Genetic Disruption of the SH3 and SAM Domains of HACS1 Enhances Adaptive Immunity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2299-2299
Author(s):  
Dingyan Wang ◽  
A. Keith Stewart ◽  
Lihua Zhuang ◽  
Nancy Xiao ◽  
Mawmaw Hlaing ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
T Cell ◽  
B Cell ◽  
Adaptive Immunity ◽  
Knockout Mice ◽  
Wild Type ◽  
T Helper ◽  
Antigen Uptake ◽  
Helper Type

Abstract HACS1 is a SH3 (Src homology 3) and SAM (sterile alpha motif) domain containing adaptor protein that is expressed in activated B and dendritic cells, is up-regulated by Interleukin 4 in the process of B cell activation and likely serves to dampen the immune response. To elucidate the function of HACS1, we generated HACS1 gene knockout mice by deletion of the SH3 and SAM domains. HACS1−/− mice were viable and fertile and had normal bone marrow B cell development and normal splenic T and B cell populations. However, adult HACS1−/− mice had increased numbers of peritoneal B1 cells (IgM+CD5+). Upon LPS plus BCR or TCR stimulations, splenic cells from HACS1−/− mice demonstrated an upregulation of activation markers with increased intensity of CD23 expression or increased population of CD69 positive cells. Purified B220+ splenic B cells from HACS1−/− mice showed increased cell proliferation upon BCR stimulation. Both T helper type 1 (Th1) and T helper type 2 (Th2) humoral responses were enhanced in HACS1−/− mice upon immunization with T cell-dependent antigen NP-KLH, which resulted in increased production of interferon-gamma (IFN-γ), anti-NP IgG2a and IL-4, as well as anti-NP IgG1 and IgE. Upon immunization with T cell-independent antigens such as TNP-LPS and TNP-Ficoll, HACS1−/− mice had increased production of anti-TNP IgM and IgG3 as compared to normal controls. The in vitro maturation of bone marrow-derived dentritic cells from HACS1−/− mice was similar to wild-type mice but HACS1−/− dentritic cells showed increased IL-12 production upon stimulation with anti-CD40 or LPS. There was no significant difference in antigen uptake by cultured dentritic cells from non-immunized wild-type or knockout mice. However, in immunized mice, an increase in antigen uptake in HACS1−/− dentritic cells was observed. We further demonstrate that the HACS1−/− B cells had increased tyrosine phosphorylation in the resting state. As deletion of the SH3 & SAM domains of HACS1 appears to generate a hypersensitive immune response, our results collectively support that HACS1 negatively regulates adaptive immunity.

scholarly journals T Helper Type 17 Immune Response Plays an Indispensable Role for Development of Iodine-Induced Autoimmune Thyroiditis in Nonobese Diabetic-H2h4 Mice

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5135-5142 ◽  
Author(s):  
Ichiro Horie ◽  
Norio Abiru ◽  
Yuji Nagayama ◽  
Genpei Kuriya ◽  
Ohki Saitoh ◽  
...  
Keyword(s):  
Immune Response ◽  
Autoimmune Thyroiditis ◽  
Th17 Cells ◽  
Intermediate Phenotype ◽  
Lymphocyte Infiltration ◽  
Wild Type ◽  
T Helper ◽  
Nonobese Diabetic ◽  
Th2 Immune Response ◽  
Helper Type

T helper type 1(Th1)/Th2 paradigm has been expanded by discovery of a novel effector T cell (Teff) subset, Th17 cells, which produce a proinflammatory cytokine IL-17. Th17 cells have recently been shown to play a major role in numerous autoimmune diseases that had previously been thought to be Th1-dominant diseases. We here studied the significance of Th17 cells in iodine-induced autoimmune thyroiditis in nonobese diabetic-H2h4 mice, a mouse model of Hashimoto’s thyroiditis in humans, which spontaneously develop antithyroglobulin autoantibodies and intrathyroidal lymphocyte infiltration when supplied with iodine in the drinking water. We observed increased numbers of Th1 and Th17 cells in spleen and accumulation of both types of Teff in the thyroid glands of iodine-fed wild-type mice, indicating that Th17 cells as well as Th1 cells constitute thyroid lesions. Furthermore, the incidence and severity of intrathyroidal lymphocyte infiltration, and the titers of antithyroglobulin autoantibodies were markedly reduced in iodine-treated IL-17−/− mice as compared with wild-type mice. Of interest, IL-17+/− mice showed an intermediate phenotype. Therefore, the present study, together with a previous report demonstrating the importance of Th1, not Th2, immune response for developing thyroiditis using mice deficient for interferon-γ or IL-4, clearly indicates that both Th1 and Th17 cells are critical Teff subsets for the pathogenesis of spontaneous autoimmune thyroiditis in nonobese diabetic-H2h4 mice.

