Development of warm autoimmune hemolytic anemia after initiation of Ruxolitinib in a patient with STAT1 gain-of-function mutation

Introduction: STAT1 gain-of-function (GOF) disease is associated with chronic mucocutaneous candidiasis (CMC) and a broad spectrum of infectious, inflammatory, and vascular manifestations. The Janus Kinase inhibitor ruxolitinib has been used successfully for CMC and autoimmune phenomena. We describe a case of warm autoimmune hemolytic anemia (WAIHA) in a patient with STAT1 GOF disease after initiating ruxolitinib. Case report: A 36-year-old man with STAT1 c.850G>A (p.Glu284Lys) mutation presented with CMC as well as recurrent viral and bacterial infections, lymphadenopathy, enteritis, nodular regenerative hyperplasia (NRH) and splenomegaly. Immune workup confirmed a combined immunodeficiency with hypogammaglobulinemia and T-cell lymphopenia. Ruxolitinib was initiated at 5 mg twice daily (due to pre-existing thrombocytopenia) with up titration over 3 months to 20 mg twice daily. He improved with weight gain, increased energy, resolution of chronic anemia, and improved lymphadenopathy and splenomegaly on imaging. Serum CXCL9 only minimally decreased from 4660 pg/ml to 3990 pg/ml. Soon after reaching ruxolitinib 20 mg twice daily, he developed JC viremia, prompting dose reduction to 15 mg BID. Within two weeks, he developed a non-COVID upper respiratory tract infection followed by fatigue, shortness of breath with ambulation, and dark urine. Emergency evaluation revealed warm antibody positive hemolytic anemia with a hemoglobin of 5 g/dL, and worsened thrombocytopenia. He was treated with blood transfusions, pulse steroids, and high-dose IVIG with stabilization but continued hemolysis. Due to the JC viremia, there was concern to give rituximab with increased PML risk. Bone marrow showed trilineage hematopoiesis, a mild increase in megakaryocytes and RBC precursors, and a loss of B-cell progenitors with retention of mature B cells. His B and T lymphocyte numbers had increased since prior to ruxolitinib, with a predominance of Tfh1-cells (58.7% of total Tfh-cells). He was started on sirolimus with a slow taper of prednisone with continued stable hemoglobin and platelets, and resolution of hemolysis after 3 months. Conclusion(s): To our knowledge, this is the first case of a STAT1 GOF patient developing WAIHA while receiving ruxolitinib therapy. Treatment choices were complicated by the risks of PML. Sirolimus combined with ruxolitinib allowed wean of corticosteroid and subsequent resolution of hemolysis.Copyright © 2023 Elsevier Inc.

Characterization of post-vaccination SARS-CoV-2 T cell subtypes in patients with different hematologic malignancies and treatments.

Background: To evaluate the benefits of SARS-CoV-2 vaccination in cancer patients it is relevant to understand the adaptive immune response elicited after vaccination. Patients affected by hematologic malignancies are frequently immune-compromised and show a decreased seroconversion rate compared to other cancer patients or controls. Therefore, vaccine-induced cellular immune responses in these patients might have an important protective role and need a detailed evaluation. Methods: Certain T cell subtypes (CD4, CD8, Tfh, γδT), including cell functionality as indicated by cytokine secretion (IFN, TNF) and expression of activation markers (CD69, CD154) were assessed via multi-parameter flow cytometry in hematologic malignancy patients (N=12) and healthy controls (N=12) after a second SARS-CoV-2 vaccine dose. The PBMC of post-vaccination samples were stimulated with a spike-peptide pool (S-Peptides) of SARS-CoV-2, with CD3/CD28, with a pool of peptides from the cytomegalovirus, Epstein-Barr virus and influenza A virus (CEF-Peptides) or left unstimulated. Furthermore, the concentration of spike-specific antibodies has been analyzed in patients. Results: Our results indicate that hematologic malignancy patients developed a robust cellular immune response to SARS-CoV-2 vaccination comparable to that of healthy controls, and for certain T cell subtypes even higher. The most reactive T cells to SARS-CoV-2 spike peptides belonged to the CD4 and Tfh cell compartment, being median (IQR), 3.39 (1.41-5.92) and 2.12 (0.55-4.14) as a percentage of IFN- and TNF-producing Tfh cells in patients. In this regard, the immunomodulatory treatment of patients before the vaccination period seems important as it was strongly associated with a higher percentage of activated CD4 and Tfh cells. SARS-CoV-2- and CEF-specific T cell responses significantly correlated with each other. Compared to lymphoma patients, myeloma patients had an increased percentage of SARS-CoV-2-specific Tfh cells. T-SNE analysis revealed higher frequencies of γδT cells in patients compared to controls, especially in myeloma patients. In general, after vaccination, SARS-CoV-2-specific T cells were also detectable in patients without seroconversion. Conclusion: Hematologic malignancy patients are capable of developing a SARS-CoV-2-specific CD4 and Tfh cellular immune response after vaccination, and certain immunomodulatory therapies in the period before vaccination might increase the antigen-specific immune response. A proper response to recall antigens (e.g., CEF-Peptides) reflects immune cellular functionality and might be predictive for generating a newly induced antigen-specific immune response as is expected after SARS-CoV-2 vaccination.

