Abstract: Defects in B-cell reconstitution upon hematopoietic stem cell (HSC) transplant (HSCT) are a common observation, yet the mechanism remains unexplained. The bone marrow (BM) stroma, including mesenchymal stromal cells (MSCs), guides HSC maintenance and B-lymphopoiesis by secreting crucial cytokines. We report acquired, permanent, selective, and complete B-cell deficiency in the context of full donor chimerism in a patient with X-linked lymphoproliferative disease, and aimed to identify the contribution of the BM microenvironment in disrupted B-cell reconstitution after HSCT. We studied longitudinal BM samples from the patient and his identical twin, both of whom underwent HSCT with the same donor with opposite outcomes in B-cell reconstitution. In the BM of the index patient, we observed progressive loss of proliferation of HSCs and a selective block at the pre-BI cell stage. In vitro modeling studies showed limited survival of patient HSCs and a relative accumulation of pre-B cells. Patient-derived MSCs failed to support survival and proliferation of HSCs and B-cell development of healthy HSCs, which was correlated with reduced CXCL12 levels. Using bulk RNA-sequencing of MSCs and in vitro functional studies, we showed global changes in the patients’ MSCs, and a progressive loss of CXCL12 expression. Indeed, survival of the patients’ HSCs improved upon supplementing the in vitro development culture with CXCL12, suggesting a contribution of defective CXCL12 signaling to the phenotype. In summary, our data show that an acquired defect in the BM-stromal microenvironment and exhaustion of HSCs and committed progenitors may cause a permanent nonpermissive state for normal B-cell development.

Virus infections elicit long-term IgG antibody and memory responses. Human cytomegalovirus (HCMV) is widespread in humans and disseminates despite the presence of virus-specific antibodies. Here, we report that the HCMV Fc gamma-binding glycoprotein 34 modulates humoral immunity by binding to IgG(+) memory B cells. gp34-B cell receptor (BCR) interaction initiates activation of the PDK1/AKT/mTOR/S6 pathway and BCR internalization in a SYK-independent manner. Prolonged stimulation also induces B-cell activation via upregulation of CD69 and CD86. In a T-cell-dependent response, however, interaction with gp34 blocks B-cell proliferation, differentiation into plasmablasts, and soluble IgG production, while stimulating TNF-alpha secretion. Through gp34 stimulation on IgG(+) B cells, neighboring IgM(+) and IgA(+) B cells are likewise impaired in proliferation, plasmablast formation, and immunoglobulin secretion. In summary, gp34 specifically interacts with IgG(+) memory B cells, inducing a hyporesponsive state across the B-cell compartment through direct and indirect regulation. This reveals a novel mode of viral evasion from B-cell responses by suppressing secondary immunity.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Detailed knowledge of the human B-cell development is crucial for proper interpretation of inborn errors of immunity and for malignant diseases. It is of interest to understand the kinetics of protein expression changes during the B cell development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human bone marrow and peripheral blood samples from healthy individuals with the aim to describe all B-cell developmental trajectories across the two tissues. We validated a 30-parameter mass cytometry panel and demonstrated the utility of “ vaevictis ” visualization of B-cell developmental stages. We used our recently developed trajectory inference tool “ tviblindi ” to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers (CD34, CD10, sIgM, IgD, CD20, CD27). Lastly, we performed analysis of the expression changes related to developmental branching points (Natural Effector versus Switched Memory B cells, marked by up-regulation of CD73).In conclusion, we developed, validated and presented a comprehensive set of tools for investigation of B-cell development.### Competing Interest StatementThe authors have declared no competing interest.

Detailed knowledge of human B-cell development is crucial for the proper interpretation of inborn errors of immunity and malignant diseases. It is of interest to understand the kinetics of protein expression changes during development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human samples from healthy individuals with the aim of describing all B-cell developmental trajectories. We validated a 30-parameter mass cytometry panel and demonstrated the utility of "vaevictis" visualization of B-cell developmental stages. We used the trajectory inference tool "tviblindi" to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers. We observed branching of the memory development, where follicular memory formation was marked by CD73 expression. Lastly, we performed an analysis of two example cases of abnormal B-cell development caused by mutations in RAG-1 and Wiskott-Aldrich syndrome gene in patients with primary immunodeficiency. In conclusion, we developed, validated, and presented a comprehensive set of tools for the investigation of B-cell development in the bone marrow compartment.

