B-cell Compartment Research Articles

Natural Protection From Type 1 Diabetes in NOD Mice Is Characterized by a Unique Pancreatic Islet Phenotype.

The NOD mouse develops spontaneous type 1 diabetes, with some features of disease that are very similar to the human disease. However, a proportion of NOD mice are naturally protected from developing diabetes, and currently, studies characterizing this cohort are very limited. Here, using both immunofluorescence and multiparameter flow cytometry, we focus on the pancreatic islet morphology and immune infiltrate observed in naturally protected NOD mice. We show that naturally protected NOD mice are characterized by an increased frequency of insulin-containing, smaller-sized, pancreatic islets. Although mice remain diabetes free, florid immune infiltrate remains. However, this immune infiltrate is skewed toward a regulatory phenotype in both T- and B-cell compartments. Pancreatic islets have an increased frequency of IL-10-producing B cells and associated cell surface markers. Resident memory CD69+CD8+ T cells show a significant shift toward reduced CD103 expression, while CD4+ T cells have increased FoxP3+CTLA4+ expression. These data indicate that naturally protected NOD mice have a unique islet signature and provide new insight into regulatory mechanisms within pancreatic islets.

A Longitudinal Analysis Reveals Early Activation and Late Alterations in B Cells During Primary HIV Infection in Mozambican Adults.

Primary HIV infection (PHI) and subsequent chronic infection alter B-cell compartment. However, longitudinal analysis defining the dynamics of B-cell alterations are still limited. We longitudinally studied B-cell subsets in individuals followed for 1 year after PHI (n = 40). Treated and untreated chronic HIV infected (n = 56) and HIV-uninfected individuals (n = 58) were recruited as reference groups at the Manhiça District in Mozambique. B cells were analyzed by multicolor flow-cytometry. Anti-HIV humoral response and plasma cytokines were assessed by ELISA or Luminex-based technology. A generalized activation of B cells induced by HIV occurs early after infection and is characterized by increases in Activated and Tissue-like memory cells, decreases in IgM-IgD- (switched) and IgM-only B cells. These alterations remain mostly stable until chronic infection and are reverted in part by ART. In contrast, other parameters followed particular dynamics: PD-1 expression in memory cells decreases progressively during the first year of infection, Transitional B cells expand at month 3–4 after infection, and Marginal zone-like B cells show a late depletion. Plasmablasts expand 2 months after infection linked to plasma viral load and anti-p24 IgG3 responses. Most of well-defined changes induced by HIV in B-cell activation and memory subsets are readily observed after PHI, lasting until ART initiation. However, subsequent changes occur after sustained viral infection. These data indicate that HIV infection impacts B cells in several waves over time, and highlight that early treatment would result in beneficial effects on the B-cell compartment.

Highly Sensitive Flow Cytometric Detection of Residual B-Cells After Rituximab in Anti-Neutrophil Cytoplasmic Antibodies-Associated Vasculitis Patients.

BackgroundB-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX.MethodsEuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA.ResultsBy employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients.ConclusionWe demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype

AbstractAlthough human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

Depletion of circulating IgM memory B cells predicts unfavourable outcome in COVID-19

Impaired immune responses have been hypothesised to be a possible trigger of unfavourable outcomes in coronavirus disease 2019 (COVID-19). We aimed to characterise IgM memory B cells in patients with COVID-19 admitted to an internal medicine ward in Northern Italy. Overall, 66 COVID-19 patients (mean age 74 ± 16.6 years; 29 females) were enrolled. Three patients (4.5%; 1 female) had been splenectomised and were excluded from further analyses. Fifty-five patients (87.3%) had IgM memory B cell depletion, and 18 (28.6%) died during hospitalisation (cumulative incidence rate 9.26/100 person-week; 5.8–14.7 95% CI). All patients who died had IgM memory B cell depletion. A superimposed infection was found in 6 patients (9.5%), all of them having IgM memory B cell depletion (cumulative incidence rate 3.08/100 person-week; 1.3–6.8 95% CI). At bivariable analyses, older age, sex, number of comorbidities, and peripheral blood lymphocyte count < 1500/µl were not correlated with IgM memory B cell depletion. A discrete-to-marked reduction of the B-cell compartment was also noticed in autoptic spleen specimens of two COVID-19 patients. We conclude that IgM memory B cells are commonly depleted in COVID-19 patients and this correlates with increased mortality and superimposed infections.

