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

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A Phase I Clinical Trial of Treatment of B-Cell Malignancies with Autologous Anti-CD19-CAR-Transduced T Cells

Blood ◽

10.1182/blood.v116.21.2865.2865 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2865-2865 ◽

Cited By ~ 1

Author(s):

James N. Kochenderfer ◽

Mark E. Dudley ◽

Maryalice Stetler-Stevenson ◽

Wyndham H. Wilson ◽

John E. Janik ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Adoptive Transfer ◽

B Cell Malignancies ◽

B Lineage

Abstract Abstract 2865 T cells can be genetically modified to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19. Adoptive transfer of autologous T cells expressing anti-CD19 CARs is an attractive new approach for treating B-cell malignancies. We have constructed a CAR that consists of the variable regions of a mouse-anti-human-CD19 antibody coupled to the signaling domains of CD28 and CD3-zeta. We have treated 5 patients with 2 doses of 60 mg/kg of cyclophosphamide and 5 doses of 25 mg/m2 of fludarabine followed by infusions of anti-CD19-CAR-transduced T cells and administration of high-dose IL-2. All of the patients received infusions of cells that produced cytokines in a CD19-specific manner. The percentage of the infused cells that expressed the anti-CD19 CAR as measured by flow cytometry ranged from 45% to 65%. The first patient enrolled on our trial has follicular lymphoma. He was treated twice. The patient obtained a partial remission (PR) from his first course of chemotherapy, 0.4×109 anti-CD19-CAR-transduced T cells, and IL-2 (reported in Kochenderfer et al. Blood First Edition); however, he subsequently developed progressive disease, and 40 weeks after his first CAR-transduced T cell infusion he received a second course of chemotherapy followed by 2×109 CAR-transduced T cells and IL-2. The second course of treatment resulted in an additional PR and was not associated with any toxicity that could be attributed to the CAR-transduced T cells. At last follow-up, a small amount residual disease detected only by positron emission tomography remained. In this first patient, the initial treatment course resulted in eradication of blood and bone marrow B-lineage cells for 39 weeks. In contrast to the prolonged eradication of B-lineage cells after the initial treatment course, the number of polyclonal blood B cells normalized 9 weeks after the second CAR-transduced T cell infusion. CAR-transduced T cells were present at a level of 0.1% of total peripheral blood mononuclear cells (PBMCs) one month after the first CAR-transduced T cell infusion. Despite the five-fold higher dose of CAR-transduced T cells administered with the second treatment, CAR-transduced T cells were not detected in the blood one month after the second CAR-transduced T cell infusion. The second patient treated on our protocol had follicular lymphoma and had received extensive prior therapy including autologous stem cell transplantation. After an initially uncomplicated course, this patient developed pneumonia caused by culture-proven influenza A virus and died 18 days after CAR-transduced T cell infusion. Quantitative PCR was used to measure the level of CAR-transduced cells in multiple tissues obtained from this patient at autopsy. CAR-transduced cells were widely distributed with the highest levels in the spleen and bone marrow. The third patient treated on our trial obtained a complete remission of advanced chronic lymphocytic leukemia (CLL) after treatment with chemotherapy, infusion of 2×109 anti-CD19-CAR-transduced T cells, and IL-2. At the time of last follow-up, three months after treatment, adenopathy had resolved, CLL cells were not detected by flow cytometry analysis of the blood and bone marrow, and the number of normal polyclonal B cells in the blood was below normal levels. This patient had a period of fever and hypotension 7 days after cell infusion that was associated with an elevated serum interferon-gamma level of 1532 pg/mL. At the time of the hypotensive episode 7 days after cell infusion, anti-CD19-CAR-transduced cells made up 2.1% of PBMCs. The fourth patient treated on our study obtained a PR of splenic marginal zone lymphoma that continues 2 months after treatment with chemotherapy, 2×109 CAR-transduced T cells, and IL-2. This patient did not have prolonged depletion of normal B cells after treatment, and he did not have any toxicity that could be attributed to the anti-CD19 CAR-transduced T cells. We recently treated a fifth patient who has CLL. Follow-up on this patient is too short to evaluate toxicity or response. In conclusion, we have shown that adoptive transfer of anti-CD19-CAR-transduced T cells with in vivo activity is feasible. The promising results obtained on this trial raise important questions for future research aimed at optimizing therapy with anti-CD19-CAR-transduced T cells. Disclosures: No relevant conflicts of interest to declare.

