD1c, CD11c, CD123, CD303), monocytes (CD14, CD16); granulocytic markers (CD15, CCR3), co-stimulatory molecules and chemokine receptors. DC2 (CD1c+) showed higher CD56, CD86, CD275, and CCR7 in RA. DC2 frequencies were much lower in RA: 3.2% of DRhi [IQR 2.41 to 4.46] in RA vs. 6.9% [IQR 3.96 to 9.08] in HC; p=0.005. CD15 was increased in all RA APC subsets (p<0.01). A distinct CD15+CD16+ population appeared in RA, representing 1.5% of leukocytes [IQR 0.68 to 3.32] (vs 0.1% in HC [IQR 0.08 to 0.46]; p<0.001) and contributed a mean of 2.34% to overall DRhi. The CD15+CD16+ subset was CD303+, CD83+ and CD275+ with much less CD123 relative to reference plasmacytoid DC (p<0.01). In conclusion, APC alterations in RA include depletion of DC2 and increased CD15. Moreover, the APC (DRhi) compartment in RA contains cells with shared dendritic cell and granulocytic features; this phenotype suggests these apparent APC may participate in the pathophysiology of rheumatoid arthritis via the presentation of self-antigen(s) to CD4+ T lymphocytes.

ObjectivesAnti-type I interferon (IFN) autoantibodies have been reported in patients with systemic lupus erythematosus (SLE). Recently, an association of these autoantibodies with severe COVID-19 was reported in the general population. We assessed whether having pre-existing anti-IFN autoantibodies was associated with COVID-19 infection in SLE patients. MethodsPatients with SLE who developed COVID-19 between April 1st to October 1st, 2020 were studied. Biobanked pre-COVID-19 plasma from these SLE subjects and healthy controls were tested for anti-IFN IgG autoantibodies by ELISA. The ability of plasma anti-IFN autoantibodies to block signal transducer and activator of transcription 1 (STAT1) phosphorylation by recombinant human IFN in vitro was assessed by flow cytometry. ResultsTen SLE subjects with COVID-19 were identified. A 40% of these subjects had stable autoantibodies against IFN for up to three years preceding COVID-19 diagnosis. A 50% of the subjects with these autoantibodies neutralized IFN induced STAT1 phosphorylation.None of the other SLE samples blocked IFN signaling. ConclusionsWe noted an increased prevalence of pre-existing anti-IFN autoantibodies in SLE patients with COVID-19 compared to the reported prevalence in lupus patients and the general population with severe COVID-19. Autoantibodies against IFN in SLE patients may be pathogenic and patients with them maybe at-risk of developing COVID-19. Key MessagesO_ST_ABSWhat is already known about this subject?C_ST_ABS- Anti-type I interferon (IFN) autoantibodies have been reported in patients with systemic lupus erythematosus (SLE) and have recently been associated with severe COVID-19 in the general population. What does this study add?- SLE subjects with COVID-19 had an increased prevalence of pre-existing anti-IFN autoantibodies compared to the reported prevalence in lupus patients and the general population with severe COVID-19. - Plasma from 50% of subjects with these autoantibodies were able to block in vitro activity of IFN. - SLE patients with pre-existing anti-IFN autoantibodies had more severe COVID-19 manifestations. How might this impact on clinical practice or future developments?- Anti-IFN autoantibodies may be pathogenic and could prove to be a helpful prognostic marker to predict which SLE patient may develop COVID-19 and inform preventive measures and management of this subset of patients.

BACKGROUND AND AIMSVaccine-mediated immune responses in patients with inflammatory bowel disease (IBD) may be influenced by IBD therapies. We investigated in-depth humoral and T-cell responses to SARS-CoV-2 vaccination in IBD patients following three COVID-19 vaccine doses. METHODSImmune responses of 100 SARS-CoV-2-uninfected IBD patients on varying treatments were compared to healthy controls (n=35). Anti-S1/2 and anti-RBD SARS-CoV-2-specific antibodies, CD4+ and CD8+ T-cell responses were measured at baseline and at five time-points after COVID-19 vaccination. RESULTSAnti-S1/2 and anti-RBD antibody concentrations at [~]1 month after second dose vaccination were significantly lower in anti-TNF-treated patients compared to non-TNF IBD patients and healthy controls (126.4 vs 262.1 and 295.5, p<0.0001). Anti-S1/2 antibodies remained reduced in anti-TNF treated patients before and after the third dose (285.7 vs 365.3, p=0.03), although anti-RBD antibodies reached comparable titres to non-TNF patients. Anti-RBD antibodies were higher in the vedolizumab group than controls after second dose (4.2 vs 3.6, p=0.003). Anti-TNF monotherapy was associated with increased CD4+ and CD8+ T-cell activation compared to combination anti-TNF patients after second dose, but comparable after third dose. Overall, IBD patients demonstrated similar CD4+/CD8+ T-cell responses compared to healthy controls regardless of treatment regimen. CONCLUSIONSAnti-TNFs impaired antibody concentrations when compared to non-TNF patients and controls after two vaccine doses. These differences were not observed after the third vaccine dose. However, vaccine induced SARS-CoV-2-specific T cell responses are robust in anti-TNF-treated patients. Our study supports the need for timely booster vaccination particularly in anti-TNF treated patients to minimise the risk of severe SARS-CoV-2 infection.

