Mechanisms of Antigen Dependent and Independent Rejection of High Grade B-Cell Lymphoma in a Murine Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4091-4091
Author(s):  
Armin Gerbitz ◽  
Madhusudhanan Sukumar ◽  
Florian Helm ◽  
Andrea Wilke ◽  
Thomas Kammertoens ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Nk Cells ◽  
Mhc Class I ◽  
Cell Lines ◽  
Interferon Gamma ◽  
Class I ◽  
High Grade ◽  
Lymphoma Cells

Abstract Abstract 4091 Poster Board III-1026 The rejection of Non Hodgkin Lymphomas expressing foreign, for example viral, antigens is compromised despite the presence of specific T-cells. The underlying immunosuppressive mechanisms are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven by parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt's lymphoma, we investigated the anti-lymphoma activity of specific T-cells. By retroviral transduction of a specific foreign antigen (chicken Ovalbumin-IRES-GFP, OVA) into cell lines from primary c-myc transgenic lymphomas we established a syngeneic model that would allow us to address the role of interferon gamma signalling in rejection of high grade lymphomas. All primary lymphoma cells displayed normal MHC class I and II levels on the surface when compared to wildtype splenic B-cells. This expression could be enhanced by treatment with interferon gamma (IFNg,100U/ml) up to 10 fold. When retrovirally OVA-transduced lymphoma cells were injected into either wildtype or GFP transgenic recipients, animals displayed a significant delay in lymphoma growth compared to non transduced or IRES-GFP transduced control cell lines. 80% of the recipient mice rejected OVA expressing lymphomas. By contrast, we observed 100% mortality when GFP expressing control lymphomas were injected in GFP transgenic recipients, which are tolerant for GFP. Developing OVA expressing lymphomas (20%) displayed a loss of GFP expression indicating a selection for antigen negative cells (p=0.001). In spleens from mice rejecting OVA-expressing lymphomas we found up to 1.8% SIINFEKL specific T-cells. To gain more mechanistic insights, we transferred OVA expressing lymphoma cells into IFNg, Stat1-/- or IFNg-/-receptor deficient recipients. Lack of STAT1-/- or IFNg-receptor on the recipient side or inability to secrete IFNg was associated with fast lymphoma progression and growth was not different when compared to non transduced, antigen negative cell lines. When IFNg-receptor or STAT1 deficient OVA expressing cell lines were transferred into wildytpe mice, rejection was not influenced. Outgrowing OVA expressing lymphomas in wildtype mice displayed a high MHC class I and II expression compared to the cell line prior to injection. MHC induction was absent in lymphomas transferred to Stat1 or IFNg deficient recipients. Depletion of NK cells by anti AsialoGM antibody in wildtype recipients resulted in a significant reduction of disease free survival (80% vs. 50%, p=0.002) and animals developed larger tumors which were eventually rejected resulting in a comparable overall survival. In peripheral blood of NK depleted mice significantly more OVA specific T-cells were detectable through pentamer staining. When lymphoma cell lines were injected into Rag1-/- mice, NK cell mediated rejection was also significantly impaired upon depletion. Our results suggest that T-cell mediated rejection of high grade B-cell lymphomas is strongly dependent on host IFNg secretion and that NK cells substantially contribute to T-cell mediated rejection. Disclosures: No relevant conflicts of interest to declare.

Host Interferon gamma Production and STAT-1 Signalling Is Crucial for Minor Antigen Mediated Rejection of High Grade Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2175-2175
Author(s):  
Madhusudhanan Sukumar ◽  
Andrea Wilke ◽  
Eva Jaeger ◽  
Josef Mautner ◽  
Joachim Ellwart ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class I ◽  
Cell Lines ◽  
Interferon Gamma ◽  
Control Cell ◽  
Specific Antigen ◽  
Class I ◽  
Cell Mediated Immunity ◽  
High Grade ◽  
Lymphoma Cells