Immunologic Characterization of Coronavirus Disease 2019 (COVID-19) Patients with Hematological Cancer: Biologic and Clinical Significance

Background: The immune system reacts to viral infection with cellular and humoral responses. Thus, myelo- and lympho-suppression caused by cancer itself as well as cytotoxic treatment may pose a challenge to COVID-19 patients with solid and hematological tumors, but severe events from initial onset of COVID-19 appear to be more frequent in blood malignancies vs other cancer types. Preliminary data showed lower neutrophil and lymphocyte counts in COVID-19 patients bearing hematological cancer, but there are conflicting results supporting that both worsening of lymphopenia during COVID-19 and its depth prior to infection had a beneficial impact on survival. Thus, greater knowledge on the immune status of hematological patients may be useful to optimize prevention, risk stratification and treatment strategies.Aim: Analyze the immune status of COVID-19 patients with or without solid and hematological cancer.Methods: We use multidimensional flow cytometry (MFC) to analyze immune profiles in peripheral blood samples of 515 COVID-19 patients at presentation. Data was analyzed with a semi-automated pipeline that performs batch-analyses of MFC data to avoid variability intrinsic to manual analysis, and unveils full cellular diversity based on unbiased clustering. In 14 cases, deep immunophenotyping of B- and T-cells was performed and six myeloid- and dendritic-cell subsets were FACSorted for transcriptome analysis using RNAseq.Results: Of the 515 COVID-19 patients, 15 and 10 had solid and hematological tumors, respectively. Those with hematological cancer showed similar frequency of hospitalization than those with solid tumors (90% and 93%, respectively), which was modestly higher to that observed in persons without an active tumor (76%). By contrast, the frequency of hematological cases requiring intensive care (50%) and dying from COVID-19 (30%) was significantly higher to that observed in patients with no active tumor (5.5% and 4%, respectively), or with solid cancer (both 0%).Based on semi-automated analysis of MFC data, we systematically quantified a total of 19 cell types in PB that included 6 myeloid and 13 lymphoid subsets. Patients with hematological malignancies displayed altered immune profiles with significantly decreased absolute numbers of classical and intermediate monocytes, immunoregulatory and cytotoxic NK cells, double-negative, double-positive, CD4 and CD56- γδ T cells, as well as of mature B cells when compared to those with no tumor.Unsupervised hierarchical analysis of RNAseq data from basophils, myeloid and plasmacytoid dendritic cells, classical and non-classical monocytes and neutrophils showed considerable clustering of samples from hematological cases. Furthermore, a variable number of differentially expressed genes was found in all six cell types between COVID-19 patients with or without blood cancer. Genes related to NF-κB and STAT transcription factors as well as genes encoding toll-like receptors and proinflammatory interleukin receptors, all of which described to be implicated in the response and evasion of innate sensing by coronaviruses, were differentially expressed in many of these cell types. Deep phenotypic characterization of T- and B-cell compartments in PB of COVID-19 patients with (N = 4) or without (N = 10) hematological cancer showed that the relative distribution of antigen-dependent maturation stages within the T-cell compartment was generally similar between both groups. However, some hematological cases displayed profound alterations in virtually all of the 16 B-cell subsets analyzed, with a notorious reduction in memory B cells expressing IgG and IgA subclasses.We next compared immune responses from presentation to last follow-up in COVID-19 patients with hematological cancer and favorable (N = 3) vs fatal (N = 3) outcome. Interestingly, we found opposite kinetics in myeloid cell types such as eosinophils and neutrophils, decreasing numbers of various T cell subsets, as well as lower mature B cells and circulating PCs at presentation together with a decrease in B cell counts in deceased cases.Conclusions: Our study exposes for the first time that hematological patients show a constellation of immune alterations that could compromise the response to the infection caused by SARS-CoV-2, suggesting an association between impaired immune responses and poorer outcomes in COVID-19 patients with hematological malignancies.DisclosuresPaiva:SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Characterization of B-Cell and Plasma Cell Compartment By Eight-Color Multiparameter Flow Cytometry in Patients with Waldenstrom Macroglobulinemia Prospectively Enrolled in the Fondazione Italiana Linfomi (FIL) BIO-WM Trial

Characterization of B-Cell and Plasma Cell Compartment By Eight-Color Multiparameter Flow Cytometry in Patients with Waldenstrom Macroglobulinemia Prospectively Enrolled in the Fondazione Italiana Linfomi (FIL) BIO-WM Trial

Recurrent Crebbp Mutations in Follicular Lymphoma Appear Localized to the Committed B-Cell Lineage