T HELPER CELL SUBSETS AND RELATED TARGET CELLS IN ACUTE COVID-19.

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2-4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells - basophiles and eosinophils - were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory" Tfh1 cell and increased "pro-inflammatory" Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naive" and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24- plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

Boosting compromised SARS-CoV-2-specific T cell immunity in liver transplant recipients by mRNA vaccination

Background and aims Liver transplant recipients (LTR) are threatened by a lower immunogenicity of SARS-CoV-2 mRNA vaccines. However, the interplay between the different branches of the adaptive immune system especially after a third (and fourth) vaccine dose is still poorly understood. Methods Our study longitudinally compares the humoral as well as the cellular response between age-matched LTR (n = 24) and healthy controls (HC, n = 19) after three to four vaccine doses. Therefore, we assessed antibody titers, analyzed the spike-specific T cell epitope repertoire, performed an in-depth characterization of spike-specific CD8 + T cells on a single-epitope level and examined the distribution of different virus-specific CD4 + T cell subpopulations. Results Compared to HC, the development of high antibody titers depended on a third vaccine dose in most LTR. In contrast, spike-specific CD8 + T cells reached a stable level already after the second vaccine dose, albeit with a lower frequency and a narrower epitope repertoire compared to HC. Concerning the CD4 + T cells, the total number of detectable responses as well as the repertoire of targeted epitopes within the spike protein did not signifcantly difer in both cohorts. However, we observed a link between the overall attenuated vaccine response and a reduced frequency of spike-reactive follicular T helper cells (TFH) in LTR. Conclusion Three doses of a COVID-19 mRNA vaccine induce an overall robust humoral and cellular memory response in most LTR. Evaluations of additional booster doses may thus consider the individual vaccine responsiveness as well as the evolution of novel variants of concern.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

MAIT cells activate dendritic cells to promote TFH cell differentiation and induce humoral immunity.

Protective immune responses against respiratory pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has been hampered by the lack of a suitable mucosal adjuvant. In this study we explore a class of adjuvant that harnesses mucosal-associated invariant T (MAIT) cells. We show evidence that intranasal immunization of MAIT cell agonists co-administered with protein, including the spike receptor binding domain from SARS-CoV-2 virus and hemagglutinin from influenza virus, induce protective humoral immunity and immunoglobulin A production. MAIT cell adjuvant activity is mediated by CD40L-dependent activation of dendritic cells and subsequent priming of T follicular helper cells. In summary, we show that MAIT cells are promising vaccine targets that can be utilized as cellular adjuvants in mucosal vaccines.

T follicular helper cell responses to SARS-CoV-2 vaccination among healthy and immunocompromised adults.

The worldwide rollout of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations in the last 2 years has produced a multitude of studies investigating T-cell responses in the peripheral blood and a limited number in secondary lymphoid tissues. As a key component to an effective immune response, vaccine-specific T follicular helper (Tfh) cells are localized in the draining lymph node (LN) and assist in the selection of highly specific B-cell clones for the production of neutralizing antibodies. While these cells have been noted in the blood as circulating Tfh (cTfh) cells, they are not often taken into consideration when examining effective CD4+ T-cell responses, particularly in immunocompromised groups. Furthermore, site-specific analyses in locations such as the LN have recently become an attractive area of investigation. This is mainly a result of improved sampling methods via ultrasound-guided fine-needle biopsy (FNB)/fine-needle aspiration (FNA), which are less invasive than LN excision and able to be performed longitudinally. While these studies have been undertaken in healthy individuals, data from immunocompromised groups are lacking. This review will focus on both Tfh and cTfh responses after SARS-CoV-2 vaccination in healthy and immunocompromised individuals. This area of investigation could identify key characteristics of a successful LN response required for the prevention of infection and viral clearance. This furthermore may highlight responses that could be fine-tuned to improve vaccine efficacy within immunocompromised groups that are at a risk of more severe disease.