Abstract To date mechanisms of T-cell mediated immunity and immune-escape in high grade lymphomas are poorly understood. Using a transgenic mouse lymphoma model, where the proto-oncogene c-myc is driven in parts of the immunoglobulin lambda locus representing a t(8;22) translocation as found in Burkitt’s lymphoma, we developed a syngeneic model to investigate the anti-lymphoma activity of lymphoma specific T-cells. By retroviral transduction of a lymphoma specific antigen (chicken ovalbumin-IRES-GFP vector) into primary cell lines from c-myc transgenic lymphomas we established a model that would allow us to investigate the contribution of interferon gamma signalling in rejection of high grade lymphoma. All lymphomas established displayed low MHC class I and II levels on the surface when compared to wildtype B-cells. This expression could be enhanced by treatment with interferon gamma (100U/ml) up to 10 fold. When retrovirally transduced lymphoma cells were injected into wildtype or GFP transgenic C57BL/6 recipients, animals displayed a significant delay in lymphoma growth compared to IRES-GFP transduced control cell lines. 50% of the recipient mice rejected OVA containing lymphomas whereas we observed a 100% lymphoma growth of IRES-GFP transduced lymphomas in GFP transgenic recipients. Developing OVA containing lymphomas displayed a loss of GFP expression indicating a selection for non transduced cells. In spleens from mice successfully rejecting OVA-containing lymphomas we found up to 1.5% (±0.12%) SIINFEKL specific T-cells. To gain mechanistic insights of lymphoma rejection, we transferred OVA transduced lymphoma cells to Stat1−/− and IFNg−/− recipients. Lack of STAT1−/− on the recipient side or inability to secrete interferon gamma was associated with fast lymphoma progression and was not different when compared to IRES-GFP transduced cell lines injected into GFP transgenic hosts. Although we found 1.6% (±0.52%) SIINFEKL T cells in spleens of lymphoma bearing STAT1−/− animals, interferon gamma production was significantly decreased. We could also show that in wild type recipients, OVA containing lymphomas displayed high MHC class I and II expression which is completely absent in lymphomas from Stat1−/− recipients. Our results suggest that rejection of high grade lymphoma by a specific minor antigen is possible and Stat1 signaling and interferon gamma production by the host is crucial for lymphoma rejection.

Targeting DKK1 for the Immunotherapy of B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 465-465
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Liang Zhang ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Specific Ctls

Abstract Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Stromal Cross-Presentation and Host Interferon-γ Signaling and Are Required for Elimination of Antigen-Loss Variants of High-Grade B Cell Lymphomas: Implications for Adoptive T Cell Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3927-3927
Author(s):  
Armin Gerbitz ◽  
Madhusudhanan Sukumar ◽  
Florian Helm ◽  
Andrea Wilke ◽  
Christian Friese ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Antigen Expression ◽  
High Grade ◽  
B Cell Lymphomas ◽  
Lymphoma Cells ◽  
T Cell Therapy ◽  
Ifn Γ ◽  
Antigen Loss

Abstract Abstract 3927 The incidence of high-grade B cell lymphomas has been increasing over the last decades in western countries for unclear reasons. Relapse after conventional chemotherapy especially in high-grade B cell lymphomas remains a very difficult clinical issue. Contrary to CML and AML, the benefit of allogeneic SCT for treatment of high-grade lymphomas is not well established. Several studies suggested a potential graft versus lymphoma (GvL) effect for acute lymphoblastic leukemia (ALL) and several types of non-Hodgkin lymphomas. To study mechanisms involved in T cell-mediated rejection of B cell lymphomas, we have developed a murine lymphoma model in which three antigens, human c-MYC protein, chicken ovalbumin (OVA) and GFP, serve as foreign antigens for rejection. Lymphomas expressing all three antigens were rejected in 60 to 70% of animals after transfer into wild type mice, whereas lymphomas expressing only human c-MYC protein were not rejected. Outgrowing OVA-expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation and loss of antigen expression, indicating immune escape. In contrast to wild type recipients of OVA-expressing lymphomas, 80 to 100% of recipient STAT1-, IFN-γ-, or IFN-γ receptor-deficient mice died due to lymphoma growth. Remarkably, lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice also invariably showed lost antigen expression. Thus, poor overall survival of IFN-γ- and IFN-γ-receptor-deficient recipient mice is not due to a lack of antigen-specific T cell killing but due to inefficient eradication of antigen-negative variants of the lymphoma. In order to address the role of the stroma in eradication of lymphoma cells we made use of B6bm1 animals that do not present the immunodominant OVA derived peptide SIINFEKL in the context of MHC class I. Since the wildtype MHC represents an allo-antigen in B6bm1 mice, B6bm1 and B6 wildtype control recipients were T-cell depleted by 30H12 anti CD90.2 antibody prior to transfer of lymphoma cells. Anti OVA immunity was restored by adoptive transfer of 1 Mio. primed CD90.1+ OT-I-T-cells one day after lymphoma transfer. T-cell depletion was continued for 28 days biweekly. Lymphoma growth was faster in bm1 recipients and disease free survival significantly reduced (A). In addition, T-cell expansion was significantly reduced (B) in bm1 recipients as analyzed by pentamer staining of OT-I-T-cells in peripheral blood (day 21 0.84%±0.2 vs. 3.53%±0.2 of lymphocytes, p=0.001) indicating an important role of stromal crosspresentation for the rejection of lymphoma cells. Our data show that mechanisms established for solid tumors hold true also for hematologic neoplasias such as B cell lymphomas. Antigen-dependent eradication of tumor antigen-loss variants makes antigen-specific T cell therapy particularly attractive as a novel therapeutic treatment option. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Self MHC class I–licensed NK cells enhance adaptive CD8 T-cell viral immunity

