Maturation and persistence of the anti-SARS-CoV-2 memory B cell response.

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

SARS-CoV-2 Omicron BA.1 breakthrough infection drives late remodeling of the memory B cell repertoire in vaccinated individuals.

How infection by a viral variant showing antigenic drift impacts a preformed mature human memory B cell (MBC) repertoire remains an open question. Here, we studied the MBC response up to 6 months after SARS-CoV-2 Omicron BA.1 breakthrough infection in individuals previously vaccinated with three doses of the COVID-19 mRNA vaccine. Longitudinal analysis, using single-cell multi-omics and functional analysis of monoclonal antibodies from RBD-specific MBCs, revealed that a BA.1 breakthrough infection mostly recruited pre-existing cross-reactive MBCs with limited de novo response against BA.1-restricted epitopes. Reorganization of clonal hierarchy and new rounds of germinal center reactions, however, combined to maintain diversity and induce progressive maturation of the MBC repertoire against common Hu-1 and BA.1, but not BA.5-restricted, SARS-CoV-2 Spike RBD epitopes. Such remodeling was further associated with a marked improvement in overall neutralizing breadth and potency. These findings have fundamental implications for the design of future vaccination booster strategies.

Human anti-smallpox long-lived memory B cells are defined by dynamic interactions in the splenic niche and long-lasting germinal center imprinting.

Memory B cells (MBCs) can persist for a lifetime, but the mechanisms that allow their long-term survival remain poorly understood. Here, we isolated and analyzed human splenic smallpox/vaccinia protein B5-specific MBCs in individuals who were vaccinated more than 40 years ago. Only a handful of clones persisted over such an extended period, and they displayed limited intra-clonal diversity with signs of extensive affinity-based selection. These long-lived MBCs appeared enriched in a CD21hiCD20hi IgG+ splenic B cell subset displaying a marginal-zone-like NOTCH/MYC-driven signature, but they did not harbor a unique longevity-associated transcriptional or metabolic profile. Finally, the telomeres of B5-specific, long-lived MBCs were longer than those in patient-paired naive B cells in all the samples analyzed. Overall, these results imply that separate mechanisms such as early telomere elongation, affinity selection during the contraction phase, and access to a specific niche contribute to ensuring the functional longevity of MBCs.

Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants.

In addition to serum immunoglobulins, memory B cell (MBC) generation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is another layer of immune protection, but the quality of MBC responses in naive and coronavirus disease 2019 (COVID-19)-recovered individuals after vaccination remains ill defined. We studied longitudinal cohorts of naive and disease-recovered individuals for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of antibody repertoires, affinity, and neutralization against variants of concern (VOCs) using unbiased cultures of 2,452 MBCs. Upon boosting, the MBC pool of recovered individuals expanded selectively, matured further, and harbored potent neutralizers against VOCs. Although naive individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity toward multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs.

Prolonged response after TPO-RA discontinuation in primary ITP: results of a prospective multicenter study.

Sustained response off treatment (SROT) after thrombopoietin receptor agonist (TPO-RA) discontinuation has been reported in immune thrombocytopenia (ITP). This prospective multicenter interventional study enrolled adults with persistent or chronic primary ITP and complete response (CR) on TPO-RAs. The primary end point was the proportion of patients achieving SROT (platelet count >30 × 109/L and no bleeding) at week 24 (W24) with no other ITP-specific medications. Secondary end points included the proportion of sustained CR off-treatment (SCROT, platelet count >100 × 109/L and no bleeding) and SROT at W52, bleeding events, and pattern of response to a new course of TPO-RAs. We included 48 patients with a median age of 58.5 years; 30 of 48 had chronic ITP at TPO-RA initiation. In the intention-to-treat analysis, 27 of 48 achieved SROT, 15 of 48 achieved SCROT at W24; 25 of 48 achieved SROT, and 14 of 48 achieved SCROT at W52. No severe bleeding episode occurred in patients who relapsed. Among patients rechallenged with TPO-RA, 11 of 12 achieved CR. We found no significant clinical predictors of SROT at W24. Single-cell RNA sequencing revealed enrichment of a tumor necrosis factor α signaling via NF-κB signature in CD8+ T cells of patients with no sustained response after TPO-RA discontinuation, which was further confirmed by a significant overexpression of CD69 on CD8+ T cells at baseline in these patients as compared with those achieving SCROT/SROT. Our results strongly support a strategy based on progressive tapering and discontinuation of TPO-RAs for patients with chronic ITP who achieved a stable CR on treatment. This trial was registered at www.clinicaltrials.gov as #NCT03119974.

