B-lymphocyte Compartment Research Articles

Adaptive immunity in different CT patterns of active tuberculosis and possible variability according to patients' geographic provenience.

BackgroundIt is still unclear if low lymphocyte levels are directly related to immunological modifications induced by the TB infection or if they depend on the general pre-existing health impairment of affected patients. Our aim was to detect eventual differences in the immunological status of patients with pulmonary TB compared to an age and sex-matched group of hospitalized patients with other bacterial community-acquired pneumonia (CAP). In addition, we tried to assess an association between alterations in the peripheral lymphocyte subsets and the development of different CT patterns of active TB and to discover differences in the immunological status and in the radiological patterns of TB presentation between patients of different geographic proveniences.MethodsThis observational study included 48 patients with TB and 48 sex- and age-matched patients affected by other bacterial CAP. The presence of HIV/AIDS, other immunocompromising conditions, and confounding chronic pulmonary comorbidities was excluded. Flow cytometry was performed on all the enrolled subjects at admission, before starting the appropriate antibiotic therapy. Patients with TB also underwent a computed tomography (CT) scan.ResultsPatients with TB showed a decrease in the absolute count of all the lymphocyte subsets compared to the CAP group. Only the reduction in the percentage of CD4+ T-lymphocytes was significant, while the percentage of CD8+ T-lymphocytes was significantly increased. Patients presenting exudative forms with atypical locations of TB showed a significant reduction in the absolute count and percentage of CD19+ B-lymphocytes compared to those affected by productive TB forms with the typical location. Despite being younger, our black Sub-Saharan Africans showed a significant reduction in the CD4+ T-lymphocytes compartment and a higher prevalence of atypical and exudative forms of TB compared with white Europeans.ConclusionTuberculosis itself may alter peripheral blood lymphocyte subsets compared to other CAP. An impaired CD19+ B-lymphocyte compartment may result in an abnormal exudative response in atypical locations and a suboptimal bacterial control. Other constitutive or environmental causes may influence immunological differences found in patients with TB, particularly in case of different geographic origins. Anyhow, flow cytometry may be of great value in evaluating the immune function of these patients.

Elucidating in vitro and in vivo phenotypic behaviour of L. infantum/L. major natural hybrids.

The clinical manifestation and course of Leishmania infections depend on factors such as species, virulence and host-immunity. Although trypanosomatids are considered to have clonal propagation, genetic hybridization has produced successful natural hybrid lineages. Hybrids displaying strong selective advantages may have an impact on pathogenesis and the eco-epidemiology of leishmaniasis. Thus, characterization of phenotypic properties of Leishmania hybrids could bring significant insight into the biology, infectivity, pathogenicity and transmission dynamics of these atypical strains. The present study focuses on phenotypic features and survival capacity of Leishmania infantum/Leishmania major hybrid isolates as compared with representative putative parental species, L. infantum and L. major. In vitro assays (growth kinetics, susceptibility to different conditions) and in vivo infection (parasite detection and histopathological alterations) showed that hybrids present higher growth capacity and decreased susceptibility to reactive oxygen species. Furthermore, evaluation of infected spleen tissue suggests that hybrids induce a stronger immune reaction than their putative parents, leading to the development of white pulp hyperplasia in B-lymphocyte compartments. Overall, these hybrids have shown high plasticity in terms of their general behaviour within the different phenotypic parameters, suggesting that they might have acquired genetic features conferring different mechanisms to evade host cells.