BackgroundVaccination against SARS-CoV-2 is a highly effective strategy to protect against infection, which is predominantly mediated by vaccine-induced antibodies. Postvaccination antibodies are robustly produced by those with inflammatory bowel disease (IBD) even on immune-modifying therapies but are blunted by anti-TNF therapy. In contrast, T-cell response which primarily determines long-term efficacy against disease progression,, is less well understood. We aimed to assess the post-vaccination T-cell response and its relationship to antibody responses in patients with inflammatory bowel disease (IBD) on immune-modifying therapies. MethodsWe evaluated IBD patients who completed SARS-CoV-2 vaccination using samples collected at four time points (dose 1, dose 2, 2 weeks after dose 2, 8 weeks after dose 2). T-cell clonal analysis was performed by T-cell Receptor (TCR) immunosequencing. The breadth (number of unique sequences to a given protein) and depth (relative abundance of all the unique sequences to a given protein) of the T-cell clonal response were quantified using reference datasets and were compared to antibody responses. ResultsOverall, 303 subjects were included (55% female; 5% with prior COVID) (Table). 53% received BNT262b (Pfizer), 42% mRNA-1273 (Moderna) and 5% Ad26CoV2 (J&J). The Spike-specific clonal response peaked 2 weeks after completion of the vaccine regimen (3- and 5-fold for breadth and depth, respectively); no changes were seen for non-Spike clones, suggesting vaccine specificity. Reduced T-cell clonal depth was associated with chronologic age, male sex, and immunomodulator treatment. It was preserved by non-anti-TNF biologic therapies, and augmented clonal depth was associated with anti-TNF treatment. TCR depth and breadth were associated with vaccine type; after adjusting for age and gender, Ad26CoV2 (J&J) exhibited weaker metrics than mRNA-1273 (Moderna) (p=0.01 for each) or BNT262b (Pfizer) (p=0.056 for depth). Antibody and T-cell responses were only modestly correlated. While those with robust humoral responses also had robust TCR clonal expansion, a substantial fraction of patients with high antibody levels had only a minimal T-cell clonal response. ConclusionAge, sex and select immunotherapies are associated with the T-cell clonal response to SARS-CoV-2 vaccines, and T-cell responses are low in many patients despite high antibody levels. These factors, as well as differences seen by vaccine type may help guide reimmunization vaccine strategy in immune-impaired populations. Further study of the effects of anti-TNF therapy on vaccine responses are warranted.

ObjectiveT helper cells develop into discrete Th1, Th2 or Th17 lineages that selectively express IFN{gamma}, IL-4/IL-5/IL-13, or IL-17, respectively and actively silence signature cytokines expressed by opposing lineages. Our objective was to compare Th1, Th2 and Th17 polarization in cell culture models using JIA patient samples. MethodsPeripheral blood mononuclear cells were isolated from JIA or healthy prepubescent children. T cell naive and memory phenotypes were assessed by flow cytometry. T cell proliferation was measured using a fluorescence-based assay. Th cell cultures were generated in vitro and IFN{gamma}, IL-17, and TNF measured by ELISA and flow cytometry. ResultsJIA Th1 cells produced increased IFN{gamma} and inappropriately produced IL-17. JIA Th17 cells produced increased IL-17. JIA Th1 cell cultures develop dual producers of IFN{gamma} and IL-17, which are Th1.17 cells. JIA Th1 cultures expressed elevated levels of both T-bet and ROR{gamma}T. RNA sequencing confirmed activation of immune responses and inappropriate activation of IL-17 signaling pathways in Th1 cultures. A subset of JIA patient samples was disproportionally responsible for the enhanced IFN{gamma} and IL-17 phenotype and Th1.17 phenotype. ConclusionsThis study reveals that JIA patient uncommitted T cell precursors, but not healthy children, inappropriately develop into inflammatory effector Th1.17 and Th17 cells under Th1 polarizing conditions. Rheumatology key messagesO_LITh1 differentiation of JIA PBMCs generates high IFN{gamma}, IL-17, and dual IFN{gamma}-IL-17 producing cells. C_LIO_LIJIA Th1 differentiation increases master transcription factor expression for Tbet and ROR{gamma}T. C_LIO_LIEnhanced JIA Th1 IFN{gamma} and IL-17 production occurs in a subset of JIA patients. C_LI

