Splenic B-cell Populations Research Articles

Prevention of Chronic Gvhd By Targeting Xbp-1 Genetically or Pharmacologically in Mice

Inhibition of the endoplasmic reticulum (ER) stress response via blockade of inositol-requiring enzyme-1α (IRE-1α) is currently a promising therapeutic strategy to treat B-cell leukemia, lymphoma, and myeloma. Because B cells play an important role in the development of chronic graft-versus-host disease (cGVHD), we hypothesize that the ER stress response contributes to B-cell function and pathogenicity in cGVHD. Here, we report that the ER stress response mediated by IRE-1α and its target X-box binding protein-1 (XBP-1) plays a critical role in cGVHD pathophysiology and represents a potential therapeutic target to prevent cGVHD.We tested the role of XBP-1 specifically in B cells by testing XBP-1 conditional knockout B cell grafts (XBP1fl/flCD19Cre+) in two mouse models of cGVHD. In the first model (B6 to BALB/c), recipients given XBP-1-deficient donor grafts showed significantly reduced cGVHD clinical scores, which were associated with reduced frequencies of donor-derived CD4 helper T cells within the lungs compared to the recipients of XBP-1fl/flCD19Cre- littermate donor grafts. XBP-1-deficient B cells produced significantly higher levels of IL-10 compared to WT control B cells after activation ex vivo. In the second model (B6 to B10.BR), the conversion of donor B cells to plasma cells (B220+CD38+CD138+) was reduced in both the spleens and lungs of recipients transplanted with XBP1fl/flCD19Cre+ grafts compared to those of the recipients given XBP1fl/flCD19Cre- grafts. Recipients given XBP1fl/flCD19Cre+ grafts also showed significantly higher total splenocytes and vastly increased splenic B-cell populations when compared with the recipients of XBP1fl/flCD19Cre- grafts.To expand on these findings, we tested if systemic XBP-1 blockade via a novel IRE-1α inhibitor, B-I09, would attenuate cGVHD. In a cutaneous model of cGVHD (B10.D2 to BALB/c), we found that prophylactic administration of B-I09 significantly reduced clinical features of cGVHD compared to vehicle controls (Fig. 1A). Validating these findings, hematoxylin and eosin stained skin sections of B-I09-treated mice had significantly lower pathology scores compared to vehicle controls (Fig. 1B). Isolated skin lymphocytes from recipients treated with B-I09 showed significant reductions in donor derived T cells and DCs compared to those treated with vehicle controls (Fig. 1C and D).Taken together, our findings reveal a novel role of the IRE-1α/XBP-1 pathway of the ER stress response in cGVHD pathophysiology and provide a readily translatable strategy to prevent the development of cGVHD in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.

Effects of the Anti-CD20 Antibody Rituximab on B Cells in Human Secondary Lymphoid Organs

Introduction: A single dose of the anti-CD20 monoclonal antibody rituximab (RTX) induces a nearly complete B-cell depletion in peripheral blood. However, there remains a residual B-cell population in secondary lymphoid organs, such as spleen and lymph nodes. An intriguing question that remains to be answered is whether these remaining B cells are modified due to binding by RTX. Methods: To mimic the in vivo situation, where B cells are exposed to RTX but only partially depleted, spleen cells obtained from organ donors were incubated in vitro with RTX and a low concentration of complement. The distribution of B-cell subsets was analyzed after 3 days of culture in the presence or absence of CpG-B. Results: Incomplete B-cell depletion followed by culture in the absence of CpG-B resulted in a IgD+CD27- (naïve): IgD-CD27+ (switched memory) B-cell ratio that was comparable to the non-depleted condition. However, after culture in the presence of CpG-B, the B cells that survived the depletion procedure showed a reduced naïve: switched memory ratio. In contrast to the total splenic B-cell population, the depletion surviving B cells did not contain IL-10 producing cells after culture with CpG-B. Moreover, B cells were also characterized ex vivo in peripheral blood and lymph nodes obtained during renal transplant surgery from patients (n=4) who had received RTX 4 weeks before transplantation. As described in literature, a complete depletion of B cells in the peripheral blood was obtained after a single dose of RTX, however in the lymph nodes the frequency of B cells was comparable to that of untreated control patients. Remarkably, the remaining lymph node B cells from RTX-treated patients showed a lower percentage of IgD+CD27- and a higher percentage of IgD-CD27+ B cells compared to lymph node B cells of untreated patients. Conclusion: Exposure of both spleen and lymph node B cells to RTX results in a shift of B cells from a naïve to a switched memory phenotype.