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing, B cell receptors (BCR) repertoire profiling, CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment, polyreactive, and derived from the natural repertoire. Furthermore, B cells with aPL specificities are not eliminated in patients with PAPS, persist until the memory and long-lived plasma cell stages, likely after defective germinal center selection, while becoming less polyreactive. Lastly, compared with the non-PAPS cells, PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS. Primary antiphospholipid syndrome (PAPS) is a clotting disorder attributed to autoreactive antibodies produced by B cells. Here the authors show, using single cell omics and B cell repertoire data, that autoreactive B cells originate from the natural B cell repertoire and escape germinal center selection to persist in PAPS patient via potential dysregulation of mTORC1 and MYC pathways.

Development of T cells is controlled by the signal strength of the TCR. The scaffold protein kinase D–interacting substrate of 220 kilodalton (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell–specific Kidins220 knockout (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stages 2 and 3 shows that Kidins220 down-regulates TCR signaling at these stages. scRNA-seq indicated that the transcription factor Aiolos is down-regulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.

OBJECTIVES:B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy. METHODS:We recruited 91 patients with AAV and performed deep phenotyping of the peripheral and bone marrow B-cell compartment by spectral flow and mass cytometry. B-cell development was studied by in vitro modelling and the role of BAFF receptor by quantitative PCR, western blot analysis and in vitro assays. RESULTS:Treatment-naïve patients with AAV showed low transitional B-cell numbers, suggesting impaired B-lymphopoiesis. We analysed bone marrow of treatment-naïve and RTX-treated patients with AAV and found reduced B-lymphoid precursors. In vitro modelling of B-lymphopoiesis from AAV haematopoietic stem cells showed intact, but slower and reduced immature B-cell development. In a subgroup of patients, after RTX treatment, the presence of transitional B cells did not translate in replenishment of naïve B cells, suggesting an impairment in peripheral B-cell maturation. We found low BAFF-receptor expression on B cells of RTX-treated patients with AAV, resulting in reduced survival in response to BAFF in vitro. CONCLUSIONS:Prolonged depletion of B cells in patients with AAV after RTX therapy indicates a B-cell defect that is unmasked by RTX treatment. Our data indicate that impaired bone marrow B-lymphopoiesis results in a delayed recovery of peripheral B cells that may be further aggravated by a survival defect of B cells. Our findings contribute to the understanding of AAV pathogenesis and may have clinical implications regarding RTX retreatment schedules and immunomonitoring after RTX therapy.

Early B cell development in the bone marrow ensures the replenishment of the peripheral B cell pool. Immature B cells continuously develop from hematopoietic stem cells, in a process guided by an intricate network of transcription factors as well as chemokine and cytokine signals. Humans and mice possess somewhat similar regulatory mechanisms of B lymphopoiesis. The continuous discovery of monogenetic defects that impact early B cell development in humans substantiates the similarities and differences with B cell development in mice. These differences become relevant when targeted therapeutic approaches are used in patients; therefore, predicting potential immunological adverse events is crucial. In this review, we have provided a phenotypical classification of human and murine early progenitors and B cell stages, based on surface and intracellular protein expression. Further, we have critically compared the role of key transcription factors (Ikaros, E2A, EBF1, PAX5, and Aiolos) and chemo- or cytokine signals (FLT3, c-kit, IL-7R, and CXCR4) during homeostatic and aberrant B lymphopoiesis in both humans and mice.

Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor alpha chain (IL-7R alpha) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7R alpha deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7R alpha deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis.

BackgroundJanus kinase (JAK) inhibitors have been approved for the treatment of several immune-mediated diseases (IMIDs) including rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other IMIDs. However, detailed studies investigating the effects of different JAK inhibitors on B cells are missing. Within this study, we therefore aimed to characterize the effect of JAK inhibition on the B cell compartment.MethodsTo this end, we investigated the B cell compartment under JAK inhibition and compared the specific effects of the different JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib (JAK1/2) as well as filgotinib (selective JAK1) on in-vitro B cell activation, proliferation, and class switch recombination and involved pathways.ResultsWhile B cell phenotyping of RA patients showed an increase in marginal zone (MZ) B cells under JAK inhibition, comparison with healthy donors revealed that the relative frequency of MZ B cells was still lower compared to healthy controls. In an in-vitro model of T-cell-independent B cell activation we observed that JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. We detected an altered B cell differentiation with a significant increase in MZ-like B cells and an increase in plasmablast differentiation in the first days of culture, most pronounced with the pan-JAK inhibitor tofacitinib, although there was no increase in immunoglobulin secretion in-vitro. Notably, we further observed a profound reduction of switched memory B cell formation, especially with JAK1/2 inhibition. JAK inhibitor treatment led to a dose-dependent reduction of STAT3 expression and phosphorylation as well as STAT3 target gene expression and modulated the secretion of pro- and anti-inflammatory cytokines by B cells.ConclusionJAK inhibition has a major effect on B cell activation and differentiation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