Background and aimThe distinction of drug-induced liver injury (DILI), drug-induced autoimmune-like hepatitis (DI-ALH) and autoimmune hepatitis (AIH) can be challenging due to overlapping clinical characteristics. Recently, polyreactive immunoglobulin G (pIgG) was identified as a novel biomarker with a higher accuracy for the diagnose of AIH than conventional autoantibodies. This retrospective multicenter study aimed to evaluate the diagnostic accuracy of pIgG to distinguish between AIH, DI-ALH and DILI and thus identify patients in need of immunosuppression. MethodsSamples from 116 patients (AIH=81, DI-ALH=12, DILI=23) were recruited and compared to a control group (non-AIH-non-DILI-LD= 596) from existing biorepositories. ResultsNo patient in the DILI-group but 98% in the AIH-and 92% in the DI-ALH-group received immunosuppressive treatment. pIgG levels were significantly higher in the AIH-group (1.9 normalized arbitrary units (nAU) compared to DILI (1.1 nAU, p<0.001) and non-AIH-non-DILI-LD (1.0 nAU, p<0.001). Median pIgG concentrations of the DI-ALH-group (1.7 nAU) were between AIH (p=.634) and DILI (p=.052). Patients that needed immunosuppressive therapy for remission induction had significantly higher pIgG concentrations compared to those with spontaneous recovery of liver injury (1.8 nAU vs. 1.1 nAU, p<.001). The overall accuracy of pIgG >1.27nAU to distinguish AIH from DILI (74%) and liver injuries with and without the need for immunosuppression (74%) was similar to that of ANA (71/74%) and SMA (74/70%) at cut-offs of [&ge;]1/40. ConclusionPolyreactive IgG can be used to predict AIH in comparison to DILI and indicate the need for immunosuppressive therapy in the work-up of immune mediated or drug-induced liver injuries.

We report SARS-CoV-2 vaccine-induced immunity and risk of breakthrough infections in patients with inflammatory bowel disease treated with infliximab, a commonly used anti-TNF drug and those treated with vedolizumab, a gut-specific antibody targeting integrin a4b7 that does not impact systemic immunity. In infliximab-treated patients, the magnitude of anti-SARS-CoV2 antibodies was reduced 4-6-fold. One fifth of both infliximab- and vedolizumab-treated patients did not mount a T cell response. Antibody half-life was shorter in infliximab-treated patients. Breakthrough SARS-CoV-2 infections occurred more frequently in infliximab-treated patients and the risk was predicted by the level of antibody response after second vaccine dose. Overall, recipients of two doses of the BNT162b2 vaccine had higher anti-SARS-CoV-2 antibody concentrations, higher seroconversion rates, shorter antibody half-life and less breakthrough infections compared to ChAdOx1 nCoV-19 vaccine recipients. Irrespective of biologic treatment, higher, more sustained antibody levels were observed in patients with a history of SARS-CoV-2 infection prior to vaccination. Patients treated with anti-TNF therapy should be offered third vaccine doses.

Type I interferons are essential for host response to viral infections, while dysregulation of their response can result in autoinflammation or autoimmunity. Among IFN (alpha) responses, 13 subtypes exist that signal through the same receptor, but have been reported to have different effector functions. However, the lack of available tools for discriminating these closely related subtypes, in particular at the protein level, has restricted the study of their differential roles in disease. We developed a digital ELISA with specificity and high sensitivity for the IFN2 subtype. Application of this assay, in parallel with our previously described pan-IFN assay, allowed us to study different IFN protein responses following cellular stimulation and in diverse patient cohorts. We observed different ratios of IFN protein responses between viral infection and autoimmune patients. This analysis also revealed a small percentage of autoimmune patients with high IFN2 protein measurements but low pan-IFN measurements. Correlation with an ISG score and functional activity showed that in this small sub group of patients, IFN2 protein measurements did not reflect its biological activity. This unusual phenotype was partly explained by the presence of anti-IFN auto-antibodies in a subset of autoimmune patients. This study reports ultrasensitive assays for the study of IFN proteins in patient samples and highlights the insights that can be obtained from the use of multiple phenotypic readouts in translational and clinical studies.