TNF-α+ CD4+ T cells dominate the SARS-CoV-2 specific T cell response in COVID-19 outpatients and are associated with durable antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ T cells are likely important in immunity against coronavirus 2019 (COVID-19), but our understanding of CD4+ longitudinal dynamics following infection and of specific features that correlate with the maintenance of neutralizing antibodies remains limited. Here, we characterize SARS-CoV-2-specific CD4+ T cells in a longitudinal cohort of 109 COVID-19 outpatients enrolled during acute infection. The quality of the SARS-CoV-2-specific CD4+ response shifts from cells producing interferon gamma (IFNγ) to tumor necrosis factor alpha (TNF-α) from 5 days to 4 months post-enrollment, with IFNγ-IL-21-TNF-α+ CD4+ T cells the predominant population detected at later time points. Greater percentages of IFNγ-IL-21-TNF-α+ CD4+ T cells on day 28 correlate with SARS-CoV-2-neutralizing antibodies measured 7 months post-infection (⍴ = 0.4, p = 0.01). mRNA vaccination following SARS-CoV-2 infection boosts both IFNγ- and TNF-α-producing, spike-protein-specific CD4+ T cells. These data suggest that SARS-CoV-2-specific, TNF-α-producing CD4+ T cells may play an important role in antibody maintenance following COVID-19.

T HELPER CELL SUBSETS AND RELATED TARGET CELLS IN ACUTE COVID-19

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2–4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells — basophiles and eosinophils — were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased "regulatory” Tfh1 cell and increased "pro-inflammatory” Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of "naïve” and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24– plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.

Tfh cells and the germinal center are required for memory B cell formation & humoral immunity after ChAdOx1 nCoV-19 vaccination.

Emergence from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been facilitated by the rollout of effective vaccines. Successful vaccines generate high-affinity plasma blasts and long-lived protective memory B cells. Here, we show a requirement for T follicular helper (Tfh) cells and the germinal center reaction for optimal serum antibody and memory B cell formation after ChAdOx1 nCoV-19 vaccination. We found that Tfh cells play an important role in expanding antigen-specific B cells while identifying Tfh-cell-dependent and -independent memory B cell subsets. Upon secondary vaccination, germinal center B cells generated during primary immunizations can be recalled as germinal center B cells again. Likewise, primary immunization GC-Tfh cells can be recalled as either Tfh or Th1 cells, highlighting the pluripotent nature of Tfh cell memory. This study demonstrates that ChAdOx1 nCoV-19-induced germinal centers are a critical source of humoral immunity.

First person - Sarantis Korniotis

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Sarantis Korniotis is first author on 'GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner', published in JCS. Sarantis conducted the research described in this article while a postdoctoral researcher in Professor Vassili Soumelis's lab at Saint-Louis Hospital, Universite de Paris, France. He is now a Senior Scientific Associate in Immunology at Intelligencia AI, Athens, Greece, investigating new signals and factors that make cells acquire regulatory or immune-suppressive properties. Copyright © 2022 Company of Biologists Ltd. All rights reserved.

Current understanding of T cell immunity against SARS-CoV-2.

As an important part of adaptive immunity, T cells are indispensable in the defense against pathogens including viruses. SARS-CoV-2 is a new human coronavirus that occurred at the end of 2019 and has caused the COVID-19 pandemic. Nevertheless, most of the infected patients recovered without any antiviral therapies, suggesting an effective immunity developed in the bodies. T cell immunity responds upon SARS-CoV-2 infection or vaccination and plays crucial roles in eliminating the viruses and generating T cell memory. Specifically, a subpopulation of CD4+ T cells could support the production of anti-SARS-CoV-2 antibodies, and cytotoxic CD8+ T cells are also protective against the infection. SARS-CoV-2-recognizing T cells could be detected in SARS-CoV-2-unexposed donors, but the role of these cross-reactive T cells is still in debate. T cell responses could be diverse across individuals, mainly due to the polymorphism of HLAs. Thus, compared to antibodies, T cell responses are generally less affected by the mutations of SARS-CoV-2 variants. Up to now, a huge number of studies on SARS-CoV-2-responsive T cells have been published. In this review, we introduced some major findings addressing the questions in the main aspects about T cell responses elicited by SARS-CoV-2, to summarize the current understanding of COVID-19.

GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner.

T follicular helper (Tfh) cells regulate humoral responses and present a marked phenotypic and functional diversity. Type 1 Tfh (Tfh1) cells were recently identified and associated with disease severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are not known. Here, using single-cell RNA sequencing (scRNAseq) and protein validation, we report that human blood CD1c+ dendritic cells (DCs) activated by GM-CSF (also known as CSF2) drive the differentiation of naive CD4+ T cells into Tfh1 cells. These Tfh1 cells displayed typical Tfh molecular features, including high levels of PD-1 (encoded by PDCD1), CXCR5 and ICOS. They co-expressed BCL6 and TBET (encoded by TBX21), and secreted large amounts of IL-21 and IFN-γ (encoded by IFNG). Mechanistically, GM-CSF triggered the emergence of two DC sub-populations defined by their expression of CD40 and ICOS ligand (ICOS-L), presenting distinct phenotypes, morphologies, transcriptomic signatures and functions. CD40High ICOS-LLow DCs efficiently induced Tfh1 differentiation in a CD40-dependent manner. In patients with mild COVID-19 or latent Mycobacterium tuberculosis infection, Tfh1 cells were positively correlated with a CD40High ICOS-LLow DC signature in scRNAseq of peripheral blood mononuclear cells or blood transcriptomics, respectively. Our study uncovered a novel CD40-dependent Tfh1 axis with potential physiopathological relevance to infection. This article has an associated First Person interview with the first author of the paper.

Effector-Memory B-Lymphocytes and Follicular Helper T-Lymphocytes as Central Players in the Immune Response in Vaccinated and Nonvaccinated Populations against SARS-CoV-2.

Vaccines have been recognized as having a central role in controlling the COVID-19 pandemic; however, most vaccine development research is focused on IgG-induced antibodies. Here, we analyzed the generation of IgGs related to SARS-CoV-2 and the changes in B- and T-lymphocyte proportions following vaccination against COVID-19. We included samples from 69 volunteers inoculated with the Pfizer-BioNTech (BNT162b2), Astra Zeneca (AZD1222 Covishield), or Sputnik V (Gam-COVID-Vac) vaccines. IgGs related to SARS-CoV-2 increased after the first vaccine dose compared with the nonvaccinated group (Pfizer, p = 0.0001; Astra Zeneca, p < 0.0001; Sputnik V, p = 0.0089). The results of the flow cytometry analysis of B- and T-lymphocytes showed a higher proportion of effector-memory B-lymphocytes in both first and second doses when compared with the nonvaccinated subjects. FcRL4+ cells were increased in second-dose-vaccinated COVID-19(-) and recovered COVID-19(+) participants when compared with the nonvaccinated participants. COVID-19(-) participants showed a lower proportion of follicular helper T-lymphocytes (TFH) in the second dose when compared with the first-vaccine-dose and nonvaccinated subjects. In conclusion, after the first vaccine dose, immunization against SARS-CoV-2 induces IgG production, and this could be mediated by TFH and effector-memory B-lymphocytes. Our data can be used in the design of vaccine schedules to evaluate immuno-bridging from a cellular point of view.

Model Immunization with Coronavirus-2 Vaccines Induce Potent Antibody Responses but Does Not Aggravate Lymphocyte Abnormalities in Primary Sjogren's Syndrome Patients