Splenic Morphological Changes Are Accompanied by Altered Baseline Immunity in a Mouse Model of Sickle-Cell Disease

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.

The Effect of Omega-3 as a Dietary Supplement in a Mouse Model of Stem Cell Transplantation (SCT)

Introduction: Several studies reported the importance of nutrition on SCT outcome. Many food components have been shown to affect the immune system and to interact with a number of drugs used in conditioning and in prophylactic treatment during SCT. Omega-3 fatty acids have immuno-modulatory effects via improving endothelial function, protection against oxidative DNA damage and by suppressing CD4+ T-cell function and proliferation. In the present study we examined the effects of omega-3 on the immune system in mice undergoing SCT.Method: Female BALB/c mice, 8 – 12 weeks, were randomised to receive either diet enriched with menhaden fish oil (omega-3) or, corn oil (rich in omega-6). Both groups were fed for two weeks prior to conditioning (Day 0 was set to be the day of transplantation). Both groups injected intra-peritoneally with 80mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200mg/kg cyclophosphamide (Cy) for two days (days –3 & -2). At day 0, half of the mice of both groups were killed and bone marrow, spleen and thymus were harvested and analysed with FACS to evaluate the lymphoid and myeloid cell phenotype. Serum was collected for analysis of cytokines. The remaining mice in both groups were transplanted with allogeneic bone marrow from 8 – 12 weeks male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107 spleen cells).Results: Mice fed an omega 3-rich diet showed significant decrease in bone marrow and spleen cellularity compared to that observed in the corn oil group. In particular, the splenic B-cell population was severely affected, showing a 50% decrease in the omega-3 group compared to that seen in corn oil group. The T-cell populations of omega-3 fed mice were also affected, with a significant decrease by 10 to 20 % in the number of CD4+ cells. In contrast, no difference was observed for CD8+ cells, compared to corn oil-fed mice. In the thymus, the number of activated T-cells was decreased by 50% in the omega-3 compared to that observed in corn oil group. NK cells were decreased by 50% in the spleens of omega-3 fed mice. In bone marrow, the myeloid cells (CD11b+) were significantly lower in omega-3 fed mice compared to corn oil-fed controls. All transplanted mice that had received an omega-3 enriched diet died within the first week. These data can be compared to our previous results where more than 60% of mice receiving standard diet survive during the first week and of which at least 20% reach day+21.Conclusion: Omega-3 administration as a dietary supplement for two weeks prior to conditioning regimen showed significant effects on the immune system in terms of a pronounced alteration in the balance of different cells lineages in both bone marrow and spleen. Rapid death of omega-3 fed animals after allogeneic transplantation was observed. Further studies are warranted to investigate the role of omega-3 on the outcome of stem cell transplantation.

Lifetime exposure to trichloroethylene (TCE) does not accelerate autoimmune disease in MRL +/+ mice

A genetically-prone murine lupus model was used to assess the developmental effects of trichloroethylene (TCE) exposure on disease symptom onset (e.g., autoantibody production and proteinuria), lymphocyte proliferation, splenic B-cell populations, and thymic and splenic T-cell populations. MRL +/+ mice were exposed to TCE (0, 1,400 or 14,000 ppb) via drinking water beginning on gestation day (GD) 0 and continuing until 12 months of age. With the exception of splenic CD4-/CD8-cells in female mice only, no alterations were observed in splenic T-cell populations, numbers of splenic B220+ cells, or in lymphocyte proliferation at 12 months of age. Furthermore, populations of all thymic T-cell subpopulations were decreased in male but not female mice following exposure to 14,000 ppb TCE. Autoantibody levels (anti-dsDNA and anti-GA) were assessed periodically from 4 to 12 months of age. Over this period, no increase in autoantibody levels as compared to control was detected, suggesting that TCE did not contribute to or accelerate the development of autoimmune disease markers following lifetime exposure. Not only does this study offer encouraging results, but it is the first study to approach the development of autoimmunity in a novel lifetime exposure paradigm, using an autoimmune prone model, at environmentally relevant exposure levels.