FBXW7 Is Critical for B1 Cell Maintenance and Function

Background: The E3 ubiquitin ligase Fbxw7 is responsible for the proteasomal degradation of several oncogenes involved in cellular proliferation, growth and survival. Notably, Fbxw7 has been characterized as a tumor suppressor in a variety of cancers, including T ALL and CLL. In the absence of Fbxw7, hematopoiesis is defective leading to abnormal development of B and T cells. In these mice, hematopoietic stem cells have impaired self-renewal due to stabilization of c-Myc. Fbxw7 targets several well-characterized molecules (c-Myc, Mcl1, p100, cyclin E, mTOR, SREBP1) with essential functions in B cells. We have recently shown that glycogen synthase kinase 3 (Gsk3), which is negatively regulated by PI3K signaling, controls the metabolic needs of B cells (Jellusova et al., 2017 Nat. Immunol.). A number of Gsk3 substrates are phosphorylated and degraded via the proteasome through recognition of Fbxw7. We sought to investigate the GSK3-FBXW7 axis in B cells and its implications for B cell malignancies.Results: To elucidate the role of Fbxw7 in B cells, we studied Fbxw7 in a conditional knockout mouse model. We crossed Fbxw7fl/flmice with Mb1Cre mice to ablate Fbxw7 during early B cell development. In the absence of Fbxw7, B cells passed through their developmental stages and showed no significant signs of maturation defects in the bone marrow. However, mature recirculating B cells were nearly absent in Fbxw7-deficient animals. Correspondingly, peripheral B cells in the spleen were reduced and showed specific decreases in follicular B cells. Histological analysis of the spleen architecture showed intact B cell follicles with presence of marginal zone B cells. In B cell malignancies, mutations of FBXW7 have been reported in CLL, for which the normal counterpart are B1 cells. We analyzed the B1 cell compartment in the peritoneal cavity in the absence of Fbxw7. Loss of Fbxw7 resulted in drastic reductions of B1 cells in the peritoneal cavity. Both B1a and B1b cells were mutually affected in these mice. To further characterize this B1 cell defect, we generated a novel acute deletion model for Fbxw7. We crossed Fbxw7fl/flmice with hCD20Cre-ERT2 and administered tamoxifen by oral gavage to induce genetic ablation. Acute deletion of Fbxw7 lead to reduction of B1 cells within a few weeks, indicating the importance of Fbxw7 for the maintenance of B1 cells. When challenged with T cell-independent immunizations the overall antibody responses where reduced. BCR-stimulation of cells in vitro showed diminished growth and signaling potential of knockout cells. These cells have reduced calcium signaling and undergo massive apoptosis. The survival defect could be partially rescued with co-stimulation of IL-4 or ectopic expression of BCL2.Conclusion: Our findings identify Fbxw7 as a critical regulator of B cell development. Specifically, Fbxw7 regulates B cell signaling, development and maintenance of B1 cells. These findings are directly relevant to the pathogenesis of CLL. Loss of function mutations of FBXW7 in CLL likely occur later during tumor evolution and potentially contribute to the specific microenvironmental niche. DisclosuresRickert:Pfizer: Employment.

Allogeneic Hematopoietic Stem Cell Transplant for GATA2 Deficiency

Background:Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with atypical mycobacterial infection (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency.Methods: We carried out myeloablative allogeneic hematopoietic stem cell transplant (HSCT) on 24 patients (mean age 24.9 years; range 16 to 46 years) with mutations in GATA2, or the MonoMAC syndrome, during the preceding three years. Three matched related donor (MRD) recipients and 13 matched unrelated donor (URD) recipients received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on day's -6, -5, -4, and -3. Eight haploidentical related donor recipients received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. The majority of the MRD and URD recipients (n=13) received tacrolimus and short course methotrexate post-transplant as graft-versus host disease (GVHD) prophylaxis; three URD, while three received post-transplant cyclophosphamide (PT/CY) on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for GVHD. All 8 haploidentical related donor recipients received PT/CY as described followed by tacrolimus/methotrexate.Results:Twenty-two of the 24 patients are alive and disease-free at a mean follow-up of 13 months (range, 2 to 36 months). One matched URD recipient died from persistent AML 110 days post-transplant, and one matched URD recipient died from GVHD 2 years post-transplant. Despite a previous history of MAC in 11 patients, including 3 patients with active MAC at the time of HSCT, there were no recurrences of MAC during or following HSCT. Similarly, 13 patients had MDS with cytogenetic abnormalities at the time of HSCT (trisomy 8, monosomy 7, 5q-, trisomy 1q), and all 13 had resolution of their cytogenetic abnormalities with no late relapses following HSCT. Three patients had lymphedema at the time of HSCT (Emberger syndrome), which was not reversed. Twenty-three of the 24 patients had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, which were severely deficient pre-transplant. One URD who received PT/CT had poor graft reconstitution and required a CD34+ donor cell boost three months post-transplant. Four of the 13 MRD and URD recipients who received tacrolimus/methotrexate developed grade III-IV acute GVHD, and 3 of the 7 who were more than one-year post-HSCT developed moderate to severe chronic GVHD. No haploidentical related donor recipient developed grade III-IV GVHD or moderate to severe chronic GVHD.Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of clinical manifestations of GATA2 deficiency with low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We are currently using PT/CY in MRD and URD recipients with normal, favorable, or intermediate cytogenetics with isolated trisomy 8 to reduce the incidence of acute and chronic GVHD. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular MDS with or without trisomy 8. DisclosuresNo relevant conflicts of interest to declare.

IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression.Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice.In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through Filipin based cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of lipid rafts formation with decreased levels of ganglioside GM1. Thereby, Inadequate CD19/BCR co-receptor signaling caused by disruption of lipid rafts homeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo.Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining lipid rafts and CD19 surface expression. IFITM3-dependent lipid raft stability and CD19 surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment.

SummaryB cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

Allogeneic Hematopoietic Stem Cell Transplant for GATA2 Deficiency

▪Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency.Methods: Eleven patients with GATA2 deficiency received a myeloablative-conditioning regimen (2 matched related donors or MRD, 4 matched unrelated donors or URD, and 5 haploidentical related donors. MRD and URD received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on days -6, -5, -4, and -3. Haploidentical related donors received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. MRD and URD recipients received tacrolimus and short course methotrexate post-transplant, while haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for graft-versus-host disease.Results: Ten of the 11 (91%) of patients are alive and disease-free at a mean follow-up of 12 months (range 1 mo to 24 mo). One URD recipient died from persistent acute myelogenous leukemia. Four patients developed graft-versus-host disease, one case Grade 4. All 10 patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant. All 10 patients had reversal of the infection susceptibility phenotype. In particular, there were no recurrences of NTM infections. Importantly, all 10 patients had correction of the cytogenetic abnormalities present pre-transplant (5 patients with trisomy 8 and 1 patient with monosomy 7).Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of GATA2 deficiency. There was a low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular myelodysplastic syndrome. DisclosuresNo relevant conflicts of interest to declare.

The molecular pathogenesis of B-cell non-Hodgkin lymphoma.

The B-cell non-Hodgkin lymphomas (B-NHL) are a diverse group of haematological malignancies which arise from the mature B-lymphocyte compartment. Recently, our understanding of the molecular pathogenesis of these disorders has greatly increased due to technological advances such as high-throughput DNA sequencing techniques. A paradigm of B-NHL pathogenesis has emerged where the normal genetic processes that are central to generating B-cell receptor diversity (somatic hypermutation and class switch/VDJ recombination) also drive the genesis of large-scale, chromosomal-level genetic lesions and smaller-scale gene-level mutations to produce the malignant phenotypes observed. Whilst a significant degree of genetic heterogeneity exists within each B-NHL subtype, the genetic lesions present within each subtype show a degree of convergence on common intracellular signalling, epigenetic and cell cycle pathways. This convergence gives an insight into the key oncogenic drivers of specific B-NHL subtypes and potential targets for therapeutic intervention. This review covers the current understanding of the causative genetic processes of B-NHL, the associated driving molecular lesions and the implications of these findings for the treatment of this group of disorders.

