scholarly journals Cancer coopts differentiation of B-cell precursors into macrophages

2021 ◽  
Author(s):  
Chen Chen ◽  
Bongsoo Park ◽  
Emeline Ragonnaud ◽  
Monica Bodogai ◽  
Le Zong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cancer Cells ◽  
Primary Source ◽  
Small Subset ◽  
Ovarian Cancers ◽  
Immature B Cells ◽  
B Cell Precursors

Abstract We recently reported that some cancers induce accumulation of bone marrow (BM) B-cell precursors in the spleen to convert them into metastasis-promoting, immunosuppressive B cells. Here, using various murine tumor models and samples from humans with breast and ovarian cancers, we provide evidence that cancer cells also coopt differentiation of the extra nodal B-cell precursors to generate macrophages (termed B-MF). We link the trans-differentiation to a small subset of CSF1R+ Pax5Low cells within BM pre-B and immature B cells and cancer-secreted M-CSF that downregulates Pax5 via CSF1R signaling. Thus, cancer generates tumor-associated macrophages (TAM) from B-cell precursors in addition to their primary source, monocytes. Based on their differences from monocyte-derived TAM, such as a superior ability to induce FoxP3+ Tregs, suppress proliferation of T cells and more efficiently phagocytize apoptotic cells, we propose that cancer generates B-MF to mediate cancer escape.

Graft-Versus-Host Disease Impairs Early B Lymphopoiesis in the Bone Marrow

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2976-2976
Author(s):  
Kristina Doser ◽  
Tina J Boeld ◽  
Martin Heidenreich ◽  
Reinhard Andreesen ◽  
Petra Hoffmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
B Cells ◽  
B Cell ◽  
Graft Versus Host Disease ◽  
B Lymphopoiesis ◽  
Cell Compartment ◽  
Graft Versus Host ◽  
B Cell Precursors