JAK inhibitor treatment for inborn errors of JAK/STAT signaling: An ESID/EBMT-IEWP retrospective study.

BACKGROUND: Inborn errors of immunity (IEI) with dysregulated JAK/STAT signaling present with variable manifestations of immune dysregulation and infections. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but initially reported outcomes were poor. JAK inhibitors (JAKi) offer a targeted treatment option that may be an alternative or bridge to HSCT. However, data on their current use, treatment efficacy and adverse events are limited. OBJECTIVE: We evaluated the current off-label JAKi treatment experience for JAK/STAT inborn errors of immunity (IEI) among European Society for Immunodeficiencies (ESID)/European Society for Blood and Marrow Transplantation (EBMT) Inborn Errors Working Party (IEWP) centers. METHODS: We conducted a multicenter retrospective study on patients with a genetic disorder of hyperactive JAK/STAT signaling who received JAKi treatment for at least 3 months. RESULTS: Sixty-nine patients (72% children) were evaluated (45 STAT1 gain of function [GOF], 21 STAT3-GOF, 1 STAT5B-GOF, 1 suppressor of cytokine signaling 1 [aka SOCS1] loss of function, 1 JAK1-GOF). Ruxolitinib was the predominantly prescribed JAKi (80%). Overall, treatment resulted in improvement (partial or complete remission) of clinical symptoms in 87% of STAT1-GOF and in 90% of STAT3-GOF patients. We documented highly heterogeneous dosing and monitoring regimens. The response rate and time to response varied across different diseases and manifestations. Adverse events including infection and weight gain were frequent (38% of patients) but were mild (grade I-II) and transient in most patients. At last follow-up, 52 (74%) of 69 patients were still receiving JAKi treatment, and 11 patients eventually underwent HSCT after receipt of previous JAKi bridging therapy, with 91% overall survival. CONCLUSIONS: Our study suggests that JAKi may be highly effective to treat symptomatic JAK/STAT IEI patients. Prospective studies to define optimal JAKi dosing for the variable clinical presentations and age ranges should be pursued.

B-cell responses to ITP treatments.

Deficiency in regulatory B cells has been suggested in immune thrombocytopenia. In this study, Stimpson et al. emphasize the importance of considering the treatments received for immunological analyses. Commentary on: Stimpson et al. Systemic immunosuppression depletes peripheral blood regulatory B cells in patients with immune thrombocytopenia. Br J Haematol 2024;204:644-648.

Efficacy and safety of targeted therapies in VEXAS syndrome: retrospective study from the FRENVEX.

OBJECTIVES: Vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease associated with somatic ubiquitin-like modifier-activating enzyme 1 (UBA1) mutations. We aimed to evaluate the efficacy and safety of targeted therapies. METHODS: Multicentre retrospective study including patients with genetically proven VEXAS syndrome who had received at least one targeted therapy. Complete response (CR) was defined by a clinical remission, C-reactive protein (CRP) ≤10 mg/L and a ≤10 mg/day of prednisone-equivalent therapy, and partial response (PR) was defined by a clinical remission and a 50% reduction in CRP levels and glucocorticoid dose. RESULTS: 110 patients (median age 71 (68-79) years) who received 194 targeted therapies were included: 78 (40%) received Janus kinase (JAK) inhibitors (JAKi), 51 (26%) interleukin (IL)-6 inhibitors, 33 (17%) IL-1 inhibitors, 20 (10%) tumour necrosis factor (TNFα) blockers and 12 (6%) other targeted therapies. At 3 months, the overall response (CR and PR) rate was 24% with JAKi, 32% with IL-6 inhibitors, 9% with anti-IL-1 and 0% with TNFα blockers or other targeted therapies. At 6 months, the overall response rate was 30% with JAKi and 26% with IL-6 inhibitors. Survival without treatment discontinuation was significantly longer with JAKi than with the other targeted therapies. Among patients who discontinued treatment, causes were primary failure, secondary failure, serious adverse event or death in 43%, 14%, 19% and 19%, respectively, with JAKi and 46%, 11%, 31% and 9%, respectively, with IL-6 inhibitors. CONCLUSIONS: This study shows the benefit of JAKi and IL-6 inhibitors, whereas other therapies have lower efficacy. These results need to be confirmed in prospective trials.