Successful Haploidentical Hematopoietic Stem Cell Transplant for GATA2 Deficiency

▪Background: GATA2 deficiency results in a clinical syndrome known variably as: MonoMAC for the lack of monocytes and atypical mycobacterial infections (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger’s syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Reconstitution of the deficient cell compartments in GATA2 deficiency with allogeneic hematopoietic stem cell transplant (HSCT) results in reversal of the infection susceptibility and represents the only curative therapy for both the myeloid disease and the virally driven malignancies seen in this disorder. However, only about one-half of patients that require HSCT will have an HLA-matched related or unrelated donor. Moreover, the use of umbilical cord blood has been reported to be suboptimal in this cohort of patients in whom infection susceptibility underlies much of the need for a transplant.Methods: We performed haploidentical, related donor HSCT with high dose post-transplant cyclophosphamide (PT/CY) in 3 patients with GATA2 deficiency in whom a suitable HLA matched donor was not available. All three patients had a first degree relative (sibling) who shared at least 1 HLA-haplotype. The first patient, a 21 year-old Chinese woman presented with Hydroa Vacciniforme like T-cell lymphoma involving her lung, bowel (resulting in multiple small bowel resections and ileostomy), and skin (with large ulcerative lesions); multiple thrombotic cerebral events; and macrophage activation syndrome requiring etoposide and high dose prednisone in the intensive care unit (ICU). The second patient, a 20 year-old Hispanic female, presented with myelodysplasia (MDS), profound neutropenia, an invasive fungal sinusits due to Fusarium sp., and recurrent bacteremias. The third patient, a 45 year-old woman, presented with Emberger’s Syndrome, multiple episodes of bacterial and fungal sepsis and hypocellular MDS. All three patients received cyclophosphamide 14.5mg/kg on day’s -6 and-5; fludarabine 30mg/m2 on day’s -6 through -2; 200cGy of total body irradiation (TBI) on day -1. Patients 2 and 3 also received busulfan 3.2 mg/kg on day’s -4 and -3 because of MDS. All 3 patients received high dose PT/CY 50mg/kg on day’s +3 and +4, and mycophenolate mofetil and tacrolimus starting on day +5.Results: All 3 patients engrafted at a mean of 18 days and all are alive at a median follow-up of 9 months (range, 5 to 13 months). All three patients achieved 100% donor myeloid and lymphoid chimerim by day +100 post-transplant. The first patient had complete resolution of her T-cell lymphoma and macrophage activation syndrome, the second patient has had complete reversal of her hematopoietic and infectious phenotype, and the third patient has had resolution of her infectious complications that required continuous IV Daptomycin for 5 years prior to transplant. In addition, all three patients had complete reconstitution of the monocyte, NK cell, and B-lymphocyte compartments that were severely deficient prior to transplant. The main toxicity was mucositis, which required intravenous narcotics in all three patients. Lastly, the first patient developed late grade I liver graft-versus-host disease (GVHD) that was responsive to cyclosporine, the second patient developed grade I acute GVHD of skin that was responsive to topical steroids, and the third patient had no evidence of acute or chronic GVHD.Conclusions: Haploidentical, related donor HSCT represents a safe and effective viable alternative for patients with GATA2 deficiency who lack an HLA-matched donor, including patients such as our first patient who was in the ICU at the time of transplant. DisclosuresNo relevant conflicts of interest to declare.

Nonmyeloablative Versus Myeloablative Allogeneic Hematopoietic Stem Cell Transplant for GATA2 Deficiency

Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger’s syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) is the only definitive therapy for GATA2 deficiency.Methods: We used matched related donors (MRD), matched unrelated donors (URD), umbilical cord blood (UCB), and haploidentical related donors in allogeneic HSCT for GATA2 deficiency. Fourteen patients received a nonmyeloablative conditioning regimen (4 MRD, 4 URD, 4 UCB, and 2 haplo donors). Five patients received a myeloablative conditioning regimen (1 MRD, 2 URD, and 2 haplo donors). In the nonmyeloablative group, MRD and MUD recipients received fludarabine and 200cGy of total body irradiation (TBI), UCB recipients received cyclophosphamide 50mg/kg, fludarabine 150 mg/m2, and 200cGy of TBI, and haploidentical related donor recipients received cyclophosphamide 29 mg/kg, fludarabine 150 mg/m2, and 200 cGy TBI. In the myeloablative group, MRD and URD received busulfan 12.8 mg/kg and fludarabine 160 mg/m2, and haploidentical related donors received the same regimen as in the nonmyeloablative regimen except for the addition of two days of busulfan 6.4 mg/kg total dose. Nonmyeloablative MRD and URD recipients received tacrolimus and sirolimus post-transplant, and myeloablative MRD and URD recipients received tacrolimus and short course methotrexate post-transplant. All haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil. Three patients in the nonmyeloablative cohort required one or more rounds of pre-transplant chemotherapy because of an increased number of blasts, whereas none of the 5 patients in the myeloablative arm required pre-transplant chemotherapy.Results: In the nonmyeloablative cohort, 8 of 14 (57%) of patients are alive at a median follow-up of 3.7 years (range 12 months to 5 years). One MRD recipient died of GVHD and one relapsed, one URD recipient rejected the donor stem cells and died, three UCB recipient died (one rejection, one early death, and one donor cell leukemia), and one haploidentical recipient died from regimen-related toxicity. All 5 patients (100%) in the myeloablative group, including two recipients of haploidentical related donors, are alive at a median follow-up of 9.2 months (range 6 to 12 months). All patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant, and all had reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of non-tuberculous mycobacterial (NTM) infections.Conclusions: Nonmyeloablative HSCT results in reversal of the hematologic and clinical manifestations of GATA2 deficiency. However, a more intensive conditioning regimen with busulfan resulted in more uniform engraftment, a reduced risk of relapse, avoidance of pre-transplant chemotherapy, and a low regimen-related toxicity. We anticipate that with the use of a high-dose regimen with busulfan, the replacement of UCB with haploidentical related donors, and HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. DisclosuresNo relevant conflicts of interest to declare.

Reconstitution of the peripheral B lymphocyte compartment in patients with ANCA-associated vasculitides treated with rituximab for relapsing or refractory disease

While in patients with rheumatoid arthritis B-cell repopulation starts within 9 months after rituximab (RTX) therapy, a delayed B-cell repopulation was reported in some RTX-treated patients with ANCA-associated vasculitides (AAV). To date, the frequency of AAV patients with impaired peripheral B-cell regeneration and the mechanisms leading to the constricted regenerative capacity are unknown. We analyzed the B-cell repopulation kinetic in 37 AAV patients treated with RTX followed by maintenance immunosuppressants. We report on serum concentrations of the B-cell-activating factor BAFF, immunoglobulins and B-cell subpopulations in patients that relapsed after RTX. B-cells were re-detectable in only one patient within 9 months after RTX. In 14 patients (41%), B-cell repopulation started later, after a mean observation time of 21 months. Only seven of these patients had detectable B-cells within the first year after RTX. Twenty patients (59%) had no B-cell reconstitution within the observation period. BAFF was increased in RTX-treated AAV patients compared to healthy controls and correlated inversely with peripheral B-cell numbers, IgG- and IgA concentrations. Immunoglobulin concentrations declined significantly after RTX and the IgG concentration correlated with B-cell numbers. Thirteen patients relapsed after RTX. Relapses occurred exclusively either after B-cell reconstitution had started or were accompanied by rising ANCA titres. In relapsed patients, the B-lymphocyte compartment consisted mainly of switched memory B-cells. Our data indicate that RTX treatment can induce secondary immunodeficiency in AAV, with hypogammaglobulinemia and long-lasting B-lymphopenia. Further studies are needed to define the pathophysiology of the impaired B-cell development in RTX-treated AAV patients.