Background: Follicular lymphoma (FL) is genetically characterized by translocations involving the BCL2 locus on chromosome 18q21. However, up to 70% of healthy individuals also carry detectable t(14;18)-positive cells, suggesting BCL2 translocation is critical but not sufficient for FL development. Chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 are almost ubiquitously mutated in FL. We previously reported the direct characterization by ultra-deep sequencing of pre-diagnostic blood and tissue specimens from 19 subjects who ultimately developed FL. CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, detected in blood a median of 7.5 years before diagnosis in patients developing FL (8/19, 42%) but not in healthy adults with or without detected BCL2 translocations (0/13, p=0.01 and 0/20, p&amp;lt;0.001, respectively) (Schroers-Martin et al, ASH Annual Meeting 2017). While the BCL2 translocation is thought to occur in pre-B-cell precursors in the bone marrow, the high prevalence of CMG mutations raises the possibility that, analogous to myeloid age-related clonal hematopoiesis, such early lesions could occur in hematopoietic stem cells (HSCs) rather than in the committed B-cell lineage. Methods: To address this question of mutational hierarchy, we studied blood, bone marrow aspirate, and lymph node samples from 6 patients with CREBBP or KMT2D mutations in their FL tumor. We analyzed flow-sorted purified hematopoietic cell populations by deep sequencing, including isotype-specific mature B-cell, mature T-cell, and precursor populations encompassing HSCs and common lymphoid progenitors (CLPs). Patients additionally underwent routine clinical sequencing of tumor biopsy and marrow specimens. Results: Bulk sequencing of a diagnostic bone marrow aspirate from patient FL002 revealed a CREBBP mutation concordant with FL tumor biopsy. To ascertain the population bearing this mutation, we sorted to high purity viable marrow aspirate cells (Fig. A). The CREBBP mutation was confirmed in the mature B-cell compartment at an AF of 40.3% but was not detected in other cell populations. To validate this finding, a similar sorting strategy was employed on viable bone marrow aspirate or peripheral blood samples from another 4 FL patients bearing CREBBP mutations (Fig B). In each case, CREBBP was absent from the CD34+ precursor population. In patient CIML004 a characteristic KMT2D stop mutation was likewise absent in precursors. An atypical case sheds additional light on the localization of early FL mutations. Patient LYM267 was diagnosed with Grade 1-2 FL bearing CREBBP and NRAS mutations. Eight years into a prolonged remission after chemoimmunotherapy, he developed cutaneous and gingival myeloid sarcoma without radiographic or histopathological evidence of FL recurrence. While the CREBBP mutation was not detected in myeloid sarcoma or bone marrow biopsies, concordant NRAS mutations and clonal VDJ rearrangements were seen in all 3 compartments (Fig. C). This unusual clonal lineage favors the occurrence of the CREBBP mutation later than the branch point between morphologically distinct lymphoid and myeloid tumors, likely in the committed B-cell lineage after pre-BCR rearrangements (Fig D). Conclusions: HSCs are believed to be the cell of origin in several lymphoid leukemias, and mouse models have demonstrated lymphoma development with induced CREBBP lesions in HSCs (Horton et al Nat Cell Bio 2017). However, in sorted hematopoietic cell populations from marrow and peripheral blood, we observed CREBBP mutations in B-cell lineages but never in CD34+/CD20- precursor populations or paired lymphoid/myeloid disease. Our data therefore are not in support of HSCs as a precursor reservoir in FL. Given that cells harboring the t(14;18) translocation in healthy individuals appear derived from the germinal center, recurrent mutations in CREBBP are likely to occur after the pre-B-cell stage. Figure Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Khodadoust:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy. Nadel:Innate Pharma: Research Funding; Institut Roche: Research Funding. Diehn:RefleXion: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy. Roulland:Celgene/BMS: Research Funding; Roche: Honoraria. Alizadeh:Pharmacyclics: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Roche: Consultancy.

Detailed immune monitoring of a pregnant woman with critical Covid-19

A primigravid woman with Covid-19 related respiratory insufficiency was admitted into a tertiary Intensive Care Unit at 23 3/7 weeks’ gestation. Highly sensitive flow cytometry of peripheral leukocytes indicated significantly suppressed naïve T- and B-cell compartments. The suppressed immune cell responses led us keep the initially started administration of corticosteroids for fetal and maternal indication at a low dose. After three weeks her B-cell response peaked, SARS-CoV-2 was cleared and clinical improvement ensued a week later. At 28 weeks’ gestation, a son of 1570 g was born by cesarean section. She was extubated two days postpartum and discharged from hospital 5.5 weeks postpartum.