Blood ◽  
2011 ◽  
Vol 117 (19) ◽  
pp. 5133-5141 ◽  
Author(s):  
Michael D. Stadnisky ◽  
Xuefang Xie ◽  
Ebony R. Coats ◽  
Timothy N. Bullock ◽  
Michael G. Brown
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Class I ◽  
Inhibitory Receptor ◽  
Class I Mhc ◽  
Mhc I

AbstractMHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I Dk, a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I Dk disparate mice. We observed that Dk-licensed Ly49G2+ NK cells stabilized and then enhanced conventional dendritic cells (cDCs) recovery after infection. Furthermore, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude, and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T-cell number differences after low-dose MCMV in Dk disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T-cell priming.

Primary Antigen-Specific B Cells: A Novel Approach to Cellular-Based Immunotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3717-3717
Author(s):  
Tahamtan Ahmadi ◽  
Nathalie Weizmann ◽  
Yvonne A. Efebera ◽  
David H. Sherr
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Mhc Class I ◽  
Class I ◽  
Protein Antigens ◽  
Cognate Protein ◽  
Activated B Cells

Abstract Background: The potential for CD40 ligand (CD40L)-activated B cells to serve as antigen-presenting cells (APC) for cell-based immunotherapy has been suggested. Unlike dendritic cells (DC), CD40L-activated B cell populations are readily expandable in vitro. In addition, antigen-specific B cells may efficiently uptake, process, and present cognate protein antigens. Nevertheless, important questions regarding the relative efficacy of CD40L-activated B cells as cell-based vaccines remain. Here, we exploited the unique ability of B cells to uptake antigen through their B cell receptor (BCR) and the propensity for CD40L-activated B cells, including antigen-specific clones, to grow in culture and to process cognate protein antigens to determine if CD40L-activated B cells represent a suitable substitute for dendritic cells for cell-based immunotherapy. Methods: As a head to head comparison between CD40L-activated B cells and mature DC, CD40L-activated B cells and bone marrow-derived DC were pulsed with MHC II- or MHC I-restricted self protein-derived (MOG; MBP) peptides and tested for their ability to induce proliferation of CD4+ or CD8+ clones. To compare processing and presentation of foreign protein antigens, C57BL/6 mice were immunized with 200 mg NP-BSA or an equivalent volume of PBS emulsified in CFA, sacrificed 10 days later and splenocytes obtained to generate antigen-specific CD40L-activated B cells and T cells. Bone marrow cells from PBS/CFA immunized mice were used to generate DCs. CD40L-activated (antigen-specific) B cells and DC were pulsed with NP-BSA, NP-CGG, or BSA and assayed for their ability to induce proliferation of primary T cells. Results: B cell populations were readily expanded by culture on CD40L transfected L cells. CD40L stimulation significantly up-regulated MHC class I and II expression and induced expression of CD80 and CD86 to levels similar to those detected on mature DCs. CD40L-activated B cells were comparable to DCs when presenting MHC class I- or II-restricted self-peptides to T cell clones. When presenting cognate protein antigen (NP-BSA or BSA) to primary T cells, CD40L-activated B cells from NP-BSA immunized mice were as efficient as DC, both of which induced a 13–15 fold increase in T cell proliferation. To determine if the hapten moiety is sufficient to increase antigen up-take and presentation, DCs and CD40L-activated B cells from NP-BSA immunized mice were pulsed with NP-CGG and used as APC for T cells from NP-BSA immunized mice. DCs induced significant responses comparable to those seen with BSA and NP-BSA. Activated B cells from NP-BSA-immunized mice induced significantly higher responses to NP-CGG than activated B cells from control PBS/CFA “immunized” mice, although these responses were lower than those generated with dendritic cells. Conclusion:CD40L-activated B cells can be readily expanded in vitroand significantly up-regulate co-stimulatory molecules CD80 and CD86 to levels comparable to mature DCs,CD40L-activated B cells present MHC class I- and II-restricted self-peptides to T cell clones as efficiently as mature DCs,Antigen-primed B cells are as efficient at presenting cognate protein antigens as DCs, Immunization with a hapten-carrier is sufficient to induce hapten-specific B cells which, when activated with CD40L, effectively present unrelated neoantigens conjugated with the hapten. The data suggest that CD40L-activated B cells represent an important alternative APC for immunotherapy, particularly when previously educated to protein or haptenic determinants.