Abstract Abstract 2976 Graft-versus-host disease (GVHD) and infectious complications are main causes of non-relapse mortality after allogeneic stem cell transplantation (SCT). Impaired immune function after SCT is usually attributed to the immunosuppressive medication applied for GVHD prophylaxis or therapy. Using a major histocompatibility complex (MHC)–mismatched murine model of GVHD (C57BL/6→BALB/c), we now examined the influence of GVHD on B cell immunity after SCT in the absence of pharmacologic immunosuppression. Lethally irradiated BALB/c (H-2d) recipients were transplanted with T cell-depleted bone marrow (TCD BM; 2.5×106) from C57BL/6 (H-2b) donors and parallel groups received CD4+CD25− conventional donor T cells (Tconv; 0.25 × 106) 2d later. Mice that received TCD BM alone (n =10) did not develop GVHD and showed a rapid and complete reconstitution of B cells in peripheral blood (PB) (25 ± 7% CD19+ B cells at d21; 55 ± 5% at d100). Mice that received additional donor Tconv cells (n =12) developed severe GVHD and completely lacked donor and host B cells in PB until their early death or throughout the observation period of 100d (p<0.001). Animals that were protected from severe GVHD by the co-infusion of donor CD4+CD25+Foxp3+ regulatory T cells (Treg; 0.25×106 Treg transplanted together with TCD BM; 2d later transfusion of 0.25 × 106 Tconv) showed a delayed, but finally full reconstitution of their B cell compartment in PB (9 ± 12% CD19+ B cells at d21; 42 ± 17% at d100). Similarly, animals without GVHD after TCD BMT and animals protected from GVHD by co-transplanted Treg cells showed a complete reconstitution of their B cell compartment in spleen and BM at d100 (spleen: 26±4, 7×106 and 31 ± 9.3×106 CD19+ B cells, respectively; BM: 2, 2 ± 0, 3×106 and 2.9 ± 0.9×106 B cells, respectively). In contrast, B cells were not only undetectable in peripheral lymphoid organs in animals with severe GVHD but also in the BM, suggesting that B cell precursors were affected. To examine whether GVHD solely impedes B cell regeneration or actively contributes to B cell eradication, GVHD was induced after B cell reconstitution at d21 after TCD BMT by donor lymphocyte infusions (DLI). Within 1wk after the transfer of 8×106 or 12×106 donor CD4+ lymphocytes, a significant reduction of B cells in PB was detected (from 30.3 ± 5.2% to 10 ± 6.9% and 36.3 ± 9.2% to 5.9 ± 1.3%, respectively; n =4). Thus, GVHD not only affected B cell reconstitution, but even eradicated stem cell-derived B cells that were syngeneic to the GVHD-inducing T cells, suggesting that GVHD-induced inflammation contributed to B cell depletion. To examine the influence of GVHD on precursor cells, serial transplants were performed. Yet, TCD BM from both, animals with and without GVHD, reconstituted their B cell compartment upon secondary transplantation (n =18; 33.1 ± 14.8% vs. 32.4 ± 17% at d100), thereby proving that the stem cell compartment was not affected. Next, we examined the effect of GVHD on precursor cells. Multipotent BM precursors (lin−, Sca-1+, c-kit+ [LSK]) were not significantly different in GVHD animals (TCD BM plus Tconv; n =12) as compared to controls (TCD BM only; n =10; 3.5×103 ± 2.8×103 vs. 5.8×103 ± 2.5×103, respectively). However, common lymphoid precursors (CLP; Lin−, FLt3+, CD127+) in the BM were significantly reduced in animals with GVHD (0.3×103 ± 0.17×103) as compared to transplant recipients without GVHD (4.4×103 ± 2.2×103, p<0.001). These results suggest that the dysregulated production of pro-inflammatory cytokines during GVHD is toxic for early B cell precursors and/or that the alloresponse destroys the BM niche for developing B cells. As IFN- γ and TNF are known to be elevated in GVHD and to impair B lymphopoiesis even in a non-transplant setting, we generated mixed chimeras using BM from wt and cytokine receptor deficient animals. Yet, a selective B cell reconstitution from receptor deficient BM was not observed in GVHD, suggesting that neither of these cytokines is exclusively responsible for its toxic effects on B cell precursors. Taken together, our results show that GVHD not solely affects immune reconstitution by the well known destruction of secondary lymphoid organs, but it disturbs early lymphoid progenitors in the BM through inflammatory, but not necessarily allo-specific immune responses. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Abnormal B-Cell Maturation Pattern in the Bone Marrow of Patients with Germline Mutations in PIK3CD

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2752-2752
Author(s):  
Alina E Dulau Florea ◽  
Raul C Braylan ◽  
Kristian T. Schafernak ◽  
Stefania Pittaluga ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Germline Mutations ◽  
Cell Maturation ◽  
Light Chains ◽  
B Cell Maturation ◽  
Precursor B Cells ◽  
B Cell Precursors