The Effect of Desensitization Protocols on Human Splenic B-Cell Populations In Vivo

Rituximab, intravenous immunoglobulin (IVIG) and rabbit antithymocyte globulin (rATG) all have been suggested to have an effect on antibody producing cells, however, supporting data are lacking. To assess the impact of these agents on splenic B-cell populations in vivo, we retrospectively examined 25 spleens removed from patients treated with these agents as part of desensitization protocols in either ABO incompatible or positive crossmatch living donor kidney transplantation. These were compared to control (CTL) spleens removed for trauma. CTLs and spleens removed at transplant after multiple pretransplant plasmaphereses (PP) plus low-dose IVIG showed similar large numbers of naïve B cells (CD20+ and CD79+), plasma cells (CD138+) and memory B cells (CD27+ cells). Adding rituximab to this PP/IVIG regimen reduced the number naïve B cells, but had no effect on memory or plasma cells. Combination treatment (PP/IVIG, rituximab and rATG) showed a trend toward the reduction of CD27+ cells, but again plasma cells were unchanged. We conclude that none of these protocols reduces splenic plasma cells in vivo. PP/low-dose IVIG does not alter splenic B cells, but the addition of rituximab decreases mature B cells. Memory B cells may be affected by combination therapy including rATG and requires further study.

Effect of early fetal splenectomy on prenatal B‐cell development in sheep

The contribution of early splenic B-cell populations to the colonization of the ileal Peyer's patch was investigated following the surgical removal of the spleen in a series of 56-day-old fetal sheep. The fetuses were killed at 140 days of gestation and the ileal Peyer's patch, the distal jejunal lymph node which drains the Peyer's patch, and a peripheral lymph node, the superficial cervical lymph node, were examined. Enzyme and immunohistochemical evaluation concluded that the distribution of B cells, T cells and stromal cells in the ileal Peyer's patch was similar in splenectomized and normal fetal sheep. Thus, the presence of the fetal spleen was not essential for the colonization of the ileal Peyer's patch and other early sites of B-cell accumulation would appear capable of generating the necessary precursor populations. Investigation of B-cell populations in lymph nodes used a combination of terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate nick-end-labelling (TUNEL) histochemistry and immunofluorescence to determine the average number of apoptotic B cells in the primary follicles of the outer cortex of splenectomized and normal lambs. A significantly increased number of apoptotic B cells was present in the distal jejunal lymph node but not in the superficial cervical lymph node of splenectomized lambs. This finding suggests that splenectomy affected prenatal B-cell development in fetal sheep and raises questions as to the regulation of B-cell lymphopoiesis in a species using a post-rearrangement organ of diversification.

Therapy with immunoglobulin suppresses myocarditis in a murine coxsackievirus B3 model. Antiviral and anti-inflammatory effects.

The treatment of some inflammatory diseases (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. Accordingly, to elucidate the mechanism underlying such actions of immunoglobulin, we examined its effects on murine coxsackievirus B3 (CB3) myocarditis. An in vitro study showed dose-dependent suppression of CB3 by immunoglobulin. Immunoglobulin 1 g.kg-1.d-1 IP was administered to CB3-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment 1 and on day 14 after virus inoculation in experiment 2. In both experiments, survival was higher in treated than in control mice; at the time of death, inflammatory also were reduced. Notably, in experiment 1, immunoglobulin administration completely suppressed the development of myocarditis. Serum-neutralizing antibody titers in the treated mice were significantly higher than those in untreated mice in experiment 1 but not in experiment 2. The circulating antibodies of the treated mice were primarily of exogenous origin in experiment 1 and of exogenous and endogenous origins in experiment 2. The analysis of splenic lymphocyte subsets revealed a marked decrease of the B cell population in the treated mice. Immunoglobulin therapy completely suppressed acute CB3 myocarditis by transferring the neutralizing antibody into the host in the acute viremic stage and induced an anti-inflammatory effect in the subsequent aviremic stage; the reduction of the splenic B-cell population may be closely associated with an anti-inflammatory effect.