High autoantibody levels are found in individuals hospitalized for COVID-19. The temporal trajectories and levels of these autoantibodies months into convalescence after SARS-CoV-2 infection are unclear. It is also unknown if the composite autoantibody signatures of convalescent SARS-CoV-2-infected individuals resemble those with diagnosed autoimmune diseases. We measured the circulating levels of 17 autoantibodies associated with autoimmune connective tissue diseases from SARS-CoV-2 hospitalized and outpatient participants, as well as from individuals with scleroderma (SSc), systemic lupus erythematosus (SLE), and uninfected pre-pandemic controls. Seven of the 17 autoantibodies measured were higher in hospitalized and/or outpatient SARS-CoV-2 individuals an average of six months after symptom onset compared with controls, with multivariate analyses revealing links between SARS-CoV-2 infection and positivity of SSB-La, Sm, Proteinase 3, Myleoperoxidase, Jo-1, and Ku reactive IgG six months post-symptom onset. Autoantibody levels from SARS-CoV-2 infected individuals were followed over time from initial symptom onset for an average of six months, and different temporal autoantibody trajectories were classified. A negative, then positive expression pattern was found for at least one autoantibody in 18% of the outpatient and 53% of the hospitalized participants, indicating initiation and durable expression of self-reactive immune responses post-infection, particularly with severe acute illness. Analysis of individual participant autoantibody expression patterns revealed similar patterns between pre-pandemic and convalescent SARS-CoV-2 infected groups that are distinct from participants with both the SSc and SLE. As autoantibody positivity can occur years prior to autoimmune disease onset, the possibility that SARS-CoV-2-associated autoantibodies are a herald of future autoimmune disorders requires further investigation. One Sentence SummaryAutoantibody levels rise after acute SARS-CoV-2 infection and remain elevated for at least six months after symptom onset in participants with mild or severe COVID-19.

Systemic Lupus Erythematosus (SLE) is characterized by a prominent increase in expression of type-I interferon (IFN)-regulated genes in 50-75% of patients. Here we investigate the presence of autoantibodies (auto-Abs) against type I IFN in SLE patients and their possible role in controlling disease severity. We report that out of 491 SLE patients, 66 had detectable anti-IFN-auto-Abs. The presence of neutralizing anti-IFN-auto-Abs correlates with lower levels of circulating IFN protein, inhibition of IFN down-stream signalling molecules and gene signatures and with an inactive global disease score. Previously reported B cell frequency abnormalities, found to be involved in SLE pathogenesis, including increased levels of immature, double negative and plasmablast B cell populations were partially normalized in patients with neutralising anti-IFN-auto-Abs compared to other patient groups. We also show that sera from SLE patients with neutralising anti-IFN-auto-Abs biases in vitro B cell differentiation towards classical memory phenotype, while sera from patients without anti-IFN-Abs drives plasmablasts differentiation. Our findings support a role for neutralising anti-IFN-auto-Abs in controlling SLE pathogenesis and highlight their potential efficacy as novel therapy.

Objectives1 To plot the trajectory of humoral and cellular immune responses to the primary (two-dose) COVID-19 mRNA series and the third/booster dose in B-cell-depleted multiple sclerosis (MS) patients up to 2 years post-vaccination; 2. to identify predictors of immune responses to vaccination; and 3. to assess the impact of intercurrent COVID-19 infections on SARS CoV-2-specific immunity. Methods60 Ocrelizumab-treated MS patients were enrolled from NYU (New York) and University of Colorado (Anschutz) MS Centers. Samples were collected pre-vaccination, and then 4, 12, 24, and 48 weeks post-primary series, and 4, 12, 24, and 48 weeks post-booster. Binding anti-Spike antibody responses were assessed with multiplex bead-based immunoassay (MBI) and electrochemiluminescence (Elecsys(R), Roche Diagnostics), and neutralizing antibody responses with live-virus immunofluorescence-based microneutralization assay. Spike-specific cellular responses were assessed with IFN{gamma}/IL-2 ELISpot (Invitrogen) and, in a subset, by sequencing complementary determining regions (CDR)-3 within T-cell receptors (Adaptive Biotechnologies). A linear mixed effect model was used to compare antibody and cytokine levels across time points. Multivariate analyses identified predictors of immune responses. ResultsThe primary vaccination induced an 11-208-fold increase in binding and neutralizing antibody levels and a 3-4-fold increase in IFN{gamma}/IL-2 responses, followed by a modest decline in antibody but not cytokine responses. Booster dose induced a further 3-5-fold increase in binding antibodies and 4-5-fold increase in IFN{gamma}/IL-2, which were maintained for up to 1 year. Infections had a variable impact on immunity. InterpretationHumoral and cellular benefits of COVID-19 vaccination in B-cell-depleted MS patients were sustained for up to 2 years when booster doses were administered.

Neutrophils are essential antimicrobial effector cells with short lifespans. During infection or sterile inflammation, accelerated production and release of immature neutrophils from the bone marrow serves to boost circulating neutrophil counts. To facilitate the study of neutrophil development and function, we optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. We obtain high yields of neutrophils, which phenotypically resemble immature neutrophils released into the circulation upon administration of GCSF to healthy donors. We show that ex vivo differentiated immature neutrophils have similar rates of ROS production but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. We demonstrate that ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and thus provide a powerful model system for investigating the properties and behaviour of immature neutrophils.