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B-Lymphocyte Stimulator (BLyS) Is Highly Expressed in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v104.11.2291.2291 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2291-2291

Author(s):

Stephen M. Ansell ◽

Deanna M. Grote ◽

Steven C. Ziesmer ◽

Thomas E. Witzig ◽

Robert A. Kyle ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

B Lymphocyte ◽

Waldenström’S Macroglobulinemia ◽

B Lymphocyte Stimulator ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia ◽

Normal Controls

Abstract Waldenstrom’s macroglobulinemia is a serious and frequently fatal illness, however many of the mechanisms leading to this disease are not yet known. It is clear, however, that there is dysregulation of the balance between cell proliferation and programmed cell death. BLyS (B-lymphocyte stimulator) is a newly identified TNF family member expressed by monocytes, macrophages, and dendritic cells. BLyS has been shown to be critical for maintenance of normal B cell development and homeostasis, and has been found to stimulate lymphocyte growth. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B-cells. Studies of the effects of BLyS on B cell physiology have shown that it also regulates immunoglobulin secretion. To determine the relevance of the BLyS receptor-ligand system in Waldenstrom’s macroglobulinemia, we examined malignant B cells from 5 patients with Waldenstrom’s macroglobulinemia for their ability to bind soluble BLyS and for the expression of the known BLyS receptors, TACI, BAFF-R, or BCMA. The malignant B cells were found to bind BLyS and express BAFF-R and TACI. BCMA expression was undetectable. We then determined the expression of BLyS in bone marrow specimens from 5 patients with Waldenstrom’s macroglobulinemia by immunohistochemistry and compared it to the expression in 5 normal bone marrow specimens. The lymphoplasmacytic cell infiltrate in the bone marrow of patients with Waldenstrom’s macroglobulinemia showed significantly increased BLyS expression. We further determined the serum BLyS levels by ELISA in stored serum specimens from patients with Waldenstrom’s macroglobulinemia (n=20), and compared them to serum BLyS levels in other patients with lymphoplasmacytic lymphoma without elevated immunoglobulin levels (n=10) and to serum levels in normal controls (n=50). Serum BLyS levels in Waldenstrom’s patients (mean: 49.6ng/ml) as well as those in patients with lymphoplasmacytic lymphoma (mean; 46.7ng/ml) were significantly higher than normal controls (mean 12.6ng/ml). In conclusion, we have demonstrated that malignant B cells from patients with Waldenstrom’s macroglobulinemia express the receptors for BLyS and can bind soluble BLyS. Furthermore, we have found that serum BLyS levels are significantly elevated in patients with Waldenstrom’s macroglobulinemia when compared to controls. Strategies to inhibit BLyS may potentially have significant therapeutic efficacy in Waldenstrom’s macroglobulinemia.

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Tumor Microenvironment In Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) Influences Occurrence of Relapses and Progression to Large Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.2684.2684 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2684-2684

Author(s):