Siglec-G Deficiency Leads to More Severe Collagen-Induced Arthritis and Earlier Onset of Lupus-like Symptoms in MRL/lpr Mice

Siglec-G is a member of the sialic acid-binding Ig-like lectin (Siglec) family expressed on all B cells. Siglec-G-deficient mice show a large expansion of the B1 cell compartment, demonstrating the crucial role of Siglec-G as an inhibitory receptor on this cellular subset. Although Siglec-G-deficient mice did not develop spontaneous autoimmunity, mice double-deficient for Siglec-G and the related Siglec protein CD22 did show autoimmunity at an older age. In this study, we addressed the question of whether loss of Siglec G on its own affects disease severity in animal models of rheumatoid arthritis and systemic lupus erythematosus. Siglec-G-deficient mice showed moderately increased clinical severity and higher inflammation of the knee joints following collagen-induced arthritis, when compared with control mice. The Siglec-G-deficient mouse was also backcrossed to the autoimmune prone MLR/lpr background. Although both Siglec-G-deficient and control MRL/lpr mice developed a lupus-like disease, Siglec-G-deficient MRL/lpr mice showed an earlier occurrence of autoantibodies; a higher lymphoproliferation of B and T cells; and an earlier onset of disease, as shown by proteinuria and glomerular damage in the kidney. Moreover, Siglec-G-deficient female mice showed a significantly reduced survival compared with female control MRL/lpr mice. Thus, the loss of the inhibitory receptor Siglec-G led to a moderate exacerbation of disease severity and early onset in both collagen-induced arthritis and spontaneous lupus nephritis in MRL/lpr mice.

C5a synergizes with IL10 and Toll-like receptors to control the peritoneal B1 cell compartment

Changes in the glycosylation of immunoglobulins have been shown to modulate immune homeostasis and disease pathology. In this sense it has been shown that highly galactosylated but not agalactosylated IgG1 immune complexes (ICs) inhibit C5aR-mediated pro-inflammatory immune responses via the assembly of FcγRIIB-Dectin-1 receptor complexes. In this study we demonstrated that Galectin-3, a galactose-binding lectin that is known to cross-link proteins on cell-surfaces via binding their N-glycans, bound to highly-galactosylated, but not agalactosylated IgG1. Further, Galectin-3 was essential for the IC-mediated inhibition of C5a-induced neutrophil chemotaxis in vitro. Taken together our results indicate that Galectin-3 mediates the interaction of ICs with the FcγRIIB-Dectin-1 receptor complex for delivering immunoregulatory signals to inhibit C5aR-mediated immune responses.

B cell homeostasis is disturbed by immunosuppressive therapies in patients with ANCA-associated vasculitides

B-lymphocytes play a pivotal role in ANCA-associated vasculitides (AAV). The homeostasis of peripheral human B-lymphocyte subpopulations is tightly regulated, but may be disturbed in autoimmune disease or following immunosuppressive therapies. To elucidate the effect of immunosuppression and the relevance of B-lymphocyte disturbances, the B-lymphocyte compartment was analysed in 61 AAV patients. After immunosuppressive treatment a general B-lymphocytopenia developed in AAV patients. Within the B-lymphocyte subpopulations transitional B cells are the first maturation stage found in the peripheral blood. Transitional B-lymphocytes were significantly lower in AAV patients after immunosuppressive therapy compared to healthy controls. Furthermore, marginal zone B cells – a B-lymphocyte population protecting against encapsulated bacteria -- were markedly lowered after immunosuppressive therapy in AAV patients. AAV patients treated with immunosuppressants had lower numbers of naïve and memory B-lymphocytes. Numbers of marginal zone B cells, memory B cells and plasmablasts correlated with concentrations of immunoglobulins. We evaluated plasmablasts for a potential correlation with disease activity. Different from what has been reported for e.g. large vessel vasculitis, absolute numbers of plasmablasts were not increased in patients with AAV and showed no correlation to disease activity. As low transitional B cells after treatment with immunosuppressants indicated an impaired early B-lymphocyte development, seven patients treated with the B cell depleting agent rituximab (RTX) because of relapsing disease activity were analysed for their B cell repopulation kinetics. In the majority of these patients repopulation of the peripheral B cell compartment by newly formed transitional B cells after RTX treatment was constricted and delayed.