Frequent altered distribution of peripheral B-lymphocyte subsets in pediatric and adolescent patients with classical Hodgkin lymphoma

Peripheral lymphopenia is a well-known negative prognostic marker in classical Hodgkin lymphoma (cHL). We characterized the peripheral B-cell compartment in a prospective cohort of 83 pediatric cHL patients. We observed significantly low total B-cell counts (<100 cells/µl) in 31 of 83 patients (37%). More specifically, there was a smaller peripheral IgDhighCD27– naïve B-cell pool among B-cell lymphopenic patients than for non-B-cell lymphopenic patients (p < 0.01). The B-cell count was lower in patients without in situ Epstein Barr Virus (EBV) expression than among those with in situ EBV expression (p = 0.03). Peripheral B-cell lymphopenia was associated with the presence of poor prognostic features, such as advanced lymphoma stage (p < 0.01) and the presence of B symptoms (p = 0.04). Of interest, B-cell lymphopenia resolved in all six studied patients in long-term remission. Our findings support that cHL tumor-associated factors interfere with the distribution of peripheral B-cell subsets.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia.

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.

Higher frequencies of functional HIV-envelope-specific memory B cells are associated with nonprogressive HIV infection in Indian population.

The HIV-1-specific antibodies are being considered for prevention and therapy in HIV infection. For effective antibody response, presence of functionally competent memory B cells (MEBs) is important; however, HIV-infection is known to alter the B-cell functionality. Very limited data are available on the HIV-specific memory B-cell population in HIV-infected Indian population. In this study, the frequencies of HIV-gp140-specific MEBs were measured in individuals with nonprogressive [long-term-nonprogressors (LTNPs), N = 20] and progressive (N = 19) HIV infection using multicolor flow cytometry. The activation and functional status of these MEBs were assessed as frequencies and mean fluorescence intensity (MFI) of the CD38 and CD40 expression, respectively. The percentages of gp140 + MEBs were higher in LTNPs than seen in progressors (P = 0.0475) and associated with higher CD4 cell count (P = 0.0312, r = 0.2833). As compared with the progressors, LTNPs also showed higher functional (CD40+) gp140 + MEBs both frequencies (P < 0.0001) and CD40 MFI (P = 0.0222), whereas the frequencies (<0.0001) and the MFI (P = 0.0047) of CD38 expression was significantly lower. Higher CD4 cell counts and lower plasma viral load values were associated with higher frequencies of CD40+ gp140 + MEBs (P < 0.0001, r = 0.4962) (P = 0.0036, r = -0.4202) and lower frequencies (P = 0.0008, r = -0.4231) and CD38 expression (MFI) (P = 0.004, r = -0.3719) (P = 0.0066, r = 0.4033). Our study suggests that LTNPs have functional HIV-specific memory B-cell compartment with reduced activation that may lead to effective HIV-specific humoral immune responses contributing to their nondisease progression status. These findings would help in better understanding of the characteristics of the HIV-specific memory B-cell population in nonprogressive HIV infection.

Early ART in Acute HIV-1 Infection: Impact on the B-Cell Compartment.

HIV-1 infection induces B cell defects, not fully recovered upon antiretroviral therapy (ART). Acute infection and the early start of ART provide unique settings to address the impact of HIV on the B cell compartment. We took advantage of a cohort of 21 seroconverters, grouped according to the presence of severe manifestations likely mediated by antibodies or immune complexes, such as Guillain-Barré syndrome and autoimmune thrombocytopenic purpura, with a follow-up of 8 weeks upon effective ART. We combined B and T cell phenotyping with serum immunoglobulin level measurement and quantification of sj-KRECs and ΔB to estimate bone marrow output and peripheral proliferative history of B cells, respectively. We observed marked B cell disturbances, notably a significant expansion of cells expressing low levels of CD21, in parallel with markers of both impaired bone marrow output and increased peripheral B cell proliferation. This B cell dysregulation is likely to contribute to the severe immune-mediated conditions, as attested by the higher serum IgG and the reduced levels of sj-KRECs with increased ΔB in these individuals as compared to those patients with mild disease. Nevertheless, upon starting ART, the dynamic of B cell recovery was not distinct in the two groups, featuring both persistent alterations by week 8. Overall, we showed for the first time that acute HIV-1 infection is associated with decreased bone marrow B cell output assessed by sj-KRECs. Our study emphasizes the need to intervene in both bone marrow and peripheral responses to facilitate B cell recovery during acute HIV-1 infection.

Updates for the treatment of EGPA

Eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg–Strauss syndrome) is the least frequent antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). Major advances of our knowledge on its pathophysiology have revealed features of both AAV and eosinophilic disorders. The development of targeted biotherapies for both diseases opened new possibilities for EGPA management. In this review, we highlight the rationale underlying the routine treatment strategy, which relies mainly on corticosteroids, with immunosuppressant adjunction for severe disease. However, novel therapies are still needed for refractory/relapsing disease and to alleviate the corticosteroid-dependence of asthma and chronic rhinosinusitis. At present, the most promising biotherapies target either eosinophil biology, like mepolizumab, an anti-interleukin-5, or the B-cell compartment, with rituximab. Recent clinical data on new treatment options are discussed and therapeutic strategies are proposed.

DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.

To accomplish the remarkable task of lifelong infection, Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programs to navigate the B-cell compartment and evade immune responses. The transforming program, comprised of highly immunogenic EBV nuclear antigen (EBNA) and Latent Membrane Proteins (LMP), is expressed in newly infected B-lymphocytes and in post-transplant lymphomas. Upon memory cell differentiation and in most EBV-associated Burkitt lymphomas (BL), all but one viral antigen are repressed for immunoevasion. To gain insights into epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR/Cas9 screen was performed in EBV+ BL cells. Here we show that the ubiquitin ligase UHRF1 and its DNA methyltransferase partner DNMT1 were critical for restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing, and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency program switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal center B-cells, the BL cell of origin, providing a molecular link between B-cell state and EBV latency program. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.

The immune system of children: the key to understanding SARS-CoV-2 susceptibility?

The immune system of children: the key to understanding SARS-CoV-2 susceptibility?

Missense mutation in non-recombinase domain of RAG1 results in B-cell developmental defect

Abstract Objective To examine how a single amino acid change to a region of RAG1 that is not related to VDJ recombination affects B-cell development. Methods We studied B-cells within a Rag1 P326G missense mutant mouse (Rag1PG) utilizing flow cytometric analysis to phenotype B-cell development and peripheral populations, as well as biochemical studies examining the B-cell Receptor (BCR) molecular complex and signaling function. We also created a mixed bone marrow chimera to determine which alterations were cell-intrinsic. Results We find that overall there is an ~50% decrease in total B-cells in the Rag1PG mouse compared to wildtype (WT) due to a Pro-B to Pre-B-cell developmental block. Peripheral B cell cytopenias are in the Follicular Zone B-cell compartment, with sparing of the Marginal Zone B-cells. There is decreased Syk expression in Rag1PG—however no differences in BCR signaling strength were appreciated in Rag1PG. In the mixed bone marrow chimera, we find a competitive advantage in the WT developing B-cells, but peripheral B cell populations survive equally well. Conclusion Despite the Rag1PG protein being a single amino acid change to a region of RAG1 not involved in recombination, we nevertheless find a developmental defect consistent with leaky SCID. Given that there is no evidence of altered BCR signaling, and the developmental block occurs at the stage in B-cell development in which VDJ recombination is first required, these data suggest a recombination defect. The mutation is in a ubiquitin ligase domain, and it is possible this alters currently unappreciated roles of RAG1, such as histone degradation to allow RAG1 access to DNA. Our findings illustrate how mutations in the non-recombinase domain of RAG1 may result in leaky SCID.

C C Chemokine Receptor 7 Differentially Regulates Trafficking of T Cells to the Bone Marrow During the Adaptive Immune Response

Abstract It has become increasingly evident that the bone marrow is an important niche for memory B cells and memory T cells that can be activated to become effector T cells during a secondary immune response. However, signaling events that promote bone marrow persistence of memory cells remain largely unknown. We have found that the C-C chemokine receptor 7 (CCR7) signaling plays an important role in regulating the presence of memory cell populations of the bone marrow. In mice homozygously deleted for CCR7 (CCR7−/−) we find that the memory immune cell repertoire is redistributed following the secondary immune response. Specifically, instead of homing to the lymph nodes or spleen, the CCR7−/− effector memory T cells, which respond quickly to antigen, localize to the bone marrow. Additionally, using a CCR7 conditional knockout mouse, we found that when CCR7 is deleted only in T cells, numbers of activated B cells and antibody titers increase, while the regulatory T cells that would normally suppress the immune response, have reduced numbers within the bone marrow. Moreover, we found that in our CCR7f/f-CD4-Cre mice in the absence of CCR7 in T cells, bone marrow activated B cells increased in numbers on day 30. This was not due to differences in the immune response to B-cell cytokine IL-4, which was not significantly increased in any of the mice tested. Instead, we observed a difference in the overall percentage of activated B cells in CCR7f/f-CD4-Cre mice compared to CD4-Cre controls. From these data we infer that CCR7 may promote differential migration of effector memory T cells into the bone marrow that have a direct effect on the composition of the memory B-cell compartment.

Nonpermissive bone marrow environment impairs early B-cell development in common variable immunodeficiency

AbstractCommon 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.