Abstract Background Autosomal dominant germline mutations in the phosphatidylinositol-3-OH kinase (PIK3CD) encoding for the PI(3)K catalytic subunit p110δ, lead to combined immunodeficiency with increased incidence of B-cell lymphomas. (Lucas CL et.al. Nature Immunology 2014). While p110δ is selectively expressed in leukocytes, it is critical for TCR and BCR signaling and lymphocyte homeostasis. Clinically, these patients may present with sinopulmonary infections, bronchiectasis, cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) viremia, lymphoproliferation and autoimmune cytopenias. Immune phenotype includes naïve CD4+ T cell lymphopenia, expanded terminally differentiated or exhausted T cells, increased circulating transitional B cells and reduced class-switched memory B cells. Herein we report immunophenotypic abnormalities in B-lymphoid maturation in the bone marrow (BM) of 5 patients with PIK3CD mutations. Methods BM from 5 patients with PIK3CD mutations (2 males, 3 females, age range: 4–15 years, median 11.5 years) were studied by flow cytometry (FC), morphology and immunohistochemistry (IHC). BM aspirate from 5 healthy age matched pediatric patients were used as controls for flow cytometric analysis of B-cell subsets and maturation. Antibodies against CD45, CD3, CD4, CD8, CD19, CD10, CD34, CD20, and surface kappa and lambda light chains were used for FC. B-lymphocyte subsets were defined as: early stage precursor B-cells (CD34+/CD19+/CD10+bright/CD20-); intermediate precursor B-cells (CD45+moderate/ CD19+/CD10+moderate/CD34-); and late stage and mature B cells (CD34-/CD10-/CD19+/CD45+bright/CD20+). The intermediate subset corresponds to transitional B cells (developmentally intermediate between immature and mature naive B cells). IHC and in situ hybridization staining were applied to biopsy sections using standard methods. Prism software was used for statistical analyses (Mann-Whitney test). Results There was no significant difference in the median percentage of early B-cell precursors (among all B-lymphocytes) between the PIK3CD patients and the age-matched controls (3.6% vs. 3.7%; p=0.8). However, all PIK3CD marrows showed expanded CD10+ intermediate precursor B-cells which were overall 2.5 times more abundant in PIK3CD marrows than in controls (94.6% vs. 37.4% of all B-cells; p<0.01). Additionally, the PIK3CD patients showed a marked reduction in mature B-cells with 29 times fewer mature CD20+/CD10- B-cells than controls (2% vs 57%; p<0.01). These differences resulted in a markedly abnormal B-cell maturation pattern in all PIK3CD patients (Figs. A and B). A subset of CD10+ and bright CD20+ B-cells expressed polytypic light chains in the PIK3CD marrows. The median CD4:CD8 T-cell ratio was 0.32 in PIK3CD marrows with markedly reduced CD4+ T-cells. BM core biopsies showed overall normal cellularity with increased lymphocytes (20-30% of the cellular marrow). IHC revealed increased CD20+ lymphocytes (15-20% of all nucleated cells) and CD10+ lymphocytes showed similar distribution suggesting coexpression with CD20. TdT and CD34 highlighted approximately 5% of all nucleated cells. CD138, and kappa and lambda light chains showed unremarkable scattered polytypic plasma cells. CD3+ and CD8+ T-cells accounted for 5-10% of BM cells and CD4+ lymphocytes were reduced. EBV was positive in one case. CMV was negative in all cases. Conclusions For the first time, we report B-cell maturation abnormalities in the bone marrow of patients with germline mutations in PIK3CD. All marrows showed an abnormal pattern of B cell maturation characterized by an absolute increase in CD10+ intermediate precursor B-cells and a marked decrease in mature B-cells. The findings suggest either a partial block in B-cell late stage maturation or other mechanism leading to increased CD10+ B-cell precursors and markedly reduced mature B-cells. Lymphoid hyperplasia and lymphoma have been described in PIK3CD patients. The increased CD10+ B cell precursors and the abnormal maturation pattern noted by flow cytometry may mimic CD10+ B-cell neoplasia (e.g. acute lymphoblastic leukemia or Burkitt lymphoma) but detailed analysis showed no morphologic or immunophenotypic evidence of B-cell neoplastic involvement in any of the five patients studied. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.

1995 ◽  
Vol 181 (4) ◽  
pp. 1519-1526 ◽  
Author(s):  
U von Freeden-Jeffry ◽  
P Vieira ◽  
L A Lucian ◽  
T McNeil ◽  
S E Burdach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Gene Targeting ◽  
B Cell Development ◽  
B Lymphopoiesis ◽  
T And B Cells ◽  
Immature B Cells ◽  
Deficient Mice

Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.

Long-Term Chimerism and B-Cell Function After Bone Marrow Transplantation in Patients With Severe Combined Immunodeficiency With B Cells: A Single-Center Study of 22 Patients

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2923-2930 ◽  
Author(s):  
Elie Haddad ◽  
Françoise Le Deist ◽  
Pierre Aucouturier ◽  
Marina Cavazzana-Calvo ◽  
Stephane Blanche ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Combined Immunodeficiency ◽  
B Cell Function ◽  
Host Origin

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B+ SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell–depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27+ memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.