COVID-19 and splenectomy: Education matters.

Whether COVID-19-related morbidity and mortality are increased in splenectomized patients is unknown. The study by Bianchi et al. suggests increased hospitalizations and mortality rates in splenectomized patients, despite observing similar infection rates when compared to the general population. Commentary on: Bianchi et al. Burden of COVID19 disease and vaccine coverages in Apulian splenectomized patients. A retrospective observational study. Br J Haematol 2023;201:1072-1080.

Combining thrombopoietin receptor agonists with immunosuppressive drugs in adult patients with multirefractory immune thrombocytopenia, an update on the French experience.

Combining drugs could be an effective option for treating multirefractory ITP, that is, patients not responding to rituximab, thrombopoietin receptor agonists (TPO-RA) and splenectomy. We conducted a retrospective, multicenter, observational study including multirefractory ITP patients who received a combination of a TPO-RA and an immunosuppressive drug. We included 39 patients (67% women, median age 59 years [range 21-96]), with a median ITP duration of 57 months [3-393] and a median platelet count at initiation of 10 × 109 /L [1-35]. The combination regimen was given for a median duration of 12 months [1-103] and included eltrombopag (51%) or romiplostim (49%), associated with mycophenolate mofetil (54%), azathioprine (36%), cyclophosphamide (5%), cyclosporin (3%) or everolimus (3%). Overall, 30 patients (77%) achieved at least a response (platelet count ≥30 × 109 /L and at least doubling baseline during at least 3 months), including 24 complete responses (platelet count >100 × 109 /L during at least 3 months) with a median time to response of 30 days [7-270] and a median duration of response of 15 months [4-63]. Severe adverse event related to ITP treatment was observed in 31%. In conclusion, this study confirms that some patients with multirefractory ITP can achieve long lasting response with this combination.

Hematologic disorder-associated Cxcr4 gain-of-function mutation leads to uncontrolled extrafollicular immune response.

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19.

Anti-CD20 monoclonal antibodies are widely used for the treatment of hematological malignancies or autoimmune disease but may be responsible for a secondary humoral deficiency. In the context of COVID-19 infection, this may prevent the elicitation of a specific SARS-CoV-2 antibody response. We report a series of 17 consecutive patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms, negative immunoglobulin G (IgG)-IgM SARS-CoV-2 serology, and positive RNAemia measured by digital polymerase chain reaction who were treated with 4 units of COVID-19 convalescent plasma. Within 48 hours of transfusion, all but 1 patient experienced an improvement of clinical symptoms. The inflammatory syndrome abated within a week. Only 1 patient who needed mechanical ventilation for severe COVID-19 disease died of bacterial pneumonia. SARS-CoV-2 RNAemia decreased to below the sensitivity threshold in all 9 evaluated patients. In 3 patients, virus-specific T-cell responses were analyzed using T-cell enzyme-linked immunospot assay before convalescent plasma transfusion. All showed a maintained SARS-CoV-2 T-cell response and poor cross-response to other coronaviruses. No adverse event was reported. Convalescent plasma with anti-SARS-CoV-2 antibodies appears to be a very promising approach in the context of protracted COVID-19 symptoms in patients unable to mount a specific humoral response to SARS-CoV-2.

[Myeloid neoplasms associated with rearrangement of PDGFRB: A rare and tricky disease]. / La néoplasie myéloïde associée à un réarrangement de PDGFRB : une pathologie rare de diagnostic difficile.

In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×109/L. Bone marrow aspiration was rich and showed a heterogeneous distribution of myeloid cells with clusters of promyelocytes and proerythroblasts associated with numerous eosinophils and spindle-shaped mast cells but without excess of blasts, dysplasia nor maturation skewing. These aspects suggested an atypical myeloproliferative neoplasm. Bone marrow biopsy was performed showing also a very high cellularity with area of myeloid and erythroid precursors associated with numerous spindle-shaped mast cells. Diagnoses of unclassified myeloid neoplasm and/or systemic mastocytosis were then proposed. Further chromosome analysis showed a t(5;8) translocation with PDGFRB rearrangement revealed in fluorescent in situ hybridization. Patient was treated with imatinib and intravenous immunoglobulin therapy allowing a significant improvement in neurological symptoms and biological results. Patient condition is currently stable after six lines of treatment. This rare hematopoietic neoplasm displays unusual histological and cytological features and can mimic other myeloproliferative neoplasm. Specific cytogenetics analysis should be considered for such cases with hypereosinophilia to select patients that may benefit from targeted therapy.