In the bone marrow, B cells and bone-resorbing osteoclasts colocalize and form a specific microenvironment. How B cells functionally influence osteoclasts and bone architecture is poorly understood. Using genetically modified mice and highthroughput analyses, we demonstrate that prolonged HIF-1α signaling in B cells leads to enhanced RANKL production and osteoclast formation. In addition, deletion of HIF-1α in B cells prevents estrogen deficiency-induced bone loss in mice.Mechanistically, estrogen controls HIF-1α protein stabilization through HSP70-mediated degradation in bone marrow B cells.The stabilization of HIF-1α protein in HSP70-deficient bone marrow B cells promotes RANKL production and osteoclastogenesis.Induction of HSP70 expression by geranylgeranylacetone（GGA） administration alleviates ovariectomy-induced osteoporosis.Moreover, RANKL gene expression has a positive correlation with HIF1 A expression in human B cells. In conclusion, HIF-1αsignaling in B cells is crucial for the control of osteoclastogenesis, and the HSP70/HIF-1α axis may serve as a new therapeutic target for osteoporosis.

SARS-CoV-2 spike mRNA vaccines(1-3) mediate protection from severe disease as early as ten days after prime vaccination(3), when neutralizing antibodies are hardly detectable(4-6). Vaccine-induced CD8(+) T cells may therefore be the main mediators of protection at this early stage(7,8). The details of their induction, comparison to natural infection, and association with other arms of vaccine-induced immunity remain, however, incompletely understood. Here we show on a single-epitope level that a stable and fully functional CD8(+) T cell response is vigorously mobilized one week after prime vaccination with bnt162b2, when circulating CD4(+) T cells and neutralizing antibodies are still weakly detectable. Boost vaccination induced a robust expansion that generated highly differentiated effector CD8(+) T cells; however, neither the functional capacity nor the memory precursor T cell pool was affected. Compared with natural infection, vaccine-induced early memory T cells exhibited similar functional capacities but a different subset distribution. Our results indicate that CD8(+) T cells are important effector cells, are expanded in the early protection window after prime vaccination, precede maturation of other effector arms of vaccine-induced immunity and are stably maintained after boost vaccination.

SARS-CoV-2 spike mRNA vaccines mediate protection from severe disease as early as 10 days post prime vaccination, when specific antibodies are hardly detectable and still lack neutralizing activity. Vaccine-induced T cells, especially CD8+ T cells, may thus be the main mediators of protection at this early stage. The details of antigen-specific CD8+ T cell induction after prime/boost vaccination, their comparison to naturally induced CD8+ T cell responses and their association with other arms of vaccine-induced adaptive immunity remain, however, incompletely understood. Here, we show on a single epitope level that both, a stable memory precursor pool of spike-specific CD8+ T cells and fully functional spike-specific effector CD8+ T cell populations, are vigorously mobilized as early as one week after prime vaccination when CD4+ T cell and spike-specific antibody responses are still weak and neutralizing antibodies are lacking. Boost vaccination after 3 weeks induced a full-fledged recall expansion generating highly differentiated CD8+ effector T cells, however, neither the functional capacity nor the memory precursor T cell pool was affected. Compared to natural infection, vaccine-induced early memory T cells exhibited similar frequencies and functional capacities but a different subset distribution dominated by effector memory T cells at the expense of self-renewing and multipotent central memory T cells. Our results indicate that spike-specific CD8+ T cells may represent the major correlate of early protection after SARS-CoV-2 mRNA/bnt162b2 prime vaccination that precede other effector arms of vaccine-induced adaptive immunity and are stably maintained after boost vaccination.

