μMT Mice Research Articles

Pertussis Toxin Inhibits Encephalitogenic T-Cell Infiltration and Promotes a B-Cell-Driven Disease during Th17-EAE.

Pertussis toxin (PTX) is a required co-adjuvant for experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin antigen. However, PTX’s effects on EAE induced by the transfer of myelin-specific T helper cells is not known. Therefore, we investigated how PTX affects the Th17 transfer EAE model (Th17-EAE). We found that PTX significantly reduced Th17-EAE by inhibiting chemokine-receptor-dependent trafficking of Th17 cells. Strikingly, PTX also promoted the accumulation of B cells in the CNS, suggesting that PTX alters the disease toward a B-cell-dependent pathology. To determine the role of B cells, we compared the effects of PTX on Th17-EAE in wild-type (WT) and B-cell-deficient (µMT) mice. Without PTX treatment, disease severity was equivalent between WT and µMT mice. In contrast, with PTX treatment, the µMT mice had significantly less disease and a reduction in pathogenic Th17 cells in the CNS compared to the WT mice. In conclusion, this study shows that PTX inhibits the migration of pathogenic Th17 cells, while promoting the accumulation of pathogenic B cells in the CNS during Th17-EAE. These data provide useful methodological information for adoptive-transfer Th17-EAE and, furthermore, describe another important experimental system to study the pathogenic mechanisms of B cells in multiple sclerosis.

Hepatocyte-Derived Igκ Exerts a Protective Effect against ConA-Induced Acute Liver Injury.

Immunoglobulin (Igκ) has been reported to be expressed in sorted liver epithelial cells of μMT mice, and the sequence characteristics of hepatocyte-derived Igκ were different from those of classical B-cell-derived Igκ. However, the physiological function of hepatocyte-derived Igκ is still unclear. The expression of Igκ was firstly identified in primary hepatocytes and normal liver cell line (NCTC1469), and hepatocyte-derived Igκ expression was elevated and displayed unique localization in hepatocytes of concanavalin A (ConA)-induced hepatitis model. Moreover, Igκ knockout mice were more sensitive to ConA-induced hepatitis and had higher serum aspartate aminotransferase (AST) levels, more severe histological injury and a greater number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells as compared with littermate controls. Furthermore, knockdown of Igκ in primary hepatocytes and NCTC1469 cells led to accelerated activation of the mitochondrial death pathway and caspase-3 cleavage in vitro, which might be related to inhibition of NF-κB signaling pathway and activation of JNK via the cytoskeleton dynamics. Taken together, these results indicate that hepatocyte-derived Igκ mediates cellular resistance to ConA-induced liver injury by inhibiting activation of caspase-3 and the mitochondrial death pathway, suggesting that Igκ plays an important role in hepatocyte survival and exerts a protective effect against ConA-induced liver injury in mice.

Regulatory B cells control airway hyperreactivity and lung remodeling in a murine asthma model

Asthma is a widespread, multifactorial chronic airway disease. The influence of regulatory B cells on airway hyperreactivity (AHR) and remodeling in asthma is poorly understood. Our aim was to analyze the role of B cells in a house dust mite (HDM)-based murine asthma model. The influence of B cells on lung function, tissue remodeling, and the immune response were analyzed by using wild-type and B-cell-deficient (μMT) mice and transfer of IL-10-proficient and IL-10-deficient B cells to μMT mice. After HDM-sensitization, both wild-type and μMT mice developed AHR, but the AHR was significantly stronger in μMT mice, as confirmed by 2 independent techniques: invasive lung function measurement invivo and examination of precision-cut lung slices exvivo. Moreover, airway remodeling was significantly increased in allergic μMT mice, as shown by enhanced collagen deposition in the airways, whereas the numbers of FoxP3+ and FoxP3- IL-10-secreting regulatory Tcells were reduced. Adoptive transfer of IL-10-proficient but not IL-10-deficient B cells into μMT mice before HDM-sensitization attenuated AHR and lung remodeling. In contrast, FoxP3+ regulatory T cells were equally upregulated by transfer of IL-10-proficient and IL-10-deficient B cells. Our data in a murine asthma model illustrate a central role of regulatory B cells in the control of lung function and airway remodeling and may support future concepts for B-cell-targeted prevention and treatment strategies for allergic asthma.

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation.