Nasir Bakshi ◽

Mansoor Aljabry ◽

Saad Akhter ◽

Irfan Maghfoor ◽

Ayman Mashi

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Hodgkin Lymphoma ◽

Clinical Course ◽

Cell Lymphoma ◽

Lp Cells

Abstract Abstract 2684 NLPHL accounts for 6.5% of all Hodgkin lymphoma cases in the West. It is characterized by a nodular or a nodular & diffuse proliferation of scattered large atypical CD20+ neoplastic B-cells referred to as lymphocyte predominant (LP) cells and typically associated with small lymphocytes mainly of B-cell type. Patients with NLPHL typically have an indolent clinical course but can frequently relapse. Progression to a higher grade lymphoma, notably T-cell/Histiocyte rich B-cell lymphoma (T/HRBCL) has been described in a relatively small number of cases. Because of its rarity, limited information is available about the role of non-neoplastic lymphocytes in NLPHL. Some studies suggest that NLPHL with T-cell rich background may behave differently than the conventional type with predominance of B-cells within the nodules. The purpose of this study was to evaluate outcomes of differential tumor microenvironment namely B-cell versus T-cell rich in patients with NLPHL. We document the clinicopathologic profiles of 29 patients with biopsy proven NLPHL, consisting of 22 male & 7 female, median age 26 years (range, 13–80 years). All patients had lymphoadenopathy & 2 cases showed extranodal involvement in addition to nodal disease. Two patients had a bulky mass, and three had stage 4 disease at presentation. The pathological diagnoses was reviewed and confirmed by an expert hematopathologist in all 29 cases. The LP cells in all cases had a prototypic immunophenotype of CD20+, CD79a+, PU.1+, Bcl-6+, CD15− CD30− & Fascin−. T/HRBCL was excluded as all cases demonstrated preservation of follicular dendritic meshwork by CD21 staining. The meshwork was expanded in 20 cases & in 9 cases it was partially disrupted evincing an irregular architectural pattern. Epstein-Barr Virus encoded RNA by in situ hybridization was negative in 8/8 cases tested. 27/29 patients received systemic multi-agent chemotherapy consisting of: doxorubicin, bleomycin, vinblastine, and dacarbacin (ABVD), 24 patients; cyclophosphamide, doxorubicin, vincristin, and prednisone (CHOP), 2 patients; Rituximab + CHOP (R-CHOP), 1 patient. 9/29 (31%) cases underwent autologous stem cell transplant. One patient in stage 2A refused therapy and one patient (stage 3A) developed significantly decreased cardiac ejection fraction following initial 2 cycles of ABVD. Both of these cases did not have adequate follow-up information available. Results: Twelve of the 29 cases (42%) were designated as having T-cell rich background population, whereas 17 (58%) were considered as conventional variant with a vast predominance of non-neoplastic small lymphocytes being B-cells. A few of the cases seemed to show admixture of both B-cells & T-cells. Comparing T-cell rich & B-cell rich background NLPHL no significant differences were detected in clinical parameters: age, sex, and stage at presentation, absolute lymphocyte count, LDH & Hb. All 27 (100%) patients in this study responded to first-line treatment: 23 with complete response & 4 with partial response. 13/27 (48%) had relapse/s. Five cases had more than one relapses. No patient died within a clinical follow-up period ranging from 18 to 84 months. When the overall survival (OS) of T-cell rich NLPHL was compared with the conventional variant there was no statistical significance between the two groups (log rank p= 0.1206). However, comparison of relapse rate showed that cases with T-cell rich background had higher relapse rate as well as greater incidence of multiple relapses as compared to B-cell rich type of NLPHL even after adjusting for the type of treatment received (log rank p= 0.003). Moreover, 2/12 (17%) T-cell rich NLPHL cases showed transformation to a high grade lymphoma (both T/HRBCL) at the time of recurrence. These findings suggest that in NLPHL a tumor microenvironment rich in T-cells rather than B-cells is characterized by an unfavorable clinical course although OS appears to be similar. These cases perhaps represent a distinctive clinicopathologic variant within the framework of NLPHL. Lately, the term ‘NLPHL with nodules resembling T/HRBCL’ has been used to express the immunobiological overlap between these two entities. It is possible that such cases could be regarded as “intermediate lymphomas” treading between NLPHL and T/HRLBCL. Further studies using gene array profiling analysis may help clarify the molecular differences between these closely related entities. Disclosures: No relevant conflicts of interest to declare.

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Flow Cytometry Primary 10 Colours Panel for Chronic Lympho Proliferative Diseases Diagnosis

Blood ◽

10.1182/blood.v118.21.5190.5190 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5190-5190

Author(s):

Jonathan Brauner ◽

Ingrid Beukinga ◽

Zoulikha Amraoui ◽

Zaina Kassengera ◽

Michel Toungouz ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Lymphocytes ◽