The Interferon-Inducible GTPase LRG-47 Regulates Hematopoietic Stem Cell Proliferation and Is Required for Maintenance of Normal Hematopoiesis during Microbial Infection.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2266-2266
Author(s):  
David Weksberg ◽  
Carl G. Feng ◽  
Alan Sher ◽  
Margaret A. Goodell
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Stem Cell ◽  
Knockout Mice ◽  
Progenitor Cell ◽  
Side Population ◽  
Constant Number ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Error Bars

Abstract Hematopoietic stem cells (HSCs) have a remarkable capacity to respond to proliferative stimuli, as they are able to reconstitute the blood following catastrophic injuries such as chemotherapy and lethal irradiation. Most work aimed at elucidating the genetic and molecular controls on this program of activation has focused on HSCs responding to these artificial stimuli, however there is a surprising paucity of information reflecting the response of HSCs to the types of stimuli encountered in a non-laboratory setting. Here we report that LRG-47, an interferon-inducible GTPase, is required for HSCs to respond to a variety of proliferative stimuli, including mycobaterial challenge. Previously studied solely in the context of the immune response to intracellular pathogens, LRG-47 is upregulated in HSCs during 5-fluorouracil-(5FU) induced proliferation, and we now show that LRG-47 −/− HSCs exhibit profound defects. LRG-47 −/− HSCs achieve only 4–8% of wild-type engraftment activity in competitive repopulation assays (Figure 1) and, strikingly, even transplantation in 25-fold excess over wild-type competitor fails to rescue this defect. We also demonstrate that LRG-47 −/− HSCs are impaired in colony-forming ability, and that LRG-47 −/− mice exhibit both a relative and absolute failure to expand the stem cell/progenitor compartments in response to 5FU (Figure 2). Intriguingly, we also show that infectious challenge with Mycobacterium avium stimulates an expansion of the progenitor cell (LSK) compartment in wild-type mice - and that LRG-47-deficient mice are unable to mount this response. These findings implicate LRG-47 as being required for effective proliferation of HSCs in response to various stimuli. Furthermore, these results imply that expansion at the progenitor cell level is a downstream effector mechanism of the cytokine-mediated immune response to infection. Ultimately, understanding the mechanisms by which HSCs sense and respond to proliferative stimuli has far-ranging applications, and our work establishes an important connection with the immune system as a regulator of this process. Infectious processes can now arguably join ex vivo HSC manipulation, mechanisms of hematologic malignancy, and transplantation medicine as areas of importance informed by an understanding of the controls on HSC activation, proliferation and quiescence. Figure 1. Competitive transplant of LRG-/- bone marrow. Whole bone marrow from wild type and LRG-47 -/- mice (CD45.2) admixed with a constant number of CD45.1 competitor cells (250,000) and transplanted into lethally irradiated recipients (CD45.1). Perecent chimerism was assessed every four weeks post-transplant (error bars = SEM). Figure 1. Competitive transplant of LRG-/- bone marrow. Whole bone marrow from wild type and LRG-47 -/- mice (CD45.2) admixed with a constant number of CD45.1 competitor cells (250,000) and transplanted into lethally irradiated recipients (CD45.1). Perecent chimerism was assessed every four weeks post-transplant (error bars = SEM). Figure 2. LRG-47 -/- fail to expand HSC compartment in response to SFU. Wild type and LRG-47 -/- mice were injected with SFU 6-days prior to side population (SP) analysis of HSC compartment. While wild-type mice showed the expected expansion of the HSC population (upper panels - gated), this response is impaired in the knockout mice (lower panels). Figure 2. LRG-47 -/- fail to expand HSC compartment in response to SFU. Wild type and LRG-47 -/- mice were injected with SFU 6-days prior to side population (SP) analysis of HSC compartment. While wild-type mice showed the expected expansion of the HSC population (upper panels - gated), this response is impaired in the knockout mice (lower panels).