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4+ and CD8+ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti-CD20 antibody-mediated B cell depletion on CD4+ and CD8+ memory T cell alloresponses after skin transplantation in wild-type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4+ T cells but enhanced that of CD8+ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT-II CD4+ anti-OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.

Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein

Abstract Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the role B cells play in pathogenesis and regulation of disease. Historical dissection of B cell function in the MS mouse model experimental autoimmune encephalomyelitis (EAE) has largely been confined to disease induced with either the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35–55) or full length recombinant human MOG protein (hrMOG), the latter representing the only available B cell-dependent EAE model. We have demonstrated that myelin proteolipid protein (PLP178–191)-specific CD8 T cells possess striking EAE-suppression capacity. However, little is known regarding the role of B cells in these contexts. Interestingly, unlike MOG35–55, where genetic absence or depletion of B cells yields a more severe disease course, PLP178–191 yields identical EAE progression in both WT and μMT mice, suggesting suboptimal B cell engagement. Given the reports that hrMOG drives a B cell-dependent demyelinating disease versus its MOG35–55 counterpart, we hypothesized that a longer PLP antigen consisting of more epitopes may better engage B cells and provide an additional model for the field. To this end, we designed a novel peptide encompassing the extracellular domains (ECD) of PLP and compared its immunogenicity and disease-inducing capacity with PLP178–191 in μMT and WT mice. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE disease compared to WT counterparts. PLPECD also induced EAE in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Pertussis toxin treatment promotes a B cell driven disease during TH17 EAE

Abstract Pertussis toxin (PTX) is an enigmatic co-adjuvant that is required for disease induction in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the functional role of PTX during adoptive transfer TH17-EAE. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested and cultured from MOG35–55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. Recipient mice were treated with PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX delayed disease onset but did not alter disease severity or incidence. We found that PTX reduced trafficking of myeloid cells and inflammatory T helper cells in the CNS. Importantly, PTX treatment increased the number of B cells in the brain with increased antigen presenting function, suggesting an altered disease toward a B cell-dependent pathology. When disease progression was compared between WT and μMT mice treated with PTX or PBS, there was no significant difference in TH17-EAE in the PBS treated groups. However, PTX treatment significantly delayed disease onset, severity and incidence in the μMT mice vs. WT. In conclusion, our current data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype and therefore promotes a B cell driven disease. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study B cell activity during multiple sclerosis.

Promotion of T Regulatory Cells in Mice by B Cells and BAFF.

In addition to promoting B cell expansion, overexpression of BAFF promotes expansion of T cells, including T regulatory (Treg) cells. To determine the relationships among BAFF, B cells, and Treg cells, a panel of C57BL/6 (B6) congenic mice was tested. Treg cells were disproportionately expanded in mice expressing a Baff transgene (B6.BTg) and were disproportionately contracted in mice deficient in BAFF (B6.Baff-/- ). In vitro suppressor activities of B6 wild-type, B6.BTg, and B6.Baff-/- Treg cells were identical, as was in vitro generation of Treg cells. In vivo proliferation of Treg cells was greatest in B6.BTg mice, whereas in vivo survival of Treg cells was lowest in B6.Baff-/- mice. B cells promoted BAFF-independent Treg cell expansion in vivo, as evidenced by the correlation between B cells and percentages of Treg cells in B6.Baff-/- mice and by the greater percentages of Treg cells in B6.Bcl2Tg mice (which harbor B cells largely independent of BAFF because of expression of a Bcl2 transgene) than in B6 wild-type mice despite the lower serum BAFF levels in the former than in the latter. Experiments with BAFF-deficient B6.Baff-/- Bcl2Tg mice, B cell-deficient B6.μMT mice, BAFF-overexpressing/B cell-deficient B6.BTg.μMT mice, and BAFF-deficient/B cell-deficient B6.Baff-/- μMT mice demonstrated that, in a host that harbors B cells, the effect of BAFF on Treg cells goes beyond its ability to expand the B cell population and is additional to the BAFF-independent effect of B cells on Treg cells. These findings may have considerable relevance to the treatment of B cell-associated autoimmune diseases.