Plasma Cells ◽

Cell Lymphoma ◽

Cd10 Expression ◽

Complete Characterisation

Abstract Abstract 5190 Objectives: Definition of the primary antibodies panel for 10 colours flow cytometry able to describe normal and clonal T, B lymphocytes and plamocytes in blood and bone marrow. Once clonalities are detected, the complete characterisation of Chronic Lymphoproliferative Diseases (CLPD) is supported by secondary panels chosen based on the results of CD5/CD10 expression for clonal B lymphocytes, CD27/CD38 for plasmatocytes and CD3/CD27 for clonal T cells. Materials and Methods: Blood and bone marrow of patients (N=50) with CLPD (mainly B-CLL). Samples are enumerated by haematology analyzer DxH 800 then 106 cells are washed three times, stained with the antibodies combination and red blood cells lysed with Versalyse (TM. Beckman Coulter). The samples were analysed on a 10 colours Navios flow cytometer (Beckman Coulter Fullerton, CA). The staining panel consists of 14 antibodies (CD45, CD8, CD4, CD5, CD3, CD19, CD38, λ, κ, CD23, CD5, CD10, CD14, CD27) conjugated with 10 different fluorochromes. The fixed gating strategy allows linking Navios analysis software to the middleware Remisol which drives the choice of the secondary panel. In some cases a third tube is performed for Ki67 or Zap-70 intra-cytoplasmic staining. Results: Monocytes are removed on the basis of their CD14/CD4 expression. B lymphocytes are CD19 positive. Normal naïve/memory B cells, hematogones and plasma cells are defined by their CD27, CD10 and CD38 expression. Eventual monoclonality is sought by analysis of the distribution of Kappa and Lambda light chains. A first classification of B cell lymphoma is achieved with the CD5 and CD10 expression of the clone (CD5+/CD10−: B-CLL MCL and few MZL, CD5−/CD10−: MZL and related, CD5−/CD10+ DLBCL and FL). Analysis of CD27, CD20 and CD23 expression allows discriminating between CD5+/CD10- lymphomas. All the 50 samples were correctly detected as CLPD and the automated Remisol choice of the second panel fit to the final diagnosis of all the cases of this small series. T lymphocytes are defined by their CD3 and CD5 expression. The analysis of CD4/CD8 balance and CD27/CD5 distribution are first line test when T cell clonality is suspected. There is a special gating to detect CD3-CD4+ T cell lymphoma and double negativity of CD4 and CD8 is a surrogate marker for gamma/delta T cells. NK cells are mentioned as not-T not-B lymphocytes, without specific staining. Conclusion/Discussion:This 10 colours 14 antibodies panel allows describing in one tube normal T and B cells, hematogones, memory and naives B cells plasma cells and detects T and B clonalities. This panel follows a similar logic than the Euroflow LST tube but with 10 colours and with Beckman Coulter's technology and antibodies. Moreover, this combination helps discriminating rapidly the CD5+/CD10- lymphomas while the complete characterisation of CD5 negative lymphomas only require less than 6 antibodies second tube. This is a paperless (all the process is driven and controlled by Remisol), fast and inexpensive diagnostic approach (always less than 20 antibodies required). Disclosures: Pradier: Beckman Coulter: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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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 ◽

10.1182/blood.v120.21.2613.2613 ◽

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.

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

Blood ◽

10.1182/blood.v120.21.4117.4117 ◽

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.

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The landscape of the tumor-infiltrating B cell and its association with T-cell and tumor microenvironment in human cancers.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.76 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 76-76

Author(s):

Young Kwang Chae ◽

William Han Bae ◽

Yeonjoo Choi ◽

Young Suk Kim ◽

Jonathan Forrest Anker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Cell Carcinoma ◽

Expression Patterns ◽

Cell Infiltration ◽

Inverse Association ◽

Human Cancers

76 Background: Compared to recent advances in our knowledge of T cell biology with success of immunotherapy, little progress has been made in understanding of the effects of B cells in tumor microenvironment and their interactions with T cells. Preclinical studies reported that B cells may have immune suppressive roles in tumor microenvironment via induction of T cell exhaustion. However, this association has not been shown in human tissues. We explored the landscape of tumor infiltrating B and T cells and their association with tumor microenvironment in various human cancers for which the FDA approved the use of immune checkpoint inhibitors. Methods: Expression patterns for 812 immune related genes from the TCGA database were utilized to define tumor infiltrating cells in 2951 patients with bladder urothelial carcinoma, renal clear cell carcinoma, skin cutaneous melanoma, lung squamous cell carcinoma, lung adenocarcinoma, and head and neck squamous cell carcinoma. Odds ratios (ORs) of the numbers of tumors with versus without activated B cell infiltration by the presence of activated CD8T cell infiltration were calculated. Results: Immune landscape of the six human cancers showed a consistent inverse association between tumor infiltrating activated B and CD8 T cells (OR = 0.18, p < 0.001). B cell infiltration was associated with increased expressions of immune checkpoints PD-L1, PD-1 and CTLA-4 and regulatory cytokines TGF-β, IL-10 and IL-35, which are known to be secreted by regulatory B cells. Angiogenic markers, such as angiopoietins, VEGF, MMP-9, CXCL10, CXCL11 and Tie2, showed differential expression patterns between B cell high and low groups. Conclusions: This is the first study that reports the inverse association between tumor infiltrating B and CD8 T cells in human tissues. The strong associations between B cell infiltration and increased expressions of suppressive cytokines and immune checkpoints suggest regulatory B cells may play a role in the T cell suppression in tumor microenvironment. Our results implicate that depleting B cells, leading to possible disinhibition of T cell activation, may be a future therapeutic option in potentiating T cell mediated immunity.