We describe a 3-year-old boy (P-FR1) from consanguineous white parents lacking immunoglobulins despite normal total B-cell numbers. Healthy until the age of 18 months, he developed recurrent respiratory infections, followed by a progressive central nervous system disease with progressive spastic tetraparesis from 24 months onward. Cerebral magnetic resonance imaging revealed enlarged outer cerebral spinal fluid spaces and hypomyelination of the white matter. A chronic viral disease was suspected. Cerebral spinal fluid analysis showed moderate pleocytosis but negative microbial PCR results (for further clinical details, see the Methods section of the Online Repository at www.jacionline.org). A biopsy for brain tissue PCRs was declined by the parents. P-FR1's brother died of Zellweger syndrome caused by a homozygous mutation in PEX16 (p.His231Arg) at the age of 14 years. P-FR1 and his sister were heterozygous for this mutation (Fig 1, A) and did not present clinical or laboratory findings of Zellweger syndrome. Exome sequencing performed on P-FR1 and his parents (see Tables E1 and E2 in the Online Repository at www.jacionline.org) revealed the frameshift mutation c.233delC in POU2AF1 (p.Thr78Lysfs∗63 [see Fig E1, A in the Online Repository at www.jacionline.org]) that was homozygous in P-FR1 and heterozygous in his parents and sister (Fig 1, A). POU2AF1 encodes Bob1 (also called OBF-1 or OCA-B). Although POU2AF1 transcripts were detected (Fig 1, B), Bob1 protein was absent in a P-FR1 EBV B-cell line (Fig 1, C and see Fig E1, B) and in HEK293T cells transfected with the mutated Bob1 compared with the wild-type Bob1 control (see Fig E1, C), indicating instability of the aberrant fusion protein. In B cells, Bob1 is a transcriptional coactivator that confers octamer-dependent specificity to the transcription factors Oct-1 and Oct-2.1Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.Nature. 1996; 383: 538-542Crossref PubMed Scopus (237) Google Scholar P-FR1 had normal B-cell numbers but disturbed B-cell differentiation, with decreased class-switched memory cells and a relative increase in atypical-memory cells (IgG+ or IgA+ CD27– [Fig 1, D and E]). His T cells were normal in terms of distribution and activation (Fig 1, D and see Fig E1, D and E) except for a reduction in circulating T-follicular helper (TFH) cells (Fig 1, D and E). P-FR1–naive B cells showed an abnormal expression pattern of surface receptors and signaling molecules, with low levels of IgD, IgM, CD79α, CD79β, and Syk (Fig 2, A and see Fig E1, F). Indeed, Bob1 can directly regulate expression of B-cell receptor (BCR) signaling molecules by interacting with octamer motifs in their promoter regions, as described for CD79β.2Malone C.S. Wall R. Bob1 (OCA-B/OBF-1) differential transactivation of the B cell-specific B29 (Ig beta) and mb-1 (Ig alpha) promoters.J Immunol. 2002; 168: 3369-3375Crossref PubMed Scopus (20) Google Scholar Low CD79β may contribute to low BCR and CD79α expression, as shown in B-cell lines.3Minuzzo S. Indraccolo S. Tosello V. Piovan E. Cabrelle A. Trentin L. et al.Heterogeneous intracellular expression of B-cell receptor components in B-cell chronic lymphocytic leukaemia (B-CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B-CLL-derived cell line.Br J Haematol. 2005; 130: 878-889Crossref PubMed Scopus (10) Google Scholar Additionally, interaction with Bob1 has been reported to control the stability of Syk.4Siegel R. Kim U. Patke A. Yu X. Ren X. Tarakhovsky A. et al.Nontranscriptional regulation of SYK by the coactivator OCA-B is required at multiple stages of B cell development.Cell. 2006; 125: 761-774Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Also, BAFF-R expression and CD22 expression were reduced in P-FR1 B cells. The abnormal pattern of surface expression was confirmed in a P-FR1–derived EBV B-cell line (see Fig E2, A in the Online Repository at www.jacionline.org). Their lentiviral reconstitution with wild-type Bob1 increased expression of CD22, BAFF-R, and IgD (see Fig E2, B and C). In P-FR1 B cells, the response to BCR stimulation, as measured by Syk and PLCγ2 phosphorylation (Fig 2, B), and response to CD40 stimulation, as measured with nuclear factor-κB and mTORC1 phosphorylation (pS6) (Fig 2, C), were compromised. Accordingly, activated P-FR1–derived B cells failed to upregulate CD86 (see Fig E3, A in the Online Repository at www.jacionline.org). Ex vivo, the P-FR1–derived B cells did not show signs of intrinsic activation, with the exception of increased S6 phosphorylation (see Fig E3, B). The formation of antibody-secreting plasmablasts in response to CD40L and IL-21 was abolished in Bob1-deficient B cells (Fig 2, D and see Fig E3, C-F), and class switch to IgG was delayed and less efficient (Fig E3, D and G), showing a dissociation between class switch and immunoglobulin secretion. The addition of IL-4 or T cell–independent activation with a Toll-like receptor 9 (TLR9) agonist did not compensate for this defective response (see Fig E3, D-G). Marginal zone–like B-cell (CD27+IgD+) formation in response to TLR9 agonist was also defective (see Fig E3, D and G). Consistent with the absence of plasmablasts, secreted