scholarly journals Notch target Hes5 ensures appropriate Notch induced T- versus B-cell choices in the thymus

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2615-2620 ◽  
Author(s):  
Barbara Varnum-Finney ◽  
Mari H. Dallas ◽  
Keizo Kato ◽  
Irwin D. Bernstein
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Lower Threshold ◽  
Wild Type ◽  
Notch Ligand ◽  
Cell Commitment ◽  
B Cell Precursors

Notch signaling establishes boundaries in the thymus by inducing T-cell commitment and inhibiting a B-cell choice. Here, we show a significant 1.6-fold increased generation of B-cell precursors in thymuses from mice deficient for Notch target Hes5 compared with wild-type littermates. We further show that culture of bone marrow–derived progenitors with increasing densities of purified immobilized Notch ligand (Delta1ext-IgG) induced increased expression of Notch targets Hes1 and Hes5, and that although Hes5-deficient progenitors responded appropriately to high densities of ligand, they misread intermediate and low densities. Together, our results suggest that to ensure an appropriate outcome in the thymus in response to a lower threshold of induced Notch signaling, induction of the additional target Hes5 is required.

scholarly journals Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 515-515
Author(s):  
Claire E. Pillsbury ◽  
Jairo A. Fonseca ◽  
Jodi Dougan ◽  
Hasan Abukharma ◽  
Gloria Gonzalez-Flamenco ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Healthy Donor ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Soluble Form ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes. Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro. Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies. We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels. Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. Figure 1 Figure 1. Disclosures Abukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

The Obligate Role of TAK1 in the Survival of Hematopoietic Stem Cells and Progenitors in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 642-642
Author(s):  
Minghui Tang ◽  
Zhenbiao Xia ◽  
Shubin Zhang ◽  
Shanshan Zhang ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Knockout Mice ◽  
Mutant Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Expression And Activity

Abstract TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P’s. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.

scholarly journals The Role of Jarid2 in Leukemic Transformation of Chronic Myeloid Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1245-1245
Author(s):  
Hamza Celik ◽  
Andrew Martens ◽  
Cates Mallaney ◽  
Elizabeth Eultgen ◽  
Alok Kothari ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
B Cell ◽  
Median Survival ◽  
Wild Type ◽  
Self Renewal ◽  
H3k27 Methylation