B cell-derived IL-1β and IL-6 drive homeostatic T cell recovery following lymphoablation

Abstract T cell reconstitution after lymphoablation in allograft recipients may lead to acute rejection and poor graft outcome. Using a mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) we showed that while T cell recovery depends on B cells, B cell MHC expression is dispensable. The goal of this study was to investigate the role of B cell-derived IL-1β and IL-6 in T cell homeostatic reconstitution after mATG lymphoablation. The NanoString analysis showed that mATG upregulated IL-1β and IL-6 gene expression in recipient B cells. To validate NanoString findings, IL-1β and IL-6 expression was measured by intracellular flow cytometry. Following heart allograft placement and mATG treatment, B cells are the main source of IL-1β and IL-6, and these cytokines mutually regulate each other’s production. Either treatment with anti-IL-1β mAb or the use of IL-1R1 KO, caspase-1 KO and MyD88 KO recipients diminished both CD4+ and CD8+ T cell recovery along with improved heart allograft survival and decreased donor-reactive IFN-γ producing cells after mATG treatment. Similarly, in vivo IL-6 neutralization with mAb or using IL-6 KO recipients delayed CD4+ and CD8+ T cell recovery, markedly prolonged heart allograft survival and decreased donor-specific T cell responses suggesting that IL-1β and IL-6 are essential for optimal T cell recovery. Adoptive B cell transfers into μMT mice showed that B cell-derived IL-1β and IL-6 supported CD8+ T cells recovery. The absence of either IL-1R or IL-6R on CD8+ T cells markedly impaired their homeostatic recovery following mATG depletion. These findings reveal the essential role of B cell-derived IL-1β and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.

FHL2 Is Essential for Spleen T Cell-Dependent B Cell Activation and Antibody Response.

Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2-/- mouse spleens displayed enlarged follicles with more B cells. When a T cell-dependent immune response was elicited using SRBC, FHL2-/- germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2-/- mice, and circulating plasma cells were also reduced in FHL2-/- This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2-/- B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell-deficient μMT mice transplanted with WT or FHL2-/- bone marrow, suggesting that the defects observed in FHL2-/- mice were not B cell intrinsic. However, spleen lysates from FHL2-/- mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell-dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.

B cell-derived anti-beta 2 glycoprotein I antibody mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm

Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysm (AAA). Hyperhomocysteinemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibody that ultimately contributes to AAA formation. ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of lipid metabolites in B cells by LC-MS/MS, which contributed to elevated anti-β2GPI IgG secretion. By use of the Toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that the culture supernatant of Hcy-activated B cells and HHcy plasma IgG independently polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- or CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG for HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice, then transferred these IgG into μMT mice and subsequently subjected them to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG had significantly exacerbated elastase- or CaPO4-induced AAA development accompanied with increased elastin degradation, MMP2/9 expression and anti-β2GPI IgG deposition in vascular lesions by immunofluorescence histochemical staining. Thus, HHcy aggravates AAA formation by activating B cells to secret anti-β2GPI IgG.

A lack of role for antibodies in regulating Helicobacter pylori colonization and associated gastritis.

Helicobacter pylori occupy a unique niche, located within the mucus layer lining the stomach, and attached to the apical surface of the gastric epithelium. As such, antibodies would be expected to play a major role in regulating infection and/or pathogenesis. However, experiments using antibody-deficient mice to study gastric helicobacter infection have yielded inconsistent results, although some pointed toward antibodies increasing colonization levels and decreasing gastritis severity. The variability in these studies is possibly due to their use of nonmatched wild-type controls. This current study presents the first evaluation of the role of antibodies in Hpylori infection by comparing antibody-deficient mice with matched wild-type siblings. Matched wild-type and antibody-deficient μMT mice were generated by heterozygous crossings. In two separate experiments, appropriately genotyped sibling littermates were infected with Hpylori for 4months and then sera and stomachs were collected. There was no difference in Hpylori colonization levels between infected μMT mice and sibling wild-type controls. Similarly, there was no significant difference in the severity of gastritis between these groups of mice, although there was a trend toward less severe gastritis in μMT mice which was supported by a significantly lower IFNγ (Th1) gastric cytokine response. Comparing matched antibody-deficient and antibody-competent mice indicates that an antibody response does not influence Hpylori colonization levels. Contrary to previous studies, these results suggest antibodies might have a minor pro-inflammatory effect by promoting gastric Th1 cytokines, although this did not translate to a significant effect on gastritis severity.

B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm.

Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation. ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining. Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

LTA1 is a safe, intranasal enterotoxin-based adjuvant that improves vaccine protection against influenza in young, old and B-cell-depleted (\u03bcMT) mice

Enterotoxin-based adjuvants including cholera toxin and heat-labile toxin (LT) are powerful manipulators of mucosal immunity; however, past clinical trials identified unacceptable neurological toxicity when LT or mutant AB5 adjuvant proteins were added to intranasal vaccines. Here, we examined the isolated enzymatic A1 domain of LT (LTA1) for intranasal safety and efficacy in combination with influenza (flu) vaccination. LTA1-treated mice exhibited no neurotoxicity, as measured by olfactory system testing and H&E staining of nasal tissue in contrast with cholera toxin. In vaccination studies, intranasal LTA1 enhanced immune responses to inactivated virus antigen and subsequent protection against H1N1 flu challenge in mice (8-week or 24-months). In addition, lung H1N1 viral titers post-challenge correlated to serum antibody responses; however, enhanced protection was also observed in μMT mice lacking B-cells while activation and recruitment of CD4 T-cells into the lung was apparent. Thus, we report that LTA1 protein is a novel, safe and effective enterotoxin adjuvant that improves protection of an intranasal flu vaccination by a mechanism that does not appear to require B-cells.

Targeting Antigens to CD180 but Not CD40 Programs Immature and Mature B Cell Subsets to Become Efficient APCs.

Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24-48 hours, Ag-specific B cells were detectable at T-B borders in the spleen; there, they proliferated in a T cell-dependent manner and induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH cells after CD180 targeting: TFH cells were induced in BAFFR-/- mice (with only transitional 1 B cells) and not in μMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, providing insights toward vaccination of mature B cell-deficient individuals and implications in treating autoimmune disorders.

Anti-CD45RB Antibody Therapy Attenuates Renal Ischemia-Reperfusion Injury by Inducing Regulatory B Cells.

Regulatory B cells are a newly discovered B cell subset that suppresses immune responses. Recent studies found that both anti-CD45RB and anti-Tim-1 treatments regulate immune responses by inducing regulatory B cells; however, the role of these cells in renal ischemia-reperfusion injury (IRI) is unknown. Using mouse models, including T cell-deficient (RAG1 knockout and TCRα knockout) mice and B cell-deficient (μMT) mice, we investigated the effects of regulatory B cells and anti-CD45RB on IRI and the mechanisms underlying these effects. Adoptive transfer of regulatory B cells before or after IRI attenuated renal IRI. Anti-CD45RB treatment with or without anti-Tim-1 before IRI increased renal infiltration of CD19+Tim-1+ regulatory B and regulatory T cells. Anti-CD45RB decreased serum creatinine levels, pathologic injury score, tubular apoptosis, and proinflammatory cytokines levels, whereas IL-10 levels increased. Following IRI, anti-CD45RB with or without anti-Tim-1 also induced regulatory B cells, improving renal function and tubular regeneration. In RAG1 knockout mice with B cell transfer, TCRα knockout mice, and wild-type mice with T cell depletion, anti-CD45RB increased regulatory B cells and attenuated IRI. However, anti-CD45RB did not attenuate IRI in RAG1 knockout mice with T cell transfer or μMT mice and induced only mild improvement in wild-type mice with B cell depletion. Furthermore, B cell-deficient mice receiving B cells from IL-10 knockout mice (but not from wild-type mice) did not show renal protection against IRI when treated with anti-CD45RB. Anti-CD45RB treatment attenuated acute renal injury and facilitated renal recovery after IRI through induction of IL-10+ regulatory B cells, pointing to anti-CD45RB as a potential therapeutic strategy in renal IRI.

Abstract 3250: B cell-produced IL-27 up-regulates PD-L1 expression in the tumor microenvironment to promote breast cancer development