scholarly journals Reduced receptor editing in lupus-prone MRL/lpr mice

2007 ◽  
Vol 204 (12) ◽  
pp. 2853-2864 ◽  
Author(s):  
Jennifer L. Lamoureux ◽  
Lisa C. Watson ◽  
Marie Cherrier ◽  
Patrick Skog ◽  
David Nemazee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Immunoglobulin M ◽  
Considerable Proportion ◽  
Receptor Editing ◽  
Cell Tolerance ◽  
B Cell Tolerance ◽  
Immature B Cells

The initial B cell repertoire contains a considerable proportion of autoreactive specificities. The first major B cell tolerance checkpoint is at the stage of the immature B cell, where receptor editing is the primary mode of eliminating self-reactivity. The cells that emigrate from the bone marrow have a second tolerance checkpoint in the transitional compartment in the spleen. Although it is known that the second checkpoint is defective in lupus, it is not clear whether there is any breakdown in central B cell tolerance in the bone marrow. We demonstrate that receptor editing is less efficient in the lupus-prone strain MRL/lpr. In an in vitro system, when receptor-editing signals are given to bone marrow immature B cells by antiidiotype antibody or after in vivo exposure to membrane-bound self-antigen, MRL/lpr 3-83 transgenic immature B cells undergo less endogenous rearrangement and up-regulate recombination activating gene messenger RNA to a lesser extent than B10 transgenic cells. CD19, along with immunoglobulin M, is down-regulated in the bone marrow upon receptor editing, but the extent of down-regulation is fivefold less in MRL/lpr mice. Less efficient receptor editing could allow some autoreactive cells to escape from the bone marrow in lupus-prone mice, thus predisposing to autoimmunity.

IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1554-1554
Author(s):  
Lucy S. Hodge ◽  
Steve Ziesmer ◽  
Frank J Secreto ◽  
Zhi-Zhang Yang ◽  
Anne Novak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Bone Marrow Microenvironment ◽  
Waldenström’S Macroglobulinemia ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Conditioning Intensity and T Cell Dose Determine Efficacy of CD19-Targeted T Cell-Mediated Tumor Eradication in an Immunocompetent Mouse Model of B-ALL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2613-2613
Author(s):  
Marco L Davila ◽  
Christopher Kloss ◽  
Renier J Brentjens ◽  
Michel Sadelain
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Immunocompetent Mouse ◽  
Cell Dose ◽  
Conditioning Intensity

Abstract Abstract 2613 Recent work by our group and others demonstrates the therapeutic potential of CD19-targeted T cells to treat patients with indolent B cell malignancies. These studies make use of T cells that are genetically engineered with chimeric antigen receptors (CARs) comprising an scFv fused to various T cell activating elements. Whereas firs-generation CARs only direct T cell activation, second-generation CARs include two signal elements, such as CD3z and CD28 signaling domains (19–28z). We and our colleagues at MSKCC are currently evaluating the safety of 19–28z-transduced T cells in patients with acute leukemia (B-ALL) in a Phase I protocol (NCT01044069). Pre-clinical studies performed to date have mostly relied on xenogeneic models utilizing immunodeficient animals, which enable the evaluation of human engineered T cells but do not recapitulate all the interactions that may affect tumor eradication by CAR-modified T cells. We have therefore developed a pre-clinical immunocompetent mouse model of B-ALL, and addressed therein the impact of conditioning and T cell dose on the eradication of leukemia by syngeneic, CAR-targeted T cells. To establish an immunocompetent mouse model of B cell leukemia, we generated a clone from the lymph node of an Eμ-myc B6 transgenic mouse. The immunophenotype and gene-expression profile of clone Eμ-ALL01 is consistent with a progenitor B cell origin. Syngeneic B6 mice inoculated with this clone develop florid acute leukemia and die approximately 2–4 weeks after injection from progressive bone marrow infiltration. We created an anti-mouse CD19 CAR comprising all murine elements, including the CD8 signal peptide, a CD19-specific single chain variable fragment, the CD8 transmembrane region, and the CD28 and CD3z signaling domains. Transduction of the murine 19–28z CAR into mouse T cells was robust and successfully retargeted the T cells to B cells. In vitro assays demonstrated that m19–28 z transduced T cells mediated effective killing of CD19-expressing target cells and the production of effector cytokines such as IFNγ and TNFα. Cyclophosphamide either alone or in combination with control syngeneic T cells is insufficient to eradicate established Eμ-ALL01 in B6 mice. However, treatment with cyclophosphamide and m19–28z-transduced T cells cured nearly all mice. Mice sacrificed six months after treatment exhibited a dramatic reduction of B cells in the bone marrow (BM), blood, and spleen. The few remaining B lineage cells found in the BM had a phenotype consistent with early pro-B cells, suggesting that endogenous reconstitution of the B cell compartment was thwarted by persisting, functional m19–28z+ T cells. Thus, T cells are retained at the site of antigen expression, which is maintained through regeneration of progenitor B cells. The persisting CD19-targeted T cells in the BM exhibited a cell surface phenotype consistent with effector and central memory cells. Using B cell aplasia as a surrogate endpoint for assessing in vivo T cell function and persistence, we evaluated how conditioning chemotherapy and T cell dose determine the level of B cell depletion induced by adoptively transferred CD19-targeted T cells. Overall, increasing the cyclophosphamide or T cell dose, increased the degree and duration of B cell depletion and the number of persisting CAR-modified T cells. Significantly, increasing the T cell dose at a set cyclophosphamide level had a lesser impact than increasing the conditioning intensity for a given T cell dose. In summary, the new Eμ-ALL01 syngeneic, immunocompetent B-ALL model we describe here is a valuable tool for modeling CD19 CAR therapies. Our results indicate that m19–28z transduced T cells are effective at eradicating B-ALL tumor cells and persist long-term, preferentially in bone marrow. Our findings further establish that conditioning intensity and T cell dose directly determine B cell elimination and long-term T cell persistence. These studies in mice will serve as an important framework to further model and perfect our studies in patients with B-ALL. Disclosures: No relevant conflicts of interest to declare.