Abstract Despite the increasing use of targeted therapies, a subset of patients with myeloproliferative neoplasms (MPN) transform to secondary acute myeloid leukemia (sAML). MPN patients who develop sAML have a dismal outcome, with a median survival of six-months. The mechanisms and pathways that contribute to transformation from MPN to sAML have not been well delineated. The most commonly mutated genes in MPN include JAK2, MPL and CALR and are likely responsible for initiation of the disease. Although these mutations have potential roles in the pathogenesis and for some cases progression to sAML, their role in sustaining the sAML clone is challenged by the finding that some patients with post-MPN sAML who harbor these mutations in their primary MPNs have no evidence of the same mutation in the leukemic blasts. Recent genome sequencing studies identified deletions of JARID2, associated with Polycomb Repressive Complex 2 (PRC2) involved in implementing global H3K27me3, only in leukemic phase of the disease, but not in chronic phase MPNs. This data suggests that JARID2 deletion could be a sAML-specific transforming event by acting as a tumor suppressor in HSCs. We show in 32D cells, Jarid2 pull-down is able to co-immunoprecipitate core PRC2 proteins, Ezh2 and Suz12, and Jarid2 depletion using shRNAs leads to reduction in global H3K27 methylation. These data suggest Jarid2 acts in concert with PRC2 in hematopoietic cells to mediate H3K27 methylation. To examine the function of Jarid2 in vivo, we generated a Jarid2 knockout mouse model (Mx1-CRE:Jarid2fl/fl; Jarid2-KO) in which Jarid2 is conditionally deleted in HSCs. Hematopoiesis in these mice was compromised with a 3-fold reduction in hematopoietic stem cell (HSC) number, defective B-cell generation in the bone marrow (BM), a differentiation block in T-cell development in thymus, and a significant reduction in peripheral blood counts. A competitive transplantation strategy was then employed to assess the potential of Jarid2-KO HSCs. One-hundred phenotypically defined Jarid2-KO HSCs (Lineage- Sca-1+ c-Kit+ CD48- CD150+) from 8-week old mice were transplanted into lethally irradiated recipient mice along with 250,000 whole bone marrow cells from genetically distinguishable wild-type mice. Preliminary analysis of these mice show that the loss of Jarid2 is deleterious for HSC function, leading to reduced lymphoid and enhanced myeloid output and failure to maintain HSC population compared to control HSCs. To further dissect the role of Jarid2 in HSC self-renewal, 18-weeks post-transplant, 100 HSCs were re-purified from the bone marrow of primary recipient mice and transplanted into the secondary recipients along with 250,000 fresh wild-type competitor cells. In this transplant setting, Jarid2-KO HSCs failed to contribute to any PB lineages (myeloid, B-cell and T-cell). Together, these data suggest that Jarid2 is essential for HSC maintenance and is required for HSC self-renewal. To study the tumor suppressor role of Jarid2 we are using mouse models of the MPN mutation FLT3ITD in combination with Jarid2 deletion to assess the function of Jarid2 as a sAML tumor suppresser. We have established a mouse model by crossing Mx1-CRE:Jarid2fl/fl mice with FLT3ITD/+ mice to generate a Mx1-CRE:Jarid2fl/fl FLT3ITD/+ strain. These mice express the germline ITD mutation under control of the endogenous murine FLT3 promoter and develop MPN with a median survival of 10 months. To mimic the genetic progression of chronic stage MPN to sAML, the genetic deletion of Jarid2 is induced in these mice by pIpC injections once MPN is established at 3-months of age. Blood counts of these mice (2 months after Jarid2 deletion, aged 5 months old) started showing the signs of worsening MPN in the absence of Jarid2 such as, high WBC counts and increased neutrophil differentials compared to control (Mx1-CRE: FLT3ITD/+). Our ultimate goal is to understand the genetic processes associated with progression of MPN to sAML, which could eventually improve treatment outcomes for patients who can be identified as at increased risk for undergoing sAML transformation. Disclosures No relevant conflicts of interest to declare.

RhoH-Deficient Mice Show Altered B Cell Populations In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2307-2307
Author(s):  
Abel Sanchez-Aguilera ◽  
Jose Cancelas ◽  
David A. Williams
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Receptor ◽  
Wild Type ◽  
Marginal Zone B Cells

Abstract RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations. Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9) Bone marrow Spleen (*) indicates p<0.05; (**), p<0.01; (***), p<0.005 RhoH+/+ RhoH−/− RhoH+/+ RhoH−/− total B cells 7.8±1.8 11.0±2.4 (**) total B cells 31.7±10.1 25.4±8.8 pro-B 0.12±0.03 0.15±0.04 (*) transitional 8.7±1.2 8.6±2.8 pre-B 2.6±0.6 3.8±0.8 (***) B-2 11.6±4.1 7.6±2.5 (*) immature 1.5±0.4 2.1±0.5 (*) marginal 3.2±1.1 3.9±1.6 mature 1.4±0.7 1.7±0.9

Expression of C/EBPβ in Bone Marrow Mesenchymal Stem Cells Is Mandatory for Early-Stage B Cell Lymphopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3457-3457
Author(s):  
Satoshi Yoshioka ◽  
Yasuo Miura ◽  
Hisayuki Yao ◽  
Yoshihiro Hayashi ◽  
Akihiro Tamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Knockout Mice ◽  
Bone Marrow Cells ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Normal Wild Type