Abstract The host immune response can be evaded by tumor cells, e.g., through upregulation of the PD-L1/PD-1 immune checkpoint, and - paradoxically - even hijacked to promote cancer progression. Here we showed that B lymphocytes, which can produce anti-tumoral antibodies/autoantibodies in breast cancer (BCa), played an important role in promoting murine BCa development, as B cell-deficient μMT mice displayed virtually abrogated tumor growth from transplanted syngeneic ERα+ breast adenocarcinoma cells, concomitant with significantly reduced PD-L1 expression and much increased cell death in residual tumors. In BCa that grew in wildtype mice and in tumor-associated ectopic lymph nodes, B cells were the major source of IL-27, which is the IL-27p28/EBI3 heterodimer and a pleotropic cytokine with both pro- and anti-inflammatory functions. Deficiency in IL-27 expression specifically in B cells also significantly reduced the BCa growth, indicating that the effect of B cells in BCa development was mainly through IL-27 production. This was effectively induced in B cells primed by TLR ligands, including those that would be released during uncontrolled cell death in the tumor microenvironment, and stimulated by CD154 and IL-21, as likely expressed by activated tumor-infiltrating CD4+ T cells. IL-27 in turn triggered JAK-dependent STAT1 and STAT3 phosphorylation in BCa cells, enhanced PD-L1 gene expression and promoted growth of these cells. In addition, among the 2040 genes compiled by the Ingenuity Pathway Analysis as involved in BCa tumorigenesis, those encoding PD-L1, IFN-γ and Hif-1α (cytokine and transcription factor known to induce PD-L1, respectively), IDO-1, chemokine receptor CXCR3 and its ligand CXCL10 were specifically upregulated in B cells by IL-27, which also induced an overall gene signature similar to what induced by IFN-γ, reflecting the activation of STAT1 and STAT3 by both cytokines. In humans, the two genes encoding IL-27 receptor subunits were highly expressed in the breast tissue, at levels comparable to those in lymphoid organs. As shown by The Cancer Genome Atlas (TCGA) and The Kaplan Meier Plotter datasets, the higher IL27p28 expression was associated with worse survival in BCa patients overall, who displayed increased circulating IL-27 levels as compared to healthy subjects, and in ER+ BCa patients after endocrine therapies. Indeed, IL-27 supported human BCa cell growth in the presence of tamoxifen, suggesting a role of B cell-produced IL-27 in the acquisition of drug resistance by BCa. Thus, TLR-fueled IL-27 induction in B cells reinforces PD-L1 expression in the tumor environment to drive BCa cell-intrinsic growth mechanisms and generation of PD-L1+ B regulatory cells involved in the immune checkpoint, leading to BCa progression and possibly anti-estrogen therapy resistance. Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Daniel Chupp, Maria Fernandez, Shuai Wu, Jingwei Wang, Justin Moroney, Julia Taylor, John Im, Carlos Rivera, Yiliao Luo, Junhao Liu, Gangadhara Sareddy, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. B cell-produced IL-27 up-regulates PD-L1 expression in the tumor microenvironment to promote breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3250.

B1 cells protect against Schistosoma japonicum-induced liver inflammation and fibrosis by controlling monocyte infiltration.

During Schistosoma infection, lack of B cells results in more severe granulomas, inflammation, and fibrosis in the liver, but the mechanisms underlying this pathology remain unclear. This study was to clarify the mechanisms underpinning the immunomodulation of B cells in mice infected with Schistosoma japonicum (S. japonicum). We found that B cell deficiency led to aggravated liver pathology, as demonstrated by increases in the size of the egg-associated granulomas, alanine transaminase levels, and collagen deposition. Compared with infected wild-type (WT) mice, infected B cell-deficient (μMT) mice showed increased infiltration of Ly6Chi monocytes and higher levels of proinflammatory cytokines and chemokines. Furthermore, B1 cells were increased significantly in the liver of WT mice following S. japonicum infection. Adoptively transferring B1 cells, but not B2 cells, to μMT mice significantly reduced liver pathology and liver infiltration of Ly6Chi monocytes. Additionally, secretion of IL-10 from hepatic B cells increased significantly in infected WT mice and this IL-10 was mainly derived from B1 cells. Adoptively transferring B1 cells purified from WT mice, but not from IL-10-deficient mice, to μMT mice significantly reduced liver pathology and liver infiltration of Ly6Chi monocytes. These reductions were accompanied by decreases in the expression levels of chemokines and inflammatory cytokines. Taken together, these data indicated that after S. japonicum infection, an increased number of hepatic B1 cells secrete IL-10, which inhibits the expression of chemokines and cytokines and suppresses the infiltration of Ly6Chi monocytes into the liver thereby alleviating liver early inflammation and late fibrosis.