Efficacy, safety and immunological profile of combining rituximab with belimumab for adults with persistent or chronic immune thrombocytopenia: results from a prospective phase 2b trial.

B-cell activating factor may be involved in the failure of B-cell depleting therapy with rituximab in immune thrombocytopenia (ITP) by promoting the emergence of splenic long-lived plasma cells. From results obtained in mouse models, we hypothesized that combining rituximab with sequential injections of belimumab could increase the rate of response at one year in patients with persistent or chronic ITP by preventing the emergence of these long-lived plasma cells. The study was a single-center, single arm, prospective phase 2b trial (RITUX-PLUS, NCT03154385) investigating the safety and efficacy of rituximab given at a fixed dose of 1,000 mg, two weeks apart, combined with five infusions of belimumab, 10 mg/kg at week 0 (W0)+2 days, W2+2 days, W4, W8 and W12 for adults with primary persistent or chronic ITP. The primary endpoint was the total number of patients achieving an overall response (complete response + response) at W52 according to a standard definition. In total, 15 non-splenectomized adults, nine (60%) with persistent IPT and six (40%) with chronic ITP, were included. No severe adverse event, infection, or severe hypogammaglobulinemia was observed. Thirteen patients achieved an initial overall response. At W52, 12 (80%) patients achieved an overall response, including ten (66.7%) with complete response. When compared with a cohort of patients receiving rituximab alone, the kinetics of B-cell repopulation appeared similar, but the number of circulating T follicular helper cells was significantly decreased with belimumab combination therapy. Combining rituximab and belimumab seems a promising strategy in ITP, with high efficacy and acceptable safety.

Anti-CD20-mediated B-cell depletion in autoimmune diseases: successes, failures and future perspectives.

B-cell depletion with anti-CD20 monoclonal antibodies is widely used for the treatment of autoimmune diseases. This review will discuss mechanisms contributing to success or failure of B-cell depletion therapy in antibody-mediated autoimmune diseases. It will also explain how key information about disease pathogeny can be provided by the different outcomes observed after B-cell depletion therapy. These findings provide the basis for future innovative therapeutic strategies aiming at an optimized B cell and/or plasma cell depletion to increase long-term disease remission.

BAFF and CD4+ T cells are major survival factors for long-lived splenic plasma cells in a B-cell-depletion context.

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4+ T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia.

Bortezomib and dexamethasone, an original approach for treating multi-refractory warm autoimmune haemolytic anaemia.

We report the off-label use of bortezomib combined with dexamethasone in eight adults with severe and multi-refractory warm auto-immune haemolytic anaemia (wAIHA). After six cycles of induction therapy, 6 of the 8 patients achieved response (3 complete response, 3 response). Response was obtained after a median of 2 (1-4) cycles. After a median follow-up of 14 (6-36) months, six patients maintained a response (bortezomib/dexamethasone maintenance, n = 4); five patients experienced at least one moderate adverse event, including peripheral neuropathy (n = 2). These results suggest that bortezomib/dexamethasone combination is a promising approach with acceptable toxicity for treating severe refractory wAIHA in adults.

Clinical spectrum, evolution, and management of autoimmune cytopenias associated with angioimmunoblastic T-cell lymphoma.

OBJECTIVE: Angioimmunoblastic T-cell lymphoma (AITL) is frequently associated with autoimmune cytopenia (AIC). Whether such patients have a particular phenotype and require particular management is unclear. METHOD: Angioimmunoblastic T-cell lymphoma patients from the multicentric database of the Lymphoma Study Association presenting with AIC during disease course were included and matched to AITL patients without AIC (1/5 ratio). RESULTS: At diagnosis, AIC patients (n = 28) had more spleen and bone marrow involvement (54% vs 19% and 71% vs 34%, P < 0.001), Epstein-Barr virus replication (89% vs 39%, P < 0.001), gamma globulin titers (median 23 vs 15 g/L, P = 0.002), and proliferating B cells and plasmablasts in biopsies, as compared to control patients (n = 136). The 28 AIC patients had 41 episodes of AIC, diagnosed concomitantly with AITL in 23 (82%) cases. After a median follow-up of 24 months (range 3-155), 10 patients relapsed, all associated with AITL relapse. CONCLUSION: Our results provide new insight into AIC associated with AITL by highlighting the significant interplay between AITL and B-cell activation leading to subsequent autoimmunity.