immunoglobulins were not detected in the supernatant of cultured P-FR1 B cells (Fig 2, E and see Fig E3, H). Lentiviral reconstitution of P-FR1–naive B cells with wild-type Bob1 (see Fig E4, A in the Online Repository at www.jacionline.org) restored plasmablast formation and immunoglobulin secretion in response to CD40L and IL-21 (Fig 2, F and G and see Fig E4, B and C), strongly suggesting that the POU2AF1 mutation was causative for the impaired B-cell development. The immunologic manifestations of P-FR1 showed some overlap with the Bob1 knockout (Bob1–/–) mice, including dysregulated surface expression of BCR, Syk, CD79β, and BAFF-R2Malone C.S. Wall R. Bob1 (OCA-B/OBF-1) differential transactivation of the B cell-specific B29 (Ig beta) and mb-1 (Ig alpha) promoters.J Immunol. 2002; 168: 3369-3375Crossref PubMed Scopus (20) Google Scholar,5Hess J. Nielsen P.J. Fischer K.D. Bujard H. Wirth T. The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.Mol Cell Biol. 2001; 21: 1531-1539Crossref PubMed Scopus (57) Google Scholar,6Samardzic T. Marinkovic D. Nielsen P.J. Nitschke L. Wirth T. BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.Mol Cell Biol. 2002; 22: 8320-8331Crossref PubMed Scopus (41) Google Scholar; defect in B-cell activation,6Samardzic T. Marinkovic D. Nielsen P.J. Nitschke L. Wirth T. BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.Mol Cell Biol. 2002; 22: 8320-8331Crossref PubMed Scopus (41) Google Scholar and impaired in vivo T-dependent responses, with a lack of germinal centers and strongly reduced IgG.1Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.Nature. 1996; 383: 538-542Crossref PubMed Scopus (237) Google Scholar However, some significant differences were also observed. Bob1–/– mice showed a 2-fold reduction in B-cell numbers, which were normal in the patient. Although serum IgM antibodies were absent in P-FR1, IgM levels were only slightly reduced in Bob1–/– mice, which was attributed to their unaffected B1 B-cell compartment,5Hess J. Nielsen P.J. Fischer K.D. Bujard H. Wirth T. The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.Mol Cell Biol. 2001; 21: 1531-1539Crossref PubMed Scopus (57) Google Scholar the existence of which is debated in humans. Furthermore, expression of CD22, which negatively regulates BCR activation, was increased in Bob1–/– mice and low in P-FR1.7Samardzic T. Gerlach J. Muller K. Marinkovic D. Hess J. Nitschke L. et al.CD22 regulates early B cell development in BOB.1/OBF.1-deficient mice.Eur J Immunol. 2002; 32: 2481-2489Crossref PubMed Scopus (21) Google Scholar This may have resulted from the different source of the B cells examined, the spleen in Bob1–/– mice, and the blood in P-FR1. Interestingly, stimulation of Bob1–/– murine B cells with LPS resulted in plasmablast formation and immunoglobulin secretion in vitro.8Corcoran L.M. Hasbold J. Dietrich W. Hawkins E. Kallies A. Nutt S.L. et al.Differential requirement for OBF-1 during antibody-secreting cell differentiation.J Exp Med. 2005; 201: 1385-1396Crossref PubMed Scopus (50) Google Scholar TLR4 is not expressed in human B cells, but stimulation with TLR9 agonist in P-FR1 was not able to restore plasmablast formation. This is the first report of a patient with a Bob1 deficiency caused by a homozygous POU2AF1 null mutation resulting in a severe B-cell–intrinsic defect. The paucity in TFH cells may derive from dysregulated Bcl-69Stauss D. Brunner C. Berberich-Siebelt F. Hopken U.E. Lipp M. Muller G. The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6.EMBO J. 2016; 35: 881-898Crossref PubMed Scopus (22) Google Scholar and/or from an altered B-cell–T-cell interaction that is essential in the first phase of TFH cell differentiation. Bob1-deficient B cells respond poorly to activating signals. BCR signaling was reduced and plasmablast development and immunoglobulin secretion seemed abrogated in the patient. These functional impairments are Bob1 dependent because reconstitution experiments restored plasmablast development, immunoglobulin secretion, dysregulated surface expression of BCR complex molecules and costimulatory and survival signaling molecules. Overall, these data point to a role for Bob1 in the late stages of human B-cell development, regulating not only activation but also survival, possibly by controlling the expression of key mediators such as BAFF-R and CD79β. In addition, Bob1 deficiency represents a unique example of agammaglobulinemia with normal B-cell numbers. As Bob1 downregulation has been found in non–immunoglobulin-secreting Hodgkin lymphoma, a careful clinical follow-up for the development of lymphoma should be performed in P-FR1. Whether the neurodegenerative symptoms are the consequence of an infection, as is frequently observed in Bruton agammaglobulinemia, or related to an additional genetic disorder remains to be determined, also because central nervous system involvement was not reported in Bob1–/– mice. We wish to thank the patient and his family, as well as the healthy volunteers, for consenting to this work. We thank the CCI Advanced Diagnostic Unit, the Genetics and Genomics Unit, and the Freeze-Biobank, University Medical Center Freiburg, for technical assistance.

Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

Bone marrow (BM) analysis show that 20% of PAD patients harbor a block at an early stage of B cell development. The aim of this work is to study the mechanisms of this developmental block. We set up a feeder free cultivation system that in healthy donors leads to the development of lymphocyte progenitors until the stage of Immature B cells. BM derived CD34+ cells are expanded in cytokine cocktail for 2 weeks. From day 14 to day 49 cells are cultivated in cytokine-free medium and analyzed once a week by flow cytometry and RNA is collected for PCR assays. BM derived CD34+ cells from 15 PAD patients and 2 Btk-deficient patients were tested in vitro for their capability to develop into IgM+ Immature B cells. CD34+ cells from patients showing a normal B cell development in vivo (6/17) developed in vitro until the stage of Immature B cells. Among PAD patients presenting a block at early B cell stage, 5/11, including the Btk-deficient patients, could not reach the Immature B cell stage, while 6/11 could develop in vitro until the stage of IgM+ B cells. Among this last group, reaching Immature B cell stage in vitro but not in vivo, 2/6 patients showed a rapid in vitro development of Immature B cells at day 14, but presented a progressive exhaustion of the common lymphoid progenitors (CLP) compartment. In patients with a block in B cell development (in vivo and in vitro), PCR analysis revealed adelayed expression of transcription factors E2A, CD79a and PAX5, reflecting an impaired lineage commitment. Combining BM phenotyping and in vitro modeling we identified four patients’ groups characterized by (1) an intrinsic B cell defect, (2) a defect in the BM microenvironment impairing early stages of B cell development, (3) a defect in repopulation of CLP and (4) normal B cell development.

The maintenance of B cell homeostasis requires a tight control of B cell generation, survival, activation, and maturation. In lymphocytes upon activation, increased sensitivity to apoptotic signals helps controlling differentiation and proliferation. The death receptor Fas is important in this context because genetic Fas mutations in humans lead to an autoimmune lymphoproliferative syndrome that is similar to lymphoproliferation observed in Fas-deficient mice. In contrast, the physiological role of TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) in humans has been poorly studied so far. Indeed, most studies have focused on tumor cell lines and on mouse models whose results are difficult to transpose to primary human B cells. In the present work, the expression of apoptosis-inducing TRAIL-R1 and TRAIL-R2 and of the decoy receptors TRAIL-R3 and TRAIL-R4 was systematically studied in all developmental stages of peripheral B cells isolated from the blood and secondary lymphoid organs. Expression of TRAIL-Rs is modulated along development, with highest levels observed in germinal center B cells. In addition, T-dependent and T-independent signals elicited induction of TRAIL-Rs with distinct kinetics, which differed among B cell subpopulations: switched memory cells rapidly upregulated TRAIL-R1 and -2 upon activation while naïve B cells only reached similar expression levels at later time points in culture. Increased expression of TRAIL-R1 and -2 coincided with a caspase-3-dependent sensitivity to TRAIL-induced apoptosis in activated B cells but not in freshly isolated resting B cells. Finally, both TRAIL-R1 and TRAIL-R2 could signal actively and both contributed to TRAIL-induced apoptosis. In conclusion, this study provides a systematic analysis of the expression of TRAIL-Rs in human primary B cells and of their capacity to signal and induce apoptosis. This dataset forms a basis to further study and understand the dysregulation of TRAIL-Rs and TRAIL expression observed in autoimmune diseases. Additionally, it will be important to foresee potential bystander immunomodulation when TRAIL-R agonists are used in cancer treatment.