IL-1β And IL-6 Facilitate T Cell Reconstitution In Heart Allograft Recipients Treated With Anti-Thymocyte Globulin

Abstract Antibody-mediated lymphocyte depletion is used in solid organ transplantation. T cell reconstitution after lymphoablation may lead to acute rejection and poor graft outcome. We investigated the mechanisms of T cell reconstitution in heart allograft recipients treated with murine anti-thymocyte globulin (mATG). B cell depletion with anti-mouse CD20 mAb significantly diminished T cell reconstitution in B6 (H-2b) recipients of BALB/c (H-2d) heart allografts treated with mATG. However, cognate T/B cell interactions were dispensable for T cell recovery suggesting the potential role of B cell derived cytokines. The NanoString gene expression analysis of isolated B cells showed that mATG upregulated IL-1β and IL-6 expression in CD4 T cell-dependent manner. Intracellular flow cytometry confirmed that B cells are the major source of IL-1β and IL-6 following mATG treatment. The proportion of IL-1β secreting B cells was increased in mATG treated compared to untreated recipients, whereas IL-6 producing B cells were not significantly affected by lymphoablation. Neutralization of either IL-1β or IL-6 with mAb significantly decreased T cell recovery and extended heart allograft survival in mATG treated recipients. To further test the role of B cell derived IL-1β, we injected either WT or Caspase-1−/− B cells into B cell deficient μMT mice followed by BALB/C heart transplantation and mATG treatment. Caspase-1 deficiency in injected B cells resulted in significantly lower CD8 T cell recovery. Our results show the novel functions of IL-1β and IL-6 in regulating homeostatic T cell recovery following mATG treatment. IL-1β and IL-6 neutralization may be a promising strategy for enhancing the efficacy of mATG lymphoablation in transplant recipients.

BeO-exposure induces B cell-mediated Noninfectious Granuloma Formation in the Lung

Abstract Susceptibility to chronic beryllium disease (CBD)is linked to HLA-DP molecules possessing a glutamic acid at the 69thposition of the β-chain (βGlu69), with the most prevalent βGlu69-containing molecule being HLA-DP2. We have previously shown that exposure of HLA-DP2 transgenic mice to beryllium oxide (BeO) results in the development of mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and were localized within ectopic lymphoid aggregates and granulomas. B cell depletion had no effect on the development of Be-specific CD4+T cells. However, B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice compared to the isotype-treated group. Furthermore, B cell recruitment to the lung was independent of antigen and dependent on MyD88 signaling through the secretion of CXCL13. Adoptively-transferred WT B cells into BeO-exposed μMT mice significantly reduced lung injury. B cells also surrounded granulomas in transbronchial biopsies from CBD patients, confirming a role for B cells in CBD. Overall, our data suggest a novel modulatory role for B cells in the formation of granulomas and lymphoid aggregates, resulting in the protection of the lung against sterile particulate exposure.

017 Immunization of dermatomyositis-specific autoantigen transcriptional intermediary factor (TIF1)-γ induces experimental myositis in mice

A number of myositis-specific autoantigens have been identified in dermatomyositis patients. While murine models of experimental myositis have been established using immunizations of muscle-specific proteins like myosin and C protein, they are not targeted in actual diseases. Here we established a new murine model of experimental myositis inducible with immunizations of TIF1-γ, one of self-antigens for myositis-specific autoantibodies. We purified recombinant whole human TIF1-γ protein using baculovirus expression systems, and immunized wild-type C57BL/6J (B6) mice with subcutaneous injections of emulsions containing the protein and complete Freund's adjuvant four times. Myositis was observed in bilateral muscles of the immunized mice 14 days after the last immunizations. The lymph node T cells from the mice proliferated when stimulated with TIF1-γ, and IgG reacting TIF1-γ were detected in the sera of the mice. Adoptive transfer of the T cells from the mice stimulated with TIF1-γ could cause myositis in naïve B6 mice. Moreover, transfer of the purified CD8+ T cells, but not the purified CD4+ T cells, caused define myositis in naïve B6 mice (the incidences: 90 % and 0 %, respectively). β2 microglobulin-deficient mice, which lack major histocompatibility complex I, and perforin-deficient mice, in which CD8+ T cells lack cytotoxicity, developed significantly weaker myositis than wild-type mice (myositis scores: 0.13 ± 0.23, 0.30 ± 0.48, and 0.91 ± 0.70, respectively). Transfer of IgG collected from TIF1-γ-immunized mice could not cause myositis in naïve B6 mice, moreover, μMT (B cell-deficient) mice developed myositis after TIF1-γ immunizations with no inferiority compared to wild-type mice. Collectively, TIF1-γ is an autoantigen with especial immunogenicity to induce experimental myositis, in which the muscle injury is directly mediated by CD8+ T cells, but not CD4+ T cells or antibodies. TIF1-γ-induced experimental myositis would be an useful tool to investigate pathology of anti-TIF1-γ antibody-associated dermatomyositis.