Objectives. The peripheral lymphocyte compartment of patients with primary Sjogren's syndrome (pSS) differs strongly from healthy individuals. Whether this altered lymphocyte composition also abnormally changes during immune reactions, especially in the context of novel mRNA-vaccines, is unknown. Methods. Peripheral blood samples from 26 pSS patients were compared to 6 healthy controls before Coronavirus-2 (CoV-2) vaccination (BNT162b2, ChAdOx1, mRNA-1273) and 7 days after secondary vaccination. Spike. 1 (S1)-receptor binding domain (RBD)-neutralizing IgG antibodies were measured in serum samples. Within peripheral blood mononuclear cells (PBMC), lymphocytes were characterized using spectral flow cytometry and B and T cell subpopulations were phenotypically analyzed. Results. Immunization induced CoV-2 specific serum antibodies in all pSS and healthy participants. When analyzing pSS and healthy individuals together, frequencies of circulating IgG+ RBD-binding antibody-secreting cells (ASC) and anti-CoV-2 serum titers correlated (r=0.42, p=0.022). Previously described alterations of peripheral B cells in pSS patients (like reduced memory B cells, increased naive and transitional B cells and higher maturity of ASCs) remained stable during vaccination. Also the subset distribution of CD4+ and CD8+ T cells mainly stayed unchanged. However, CD4+CXCR5-PD-1+ T cells phenotypically mimicking peripheral helper TPH cells increased in pSS patients comparing pre- and post-vaccination (p=0.020), while circulating CD4+CXCR5+PD-1+ follicular helper TFH cells declined (p=0.024). Conclusions. An immune reaction induced by vaccination with the novel mRNA technology yields adequate antibody production and vaccine specific lymphocytes in pSS patients and controls. However, no major changes within the typical composition of lymphocyte subpopulations of pSS patients were observed despite small changes in TPH and TFH subsets.

Characteristics and Roles of T Follicular Helper Cells in SARS-CoV-2 Vaccine Response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is critical to controlling the coronavirus disease 2019 (COVID-19) pandemic. However, a weak response to the vaccine and insufficient persistence of specific antibodies may threaten the global impact of mass vaccination campaigns. This study summarizes the internal factors of the body that affect the effectiveness of the SARS-CoV-2 vaccine. T follicular helper (Tfh) cells support germinal center B cells to produce vaccine-specific immunoglobulins. A reduction in the Tfh cell number and a shift in the subset phenotypes caused by multiple factors may impair the production and persistence of high-affinity antibodies. Besides efficacy differences caused by the different types of vaccines, the factors that affect vaccine effectiveness by intervening in the Tfh cell response also include age-related defects, the polarity of the body microenvironment, repeated immunization, immunodeficiency, and immunosuppressive treatments. Assessing the phenotypic distribution and activation levels of Tfh cell subsets after vaccination is helpful in predicting vaccine responses and may identify potential targets for improving vaccine effectiveness.

Characterization of the humoral response to Orf virusvectored immunization

Recombinant Orf virus (rORFV) based vectors are under clinical development for COVID-19 vaccination. Little is known, however, about the cellular correlates of antibody responses to this poxviral vector platform. To monitor antigen-specific B cell responses to vaccination, we adoptively transferred to mice indicator populations of monoclonal B cells recognizing the glycoproteins (GPs) of either vesicular stomatitis virus or lymphocytic choriomeningitis virus and epitope variants thereof. Immunizations of mice with rORFV expressing the respective GPs stimulated the transferred B cells to engage in a protracted germinal center (GC) response, which was maintained longer-term when the delivered antigen was of lower affinity. GPspecific CD8 and CD4 T cells responses were also induced, and the latter included T follicular helper cells (Tfh). These T cell responses contracted over time but re-expanded upon homologous rORFV booster vaccination, alongside with an augmentation in antigen-specific memory B cells. Pre-existing rORFV-specific anti-vector immunity suppressed CD8 T cell responses to ORFV-vectored cargo whereas CD4 T cell and B cell responses were unaffected. Importantly, rORFV-based vaccination conferred long-term antibodymediated protection against VSV challenge. This study demonstrates the versatility of rORFV-vectored vaccination including its capacity to induce substantial GC B cell as well as Tfh responses. Limited interference by anti-vector immunity should facilitate the challenging task of maintaining protective antibody immunity by prime - boost vaccination.

RHEUMATOID ARTHRITIS DISEASE ACTIVITY ASSESSED by PATIENT-REPORTED OUTCOMES and FLOW CYTOMETRY before and after AN ADDITIONAL DOSE of COVID-19 VACCINE