Hepatic Stellate Cell Conditioned Myeloid Cells Provide a Novel Therapy for Prevention of Factor VIII Antibody Formation in the Hemophilia A Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4117-4117
Author(s):  
Sumantha Bhatt ◽  
Kathleen Brown ◽  
Feng Lin ◽  
Michael P Meyer ◽  
Margaret V. Ragni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Myeloid Cells ◽  
Gm Csf ◽  
Cox 2

Abstract Abstract 4117 Background: Hemophilia is an X-linked bleeding disorder resulting from a mutation in coagulation factor VIII (F.VIII). A major drawback of current plasma-derived or recombinant F.VIII therapy is the formation of F.VIII antibodies (inhibitors). Inhibitor formation is a T cell-dependent, B cell-mediated immune response to foreign infused F.VIII. Myeloid derived suppressor cells (MDSCs) are potent suppressors of T cell and B cell responses and are currently under study for therapeutic applications in transplantation and autoimmune diseases. However, the mechanisms of MDSC development and function remain unknown, and in vitro propagation of MDSCs has been a challenge. We hypothesized that MDSCs might be effective in inhibiting F.VIII inhibitor formation in the hemophilia A model. Methods: We developed a novel method for generating MDSCs in vitro by culturing bone marrow cells from hemophilia A mice with hepatic stellate cells (HSCs), hereafter referred to as HSC-conditioned myeloid cells (H-MCs). DCs were propagated from the bone marrow with GM-CSF and IL-4, whereas H-MCs were propagated from the bone marrow with GM-CSF and HSCs. Granulocyte contaminants were removed on day 2 and the remaining monocytic populations were harvested on day 5. Expression of cell surface antigens was analyzed by flow cytometry. Arginase1 and iNOS levels were compared by qPCR, with or without LPS stimulation. The in vitro suppressive capacity of the H-MCs was determined by a mixed leukocyte reaction culture. Splenic T cells from hemophilia A mice were stimulated by irradiated DCs (at a 1–20 ratio, APC to T cell) and recombinant F.VIII. Additional irradiated DCs or H-MCs were added in graded numbers as regulators. The proliferative response was determined by 3H-thymidine incorporation. The phenotype of cultured CD4+ T cells was characterized by intracellular staining for Foxp3 and IFN-gamma and analyzed by flow cytometry. Inhibition of B cells by H-MCs was determined by a CFSE dilution assay. Purified splenic B cells were labeled with CFSE and stimulated by Ig-M and IL-4. APCs (spleen cells) or H-MCs were added at a ratio of 1:10 (APC to B cell). The proportion of proliferating B cells was determined by CFSE dilution of B220 stained cells. In the COX-2 suppression assay, CFSE labeled B cells were treated with varying concentrations of the selective inhibitor of COX-2, NS398. The suppressive effect of H-MCs on B cells in vivo was determined by simultaneously administering H-MCs (I.V) and F.VIII (I.V.) to hemophila A mice on day 0 and rechallenging with recombinant F.VIII on days 2 and 4. WT B6 mice and hemophilia A mice without H-MC transfer served as controls. Plasma anti-F.VIII antibody titers were measured on day 12 by a modified ELISA assay. Results: H-MCs expressed low levels of costimulatory molecules but high levels of the inhibitory molecule B7-H1 and immunoregulatory enzyme arginase-1. In contrast, DCs expressed high levels of costimulatory molecules and MHC class II. In vitro studies demonstrated that the H-MCs