Aberrant B‐lymphocyte responses in lupus: inherent or induced and potential therapeutic targets

Lupus is a prototype autoimmune disease of unknown aetiology. The disease is complex; manifest diverse clinical symptoms and disease mechanisms. This complexity has provided many leads to explore: from disease mechanisms to approaches for therapy. B-lymphocytes play a central role in the pathogenesis of the disease. However, the cause of aberrant B-lymphocyte responses in patients and, indeed, its causal relationship with the disease remain unclear. This article provides a synopsis of current knowledge of immunological abnormalities in lupus with an emphasis on abnormalities in the B-lymphocyte compartment. There is evidence for abnormalities in most compartments of the immune system in animal models and patients with lupus including an ever expanding list of abnormalities within the B-lymphocyte compartment. In addition, recent genome-wide linkage analyses in large cohorts of patients have identified new sets of genetic association factors some with potential links with defective B-lymphocyte responses although their full pathophysiological effects remain to be determined. The accumulating knowledge may help in the identification and application of new targeted therapies for treating lupus disease. Cellular, molecular and genetic studies have provided significant insights into potential causes of immunological defects associated with lupus. Most of this insight relate to defects in B- and T-lymphocyte tolerance, signalling and responses. For B-lymphocytes, there is evidence for altered regulation of inter and intracellular signalling pathways at multiple levels. Some of these abnormalities will be discussed within the context of potential implications for disease pathogenesis and targeted therapies.

Allogeneic Hematopoietic Stem Cell Transplant Reverses the Phenotype of GATA2 Deficiency.

AbstractAbstract 3091 Background:Recently, sporadic and heritable mutations in the zinc finger transcription factor GATA2 were shown to be responsible for four different syndromes in young adults coupling opportunistic infection with a predilection to develop myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). These four syndromes are: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome, lymphedema and MDS with monosomy 7; and familial MDS/AML. Life-threatening infections, and the transformation to AML, either alone or together, constitute a rationale for allogeneic hematopoietic stem cell transplant (HSCT) for GATA2 deficiency. Methods:We evaluated matched related, unrelated, and umbilical cord blood as donor sources for nonmyeloablative conditioning for HSCT in GATA2 deficiency. Twelve patients with GATA2 deficiency underwent allogeneic transplant: 4 received peripheral blood stem cells (PBSCs) from matched related-donors (MRD), 4 received PBSC from matched unrelated-donors (MUD), and 4 received umbilical cord blood (UCB) units. Recipients of MRD and MUD transplant received fludarabine and 200cGy of total body irradiation (TBI), while UCB recipients received cyclophosphamide 50 mg/kg, fludarabine, and 200cGy of TBI. All patients received tacrolimus and sirolimus for GVHD prophylaxis.This cohort of patients had considerable pre-transplant morbidity: two patients required baseline oxygen for pulmonary alveolar proteinosis, one of whom was on a ventilator at the time of transplant; one patient had active hepatitis C that was not responding to therapy; one patient had RAEB-2; two patients were platelet transfusion-dependent; one patient had recurrent strokes and culture negative endocarditis two months before transplant. Results:Median follow-up for patients was 14.4 months (range 0.2–38.2 months). Ten of 11 patients engrafted at a median of 10 days (range 0–76); engraftment was not evaluable in a recipient of an UCB transplant due to death early in the post-transplant period. One rejection occurred in a recipient of a double UCB transplant who had been heavily transfused pre-transplant. All patients who engrafted had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant, and all had reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of NTM infection and extensive human papilloma virus infections regressed starting around 6 months post transplant. Two patients required a single cycle of pre-transplant chemotherapy for RAEB-1 and RAEB-2, respectively. In both patients the monosomy 6 and monosomy 7 clones have not recurred, now 2.5 years and 9 months following single UCB and MUD transplant, respectively.Three patients died, two early after transplant. One recipient of UCB, who had pre-transplant hepatitis C, died on day+7 of fulminant liver failure and sepsis. A second patient, who was intubated at the time of transplant because of severe pulmonary alveolar proteinosis and pulmonary hypertension, died on day + 88 of grade IV GVHD after MRD transplant. The third patient died 9 months after transplant of sepsis. One recipient of a MRD relapsed from her MDS at 1 year following transplant and was retransplanted using a myeloablative conditioning regimen. She is alive with no evidence of MDS at 3 months. Acute GVHD developed in 6 patients, five of which were steroid-responsive. One patient developed chronic GVHD. Other complications included immune mediated cytopenias that responded to rituximab and eltrombopag. Conclusions:Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B and NK cell populations and reversal of the infection susceptibility phenotype. The TRM in this cohort of patients with severe comorbidities was 25%. Now that genetic testing for GATA2 mutations is available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in the course of disease, before significant organ damage or clonal evolution of MDS to AML. Disclosures:No relevant conflicts of interest to declare.