Background: The Centers for Disease Control and Prevention recommends an additional dose (AddDose) of COVID-19 vaccine for moderately/severely immunosuppressed individuals following an initial vaccine series. The American College of Rheumatology suggests that patients interrupt use (hold) certain DMARDs around the time of COVID-19 vaccination to improve immunogenicity. Whether holding DMARDs around an AddDose of COVID-19 vaccine affects RA disease activity or affects frequencies of lymphocyte populations that may be associated with RA disease activity remains unknown. Objectives: To test whether RA disease activity and frequencies of lymphocyte populations change pre-vs. post-AddDose of COVID-19 vaccine, overall and stratifed by holding vs. continuation of DMARDs around the AddDose. Methods: Prospective observational cohort study of patients with RA who had completed an initial COVID-19 vaccine series (2 doses of mRNA vaccine or 1 dose of adenovirus vector vaccine). Subjects enrolled July-November 2021, prior to receiving an AddDose. Subjects held or continued DMARDs around the AddDose based on discussion with their rheumatologist and/or personal decision-making. RA disease activity was assessed weekly using the validated patient-reported RA Disease Activity Index-5 (RADAI-5) from enrollment through 4 weeks post-AddDose. We compared mean RADAI-5 pre-vs. post-AddDose using generalized estimating equations to account for correlated data among individual subjects. We aimed to enroll 60 subjects to achieve 91% power to detect a 15% non-inferiority margin in mean RADAI-5 post-vs. pre-AddDose. A subset of subjects with seropositive RA provided blood for fow cytometry at enrollment and week 4 post-AddDose. Frequencies of lymphocyte populations (T peripheral helper [Tph] cells, T follicular helper [Tfh] cells, age-associated B cells [ABC], and plasmablasts) were compared pre-vs. post-AddDose using Wilcoxon paired tests with Bonferroni correction. Results: Among 71 subjects, mean age was 62 (SD 12) years, 85% were female, and 87% had seropositive RA. Methotrexate (42%) and TNF inhibitors (38%) were the most common DMARDs;21% were taking prednisone. One subject reported COVID-19 infection prior to the AddDose. The mean RADAI-5 was 3.20 (SD 0.23) pre-AddDose compared to 3.25 (SD 0.23) after (difference of 1.6%, p=0.51). Figure 1 displays mean RADAI-5 in 35 (49%) subjects that held at least 1 DMARD and 36 (51%) subjects that continued all DMARDs around the AddDose. Mean change in RADAI-5 between pre-vs. post-AddDose did not signifcantly differ based on whether subjects held vs. continued DMARDs (p for interaction = 0.16). Frequencies of Tph, Tfh, ABC, and plasmablast populations did not signifcantly differ between the pre-and post-AddDose timepoints in subjects that held at least 1 DMARD (n=16) or subjects that continued all DMARD (n=11) (Figure 1). Conclusion: RA disease activity, measured weekly with a validated patient-reported outcome, is stable around the time of an AddDose of COVID-19 vaccine. Lymphocyte subsets of interest in RA were also similar before and after the AddDose, supporting the observation of stable patient-reported RA disease activity. Holding DMARDs was not associated with greater RA disease activity following the AddDose.

T HELPER CELL SUBSETS AND RELATED TARGET CELLS IN ACUTE COVID-19

Current review presents a brief overview of the immune system dysregulation during acute COVID-19 and illustrates the main alterations in peripheral blood CD4+ T-cell (Th) subsets as well as related target cells. Effects of dendritic cell dysfunction induced by SARS-CoV-2 exhibited decreased expression of cell-surface HLA-DR, CCR7 as well as co-stimulatory molecules CD80 and CD86, suggesting reduced antigen presentation, migratory and activation capacities of peripheral blood dendritic cells. SARS-CoV-2-specific Th cells could be detected as early as days 2–4 post-symptom onset, whereas the prolonged lack of SARS-CoV-2-specific Th cells was associated with severe and/or poor COVID-19 outcome. Firstly, in acute COVID-19 the frequency of Th1 cell was comparable with control levels, but several studies have reported about upregulated inhibitory immune checkpoint receptors and exhaustion-associated molecules (TIM3, PD-1, BTLA, TIGIT etc.) on circulating CD8+ T-cells and NK-cells, whereas the macrophage count was increased in bronchoalveolar lavage (BAL) samples. Next, type 2 immune responses are mediated mainly by Th2 cells, and several studies have revealed a skewing towards dominance of Th2 cell subset in peripheral blood samples from patients with acute COVID-19. Furthermore, the decrease of circulating main Th2 target cells — basophiles and eosinophils — were associated with severe COVID-19, whereas the lung tissue was enriched with mast cells and relevant mediators released during degranulation. Moreover, the frequency of peripheral blood Th17 cells was closely linked to COVID-19 severity, so that low level of Th17 cells was observed in patients with severe COVID-19, but in BAL the relative number of Th17 cells as well as the concentrations of relevant effector cytokines were dramatically increased. It was shown that severe COVID-19 patients vs. healthy control had higher relative numbers of neutrophils if compared, and the majority of patients with COVID-19 had increased frequency and absolute number of immature neutrophils with altered ROS production. Finally, the frequency of Tfh cells was decreased during acute COVID-19 infection. Elevated count of activated Tfh were found as well as the alterations in Tfh cell subsets characterized by decreased “regulatory” Tfh1 cell and increased “pro-inflammatory” Tfh2 as well as Tfh17 cell subsets were revealed. Descriptions of peripheral blood B cells during an acute SARS-CoV-2 infection werev reported as relative B cell lymphopenia with decreased frequency of “naïve” and memory B cell subsets, as well as increased level of CD27hiCD38hiCD24– plasma cell precursors and atypical CD21low B cells. Thus, the emerging evidence suggests that functional alterations occur in all Th cell subsets being linked with loss-of-functions of main Th cell subsets target cells. Furthermore, recovered individuals could suffer from long-term immune dysregulation and other persistent symptoms lasting for many months even after SARS-CoV-2 elimination, a condition referred to as post-acute COVID-19 syndrome.