Stochastic pairing of heavy-chain and kappa light-chain variable gene families occurs in polyclonally activated B cells.

Frequencies of 25 immunoglobulin heavy-chain and kappa light-chain variable (VH + V kappa) gene-family pairings expressed in splenic B-cell populations were determined by hybridization of VH- and V kappa-family-specific DNA probes to mitogen-induced B-cell colonies from C57BL/6 mice or hybridomas derived from BALB/c and NZB mice. Both analyses support the conclusion that VH and V kappa gene families pair without bias; as would be expected for random association, the frequencies of specific VH + V kappa pairs may be estimated by the product of the independent VH and V kappa frequencies. Based upon the frequencies at which 9 VH and 12 V kappa gene families are expressed, we calculated the expected usage for approximately 100 VH + V kappa family pairings in neonatal and adult C57BL/6 mice. Variability in the expression of such VH + V kappa pairings is considerable; pairs representing greater than 10% to less than 0.01% of the splenic B-cell population occur. This variability is most pronounced in the neonate, where 6 VH + V kappa family pairs account for nearly 40% of all mitogen-reactive B cells. As the neonate matures, the distribution of frequencies for VH + V kappa pairings becomes more nearly uniform. This process may underlie the patterned acquisition of humoral immune responsiveness.

Neither interleukin 2 nor gamma interferon directly promote growth or differentiation of mouse B cells.

The roles of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) as direct mediators of B-cell growth and differentiation were analysed. Products of cloned genes were used in both cases. The use of flow cytometric assays coupled with density fractionation of responding splenic B-cell populations enabled both the characterization of B cells responding to various stimuli and the estimation of their frequency. B cells responding to non-IL-2 related lymphokines promoting growth and differentiation were restricted to low buoyant density fractions. In addition, these cells expressed densities of IL-2 receptor determinants comparable to those found on T cells, although, IL-2 did not support their growth or differentiation. The inability to demonstrate any direct effect of either IL-2 or IFN-gamma on B cells in any state of activation suggests that their physiological roles are mediated through additional cell types.

Differences between virgin and memory IgM-antibody-forming-cell precursor B-cells, and correlations with the heterogeneity present in B-cell populations from unimmunized mice

Previous in vitro studies showed that a large proportion of DNP-reactive B-cells in the spleens of unimmunized mice, unlike B-cells reactive to fowl gamma globulin (FGG), did not adhere to glass-bead columns. B-cell reactivity was assessed by challenge with dinitrophenylated-polymerized flagellin (DNP-POL) or FGG in the presence of POL, both responses being thymus-independent. Now we have shown that when donor mice are immunized with FGG, a greater proportion of FGG-reactive B-cells becomes non-adherent and the column filtrates give an IgM anti-FGG response. This indicates then that the adherence properties of the IgM-producing antibody-forming-cell (AFC)-precursor differed in virgin and immunized B-cell populations. In vitro testing of the thoracic duct cell (TDC) population of normal and immunized mice revealed parallels between thoracic duct B-cells and the non-adherent splenic B-cell population. Thus while the in vitro anti-DNP response of thoracic duct lymphocytes from normal mice reached levels little below those of spleen cells, the anti-FGG response was much lower. However when TDC were taken from mice immunized with FGG, the anti-FGG responses were much higher. Thus a parallel was demonstrated both in terms of non-adherence to glass and presence in the thoracic duct, of a portion of the DNP-reactive B-cell population in unimmunized mice and the FGG-reactive population in FGG-immunized mice. This suggested that the non-adherent DNP-reactive B-cells in unimmunized mice may represent B-cells with experience of cross-reacting antigens. The significance of this heterogeneity of the B-cell populations reactive to certain antigens in unimmunized mice is discussed with particular reference to recent apparently contradictory findings in regard to B-cell activation.