markedly inhibited antigen specific T cell proliferation induced by dendritic cells in response to recombinant F.VIII (Fig. 1). H-MCs altered the T cell response in hemophilia A mice by promoting the expansion of regulatory T cells and inhibiting IFN-γ producing CD4+ T cells. When the H-MCs were cocultured with B cells isolated from hemophilia A mice, in the presence of Ig-M and IL-4, the H-MCs abrogated B cell activation and proliferation directly (Fig. 2). H-MCs may be modulating the B cell response through the Cox-2 pathway, as inhibition of Cox-2 through NS398 led to the restoration of B cell proliferation. More importantly, adoptive transfer of H-MCs into hemophilia Amice, at the time of F.VIII infusion, markedly suppressed anti-F.VIII antibody formation (Fig. 3). Conclusion: These results suggest that HSC conditioned myeloid cells may represent a potential therapeutic approach to induction of immune tolerance in patients with hemophilia A andother immune disorders. Disclosures: No relevant conflicts of interest to declare.

Index of Bone Marrow Output and Imbalance of B-Lymphocyte Homeostasis before and after Transplantation Correlate Differently with Graft-Versus-Host Disease and Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3150-3150
Author(s):  
Crisitina Skert ◽  
Simone Perucca ◽  
Imberti Luisa ◽  
Chiarini Marco ◽  
Michele Malagola ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Acute Gvhd ◽  
Chronic Gvhd ◽  
Effector Memory ◽  
Memory Cells ◽  
Immunological Parameters ◽  
Immature B Cells ◽  
Effector Memory Cells

Abstract Introduction The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT. Methods Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Results Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006). Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (p<0,05): -values of regulatory effector memory T-cells at +30, percentage of CD8+TEMRA cells at +90, values of immature B cells and levels of KRECs at +180 (positive correlation) -percentage of CD4+ central memory and CD8+ effector memory cells at +90 (negative correlation). The relapse rate (27%; median time: 5,5 months, range 3-12) was used as clinical index of ineffective GVT. The following cluster of immunological parameters at day +90 correlated positively with relapse: CD8+ effector memory cells, immature B cells, naïve, switched memory B cells, memory double negative (IgD-CD27-) B cells (p=0,006). Discussion Different clusters of immunological parameters at different time points were evidenced as predictors of GVHD and ineffective GVT, allowing a clear-cut distinction between these immunological reactions. Changes in pre- and post-transplant B-lymphopoietic microenvironment and specific imbalances in the subset of B-cells may be involved in acute and chronic GVHD development. The atypical association of regulatory T-cells with GVHD may be explained by the relative efficiency of different subsets of regulatory T-cells (naïve>effector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward "senescent" memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals A monoclonal antibody that recognizes B cells and B cell precursors in mice.

1981 ◽  
Vol 153 (2) ◽  
pp. 269-279 ◽  
Author(s):  
R L Coffman ◽  
I L Weissman
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
B Cell Precursors

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

Sign in / Sign up

Export Citation Format

Share Document