Abstract Abstract 3457 The interplay between hematopoietic cells and bone marrow microenvironment organized by mesenchymal stem cells is important for the maintenance of hematopoiesis. With respect to B cell lymphopoiesis, several constituents of bone marrow microenvironment specific for B cells (“B cell niche”) have been identified, including CXCL12/stromal cell-derived factor-1 (SDF-1)-abundant reticular cells as cellular factors, and CXCL12/SDF-1, interleukin (IL)-7, stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt3-L), and nuclear factor kappa-B ligand (RANKL), as essential humoral factors. However, the precise mechanism through which mesenchymal stem cells in the bone marrow microenvironment support B cell lymphopoiesis, especially the role of transcription factors, remains unknown. We show that the mesenchymal stem cells lacking a transcription factor, CCAAT enhancer binding protein (C/EBP) b, are functionally abnormal, which contribute to the impairment of B cell lymphopoiesis in C/EBPb knockout mice. In C/EBPb knockout mice, the number of B cells, in particular, B220+CD43+ precursor B cells, was significantly decreased in bone marrow compared with that in wild-type littermates (Figure 1A and 1B). As shown in Fig. 1A, the percentage of total B220+ B cells was decreased at 19.1 ± 7.1% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 26.5 ± 7.3%: *P<0.05). The percentage of B220+CD43+ precursor B cells was also decreased at 5.2 ± 1.5% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 7.4 ± 1.6%: **P<0.01). Intriguingly, in vivo bone marrow transplantation experiments demonstrated that the bone marrow cells derived from C/EBPb knockout mice were engrafted in lethally-irradiated (10 Gy) wild-type mice with equivalently B cell recovery compared to the bone marrow cells from normal wild-type mice. Conversely, when normal wild-type c-kit+ Sca-1+ lineages− hematopoietic stem cells (KSL cells) were co-cultured with C/EBPb deficient mesenchymal stem cells in vitro (KO), they showed impaired B cell differentiation compared to the co-culture with normal wild-type mesenchymal stem cells (WT, Figure 1C). Mechanistically, the CXCL12/SDF-1 production by C/EBPb deficient mesenchymal stem cells was reduced compared with that by wild-type mesenchymal stem cells (KO, n = 5, 4.47 ± 1.16 ng/mL; WT, n = 5, 9.90 ± 1.93 ng/mL; **P < 0.01). These results suggest a possibility that abnormal C/EBPƒÀ deficient mesenchymal stem cells in bone marrow microenvironment contribute to impaired B cell lymphopoiesis in C/EBPb knockout mice. We further found that C/EBPb deficient mesenchymal stem cells displayed several functional abnormalities. First, calcium accumulation was significantly reduced in 4 week osteogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells compared to cultures of wild-type mesenchymal stem cells. This occurred along with the down-regulated expression of the principal osteogenic master molecule runt-related transcription factor 2 (Runx2). Second, lipid deposition was significantly reduced in 1 week adipogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells. The expression of adipogenic markers, including peroxisome proliferator-activated receptor b (PPARb) was significantly reduced in adipogenic cultures of C/EBPb-deficient mesenchymal stem cells compared with cultures of wild-type mesenchymal stem cells Finally, the number of colony-forming unit fibroblast (CFU-F) was higher in the bone marrow of C/EBPb knockout mice than in that of wild-type mice. Collectively, C/EBPb-deficient mesenchymal stem cells have aberrant multi-differentiation capability and increased proliferation activity compared with wild-type mesenchymal stem cells, further supporting that C/EBPb-deficient mesenchymal stem cells were functionally abnormal. Altogether, this work demonstrates that impaired B cell lymphopoiesis in C/EBPb knockout mice is attributed to abnormal mesenchymal stem cells in bone marrow microenvironment, at least in a steady-state, an effect that is due in part to the impaired CXCL12/SDF-1 production. Disclosures: No relevant conflicts of interest to declare.

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

2019 ◽  
Vol 30 (10) ◽  
pp. 1870-1885 ◽  
Author(s):  
Taishi Fang ◽  
Tai Yeon Koo ◽  
Jae-Ghi Lee ◽  
Joon Young Jang ◽  
Yixuan Xu ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Knockout Mice ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Regulatory B Cells ◽  
Wild Type ◽  
Renal Ischemia Reperfusion Injury

BackgroundRegulatory 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.MethodsUsing 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.ResultsAdoptive 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.ConclusionsAnti-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.

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