Increased circulating apoptotic lymphocytes in children with Down syndrome

Down syndrome (DS) resembles immunodeficiency with increased infections, auto-immune diseases, and hematological malignancies. Until now, immunological studies in DS mainly focused on T-lymphocytes. We recently described a profound B-lymphocytopenia in children with DS. This could be caused by increased apoptosis. Therefore, we determined expression of flowcytometric markers for apoptosis [Annexin-V (AV) and propidium iodide (PI)] on peripheral lymphocytes in 72 children with DS and 32 age-matched controls (AMC). Within the total lymphocyte compartment, apoptosis was more pronounced in DS; it increased with age. Moreover, apoptosis was highest within the B-lymphocyte compartment which may be a contributing factor to the B-lymphocytopenia found in DS. Pediatr Blood Cancer 2012; 59: 1310-1312. © 2012 Wiley Periodicals, Inc.

Gene profiling of CD11b+ and CD11b− B1 cell subsets reveals potential cell sorting artifacts

To the Editor: In a recent issue of The Journal of Experimental Medicine , [Griffin and Rothstein][1] described two subsets within the human B1 cell compartment: a major CD11b− subset (9/10th of the population) and a minor CD11b+ subset (1/10th of the population), with the latter being increased

Age‐matched Reference Values for B‐lymphocyte Subpopulations and CVID Classifications in Children

Age-matched reference values are generally presented with 5th and 95th percentiles as 'normal' reference range. However, they are mostly determined in relatively small groups, which renders this presentation inaccurate. We determined reference values for B-lymphocyte subpopulations in healthy children with the statistical method of tolerance intervals that deals far better with the relatively small numbers tested, and compared these to the cut-off values used in the currently used EUROclass classification for common variable immunodeficiency disorders (CVID) in children. CVID is a heterogeneous group of primary immunodeficiency diseases characterized by low serum immunoglobulin levels and inadequate response to vaccination. Disease-modifying heterozygous amino acid substitutions in TACI are found in around ±10% of CVID patients. Interestingly, we found that age is the primary determinant of TACI-expression on B-lymphocytes, independent of switched memory B-lymphocyte numbers. Immunophenotyping of B-lymphocyte subpopulations is increasingly used to classify patients with CVID into subgroups with different clinical prognosis according to the composition of their B-lymphocyte compartment. These classifications were mainly developed with data obtained in adults. Because of the maturing paediatric immune system, they may not be equally applicable in children: our and other age-matched reference values show great changes in the composition of the B-lymphocyte compartment during development. Although the greatest changes in B-lymphocyte subpopulations occur below the age of 2 years, when the diagnosis of CVID cannot yet be made, it is likely that a classification developed in adults cannot be used to classify the prognosis of children.

A hypomorphic IgH-chain allele affects development of B-cell subsets and favours receptor editing

The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene-targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin μ heavy chain (μHC) mRNA levels and hence lower pre-BCR and BCR levels. Hypomorphic expression of μHC leads to augmented selection processes at all stages of B-cell development, noticeably at the expansion of pre-B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B-lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B-cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre-BCR and BCR receptor levels for the normal development of B-lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.