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New Immunophenotypic Profile of Waldenstrom’s Macroglobulinemia: Interest of CD80 and CD86 Staining

Blood ◽

10.1182/blood.v112.11.5277.5277 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5277-5277

Author(s):

Andrea Toma ◽

Magali Le Garff-Tavernier ◽

Martine Brissard ◽

Patrick Bonnemye ◽

Lucille Musset ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Population ◽

Light Chain ◽

Waldenström’S Macroglobulinemia ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia ◽

Immunophenotypic Analysis

Abstract The immunophenotypic characterization is an essential tool in the diagnosis of hematological malignancies but the immunophenotypic features in Waldenstrom’s macroglobulinemia (WM) remain not clearly defined. We studied 96 cases of WM diagnosed by monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow infiltration, and we compared results to 33 cases of other chronic B-cell lymphoproliferative disorders (LPD), including marginal zone (MZL)(n=23), mantle cell (MCL)(n=8) and follicular (FL)(n=2) lymphomas. Patients with a Matutes score >3 (chronic lymphocytic leukemia) and with pathognomonic immunophenotype (hairy cell leukemia) were excluded. Immunophenotypic analysis was performed by flow cytometry using six-colour staining (FACS Canto II, Becton Dickinson). In WM and LPD groups, a monoclonal B-cell population was identified in blood (31 and 28 patients, respectively), blood and bone marrow (28 and 4 patients) or bone marrow samples (23 and 1 patients). Overall, 61% of WM patients showed a monoclonal B-cell population in blood. Neoplastic cells of WM and LPD patients with blood and/or bone marrow involvement expressed a monoclonal immunoglobulin light chain kappa (in 70% and 73% of cases respectively) or lambda (30% and 27%). The intensity of expression of the light chain was heterogeneous in both groups (high, normal or low expression in 43%, 27% or 30% of WM, and in 52%, 33% or 15% of LPD, respectively). All pan-B antigens (CD20, CD19, CD79b) were positive for at least 97% of patients. Results obtained with other antigens in WM compared to LPD were: CD10 = 10% vs 7% of patients, CD23 = 33% vs 56%, CD5 = 14% vs 26%, FMC7 = 76% vs 89%, CD38 = 56% vs 41%, CD25 = 86% vs 84%, CD43 = 12% vs 16%, and CD11c = 10% vs 36%. The intensity of expression of these antigens was heterogeneous in both groups. Among the antigens only tested in the WM group, CD1c and CD27 were positive for 70% of patients, IgM and IgD for 95% of patients, and CD103 as well as CD117 were negative in all cases. No difference was found between blood and bone marrow for all previous antigens. Plasma cells (CD38/CD138 positive cells) were found at low levels (less than 2.5% of B-cells) for 46% of WM in blood and/or bone marrow samples. Among the 10 WM patients tested for ZAP-70 expression, 9 were negative and 1 showed a low intensity expression. These results confirm that the immunophenotypic analysis usually performed with standard antigens does not allow defining a typical profile of WM. In order to tentatively identify the WM among the B-cell malignancies, we studied the expression of molecules known to be involved in B-cell development or in costimulatory pathways of antigenic activation, namely CD69, CD83, CD80 and CD86. We first analyzed blood samples of 24 WM patients showing a peripheral monoclonal B-cell population. CD80 was positive (> 20% of B-cells) in all cases and CD83, CD69 and CD86 were always negative. Among these WM patients, 13 were also studied for the bone marrow phenotype. No difference was found between blood and bone marrow phenotype in 11/13 WM cases. We then studied 11 LPD with blood tumoral involvement (MZL(n=7), MCL(n=2) and FL(n=2)). In these LPD, CD69 and CD83 were always negative and, in most cases (9/11 patients), CD80 and CD86 were also negative. Interestingly, CD80 was found positive in 2 patients with MZL, but the CD80 positivity was always associated to the CD86 positivity. Altogether, these data suggest that the inclusion of CD80 and CD86 in the panel of cytometric analysis allow to discriminate WM from other B-LPD with peripheral blood involvement.