HUMORAL AND T CELL RESPONSES TO COVID-19 VACCINATION IN INFLAMMATORY BOWEL DISEASE

BACKGROUND: The characteristics of SARS-CoV-2 vaccine-induced immunity in inflammatory bowel disease (IBD) patients on immune modifying agents has not been clearly defined due to their exclusion in vaccine trials. Emerging results suggest infliximab impairs antibody response compared to vedolizumab. However there has not been direct comparison to controls. We evaluated this with both humoral and T cell response in IBD patients. METHODS: Antibody and T cell response were analysed in IBD patients who received BNT162b2 (Pfizer–BioNTech) or ChAdOx1 nCoV-19 (Oxford–AstraZeneca) vaccination from a single Australian centre. The control group were healthcare workers (HCW) without IBD. Blood samples were taken at 4 time points: at baseline V0 (before vaccination);V1 (7- 14 days after vaccine 1);V2 (7-14 days after vaccine 2);V3 (21-42 days after vaccine 2). Antibodies to the S1/2 IgG subunit and receptor-binding protein (RBD) were measured and reported here. RESULTS: 88 (28 ulcerative colitis, 50 Crohn's disease) IBD patients were included and compared to 53 healthy controls (Table 1). IBD patients medications included 6 5ASA (6.8%), 6 immunomodulator monotherapy (6.8%), 14 anti-TNF monotherapy (15.9%), 32 anti-TNF combination therapy with immunomodulator (36%), 16 IL12/23 (18%) and 13 vedolizumab (14%). Pre-vaccine baseline sera showed absence of anti-RBD antibodies in all participants. 84 patients (87%) received BNT162b2 and 4 (4.5%) received ChAdOx1 nCoV-19 vaccines. Geometric mean [SD] anti-S1/2 antibody concentrations at 4 weeks after second vaccination (V3) were significantly lower in IBD TNF treated patients (162.6[1.7]) compared to IBD non TNF treated patients (325.2[1.3]), and healthy controls (325.2[1.3]), p<0.0001 (Figure 1). There was no difference between non-TNF treated patients including those on vedolizumab or IL12/23 compared to controls. Similarly there was a significant difference between anti-RBD IgG titres between TNF and non-TNF IBD patients at V3 but not when compared to controls. There was no difference in RBD IgG and anti-S1/2 antibodies between anti-TNF monotherapy and combination therapy. All healthy controls and most IBD patients seroconverted at V3. 2 patients that failed to seroconvert were on steroid. CONCLUSION: TNF agents influence SARS-CoV-2 vaccine-induced antibody response in IBD patients, with lower anti-S1/2 IgG concentrations compared to non-TNF IBD patients and healthy controls. However, there was no difference in RBD IgG concentrations. It is unclear whether these subtle differences in antibody response in IBD patients on TNF agents is biologically meaningful, as most seroconverted after second dose vaccination. They may translate to differences in antibody longevity, but this is yet to be demonstrated. Neutralising antibody and T cell (CD4+/CD8+/follicular T cell) data from this study to come. (Table Presented) (Figure Presented)