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T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests

10.1101/2021.05.20.21257541 ◽

2021 ◽

Author(s):

Prabhakar Putheti ◽

Vijay Sharma ◽

Rex Friedlander ◽

Arvind Menon ◽

Darshana Dadhania ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Rank Correlation ◽

Class I ◽

Igg Antibodies ◽

Hla Antibodies ◽

Hla Class I Antigens

Background. A T cell positive and B cell negative (T+B-) flow cytometry crossmatch (FCXM) result remains a conundrum since HLA-class I antigens are expressed on both T and B cells. We investigated the frequency, HLA specificity of the antibodies and mechanisms for the T+B- FCXM result. Methods. We analyzed 3073 clinical FCXM tests performed in an American Society of Histocompatibility and Immunogenetics accredited histocompatibility laboratory. The sera associated with the T+B- FCXM were also tested for donor HLA IgG antibodies using LABScreen single antigen assays. Results. Among the 3073 FCXM tests, 1963 were T-B-, 811 were T-B+, 274 were T+B+, and 25 were T+B-. IgG antibodies directed at donor HLA-A, B, or Cw locus determined antigens (DSA) were identified in all 25 sera and the summed mean fluorescence intensity (MFI) of DSA ranged from 212 to 53,187. Correlational analyses identified a significant association between the summed MFI of class I DSA, and the median channel fluorescence (MCF) of T cells treated with the recipient serum (Spearman rank correlation, rs=0.34, P=0.05) but not with the MCF of B cells (rs=0.23, P=0.24). We identified that differential binding of anti-HLA antibodies to T cells and B cells and the B cell channel shift threshold used to classify a B cell FCXM are potential contributors to a T+B- FCXM result. Conclusions. Our analysis of 3073 FCXM, in addition to demonstrating that HLA antibodies directed at HLA-A, B or Cw locus are associated with a T+B- result, identified mechanisms for the surprising T+B- FCXM result.

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IL-21 in the Bone Marrow Microenvironment Contributes to IgM Secretion and Proliferation of Malignant Cells in Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v118.21.770.770 ◽

2011 ◽

Vol 118 (21) ◽

pp. 770-770

Author(s):

Lucy S. Hodge ◽

Steve Ziesmer ◽

Thomas E. Witzig ◽

Anne J. Novak ◽

Stephen M. Ansell

Keyword(s):

Bone Marrow ◽

B Cell ◽

Cell Line ◽

Cellular Proliferation ◽

Bone Marrow Microenvironment ◽

Waldenström’S Macroglobulinemia ◽

Waldenstrom's Macroglobulinemia ◽

Waldenstrom’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia ◽

Dependent Mechanism

Abstract Abstract 770 Background: Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results: When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p<0.0005) IgM secretion in WM as measured by ELISA (MWCL-1 5,956 +/− 393 ng/mL vs. 10,013 +/− 730 ng/mL; CD19+138+ WM 504 +/− 33 ng/mL vs. 811 +/− 32.5 ng/mL). Annexin/PI staining was used to assess viability, but no apoptotic effects were associated with IL-21 in WM. To better understand the mechanisms through which IL-21 increases cellular proliferation and IgM secretion in WM, we characterized STAT activation in response to this cytokine. In MWCL-1 cells, IL-21 significantly increased the phosphorylation of both STAT1 and STAT3, and to a lesser extent, STAT5. Treatment with a STAT3 inhibitor completely abolished the effects of IL-21 on cellular proliferation and IgM secretion suggesting IL-21 mediates its biologic activity through a STAT3-dependent mechanism. The expression of transcription factors involved in B-cell differentiation was also measured in MWCL-1 cells treated with IL-21. Both BLIMP-1 and Bcl-6 levels significantly increased upon addition of IL-21, whereas PAX5 was significantly decreased. IL-21 had no effect on the expression of XBP-1, which is involved in regulating Ig secretion, suggesting that the increase in IgM secretion in MWCL-1 cells may occur secondary to the increase in proliferation, as opposed to an actual increase in the production of IgM. Lastly, IL-21 significantly enhanced IL-10 secretion from MWCL-1 cells (669 +/− 152 pg/mL vs. 1,948 +/− 279 pg/mL, p=0.0002). While the interplay between IL-10 and IL-21 in WM remains to be examined, IL-10 is known to be involved in normal B-cell development and may have synergistic effects with IL-21 in malignant WM cells. Overall our data indicate that IL-21 in the bone marrow microenvironment significantly affects the biology of WM tumor cells through a STAT3-dependent mechanism. Disclosures: No relevant conflicts of interest to declare.

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