Quantifying Capsid Peptide:MHC I Complexes Following Adeno-Associated Virus (AAV) Transduction

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3737-3737
Author(s):  
Gary C. Pien ◽  
Nicole C. Hasbrouck ◽  
Marcela V. Maus ◽  
Federico Mingozzi ◽  
Katherine A. High
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Mhc Class I ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Factor Ix ◽  
Class I ◽  
Adeno Associated Virus

Abstract Recent data from a clinical trial of adeno-associated virus (AAV) 2-mediated hepatic gene transfer of factor IX into hemophilia B subjects demonstrated the presence of AAV2-capsid-specific CD8+ T cells and identified an immunodominant AAV2 epitope. The expansion and contraction of this CD8+ T cell population coincided with the rise and fall of an asymptomatic elevation in liver transaminases, and correlated with the expression and subsequent loss of factor IX expression. These observations suggest that CD8+ T cells directed against AAV capsid antigen clear the transduced hepatocytes. Indeed, others in our laboratory have directly shown the lysis of AAV-transduced hepatocytes by CD8+ T cells in vitro. However, the stoichiometry and kinetics of AAV capsid degradation and processing for MHC class I presentation are presently unknown. We now report the generation of soluble AAV capsid-specific T cell receptor (TCR) tetramers and their use in quantifying capsid peptide:MHC I complexes presented on cell surfaces. CD8+ T cell clones specific for AAV2-derived peptide 74 (VPQYGYLTL) presented in the context of HLA-B*0702 were isolated and used to generate TCR cDNA. This cDNA was transfected into CHO cells to produce soluble TCR α and β chain fusion proteins. The soluble TCR was biotinylated, then tetramerized to streptavidin-conjugated fluorochrome for use in flow cytometric analyses. Cell lines expressing HLA-B*0702 were loaded with cognate AAV2 peptide, a cross-reactive AAV1 peptide (IPQYGYLTL), or irrelevant HLA-B*0702 epitopes derived from HIV-1 gp120 (IPRRIRGGL) or EBV nuclear antigen (RPPIFIRRL). Staining with TCR-tetramers reveals that this reagent binds AAV-derived peptides specifically. In addition, this reagent exhibits HLA-specificity, as cognate peptide loaded onto cells which express alternate HLA alleles was not detected. These results demonstrate that TCR-tetramers retain their antigen and MHC context specificities. Capsid peptide titration experiments using HLA-B*0702-bearing JYA2B7 and SK-MES-1 cell lines reveal that TCR-tetramers exhibit a limit of detection of approximately 105 cell surface peptide:MHC I complexes. Efficiency of detection is estimated at 1 peptide:MHC I complex for every 1010 peptide molecules added to cell culture. Preliminary studies indicate that the number of presenting complexes wanes by 48 hours following peptide loading. In contrast, AAV1-mediated transduction experiments demonstrate that capsid peptide:MHC I complexes are detectable after 48 hrs. In conclusion, these results are in agreement with our central hypothesis that AAV capsid is degraded upon transduction and gains access to MHC Class I antigen presentation pathways. Studies with the TCR-tetramers will allow definition of kinetics and capsid dose-dependence of antigen presentation, defining parameters of cell vulnerability to host immune-mediated clearance of transduced cells.

TGF-β Is Selectively Expressed on Lymphoma B Cells and Regulates the Differentiation of Intratumoral T Cells in B-Cell Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1586-1586
Author(s):  
Zhi-Zhang Yang ◽  
Deanna Grote ◽  
Steven C. Ziesmer ◽  
Thomas E. Witzig ◽  
Anne J. Novak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Lymphoma Cell ◽  
Lymphoma Cells

Abstract Abstract 1586 Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19+ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4+, CD4+CD45RA+ or CD4+CD45RO+ intratumoral T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response. Disclosures: No relevant conflicts of interest to declare.

CD3/Bars: A Novel Bispecific Format for the Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3516-3516
Author(s):  
Moritz Bewarder ◽  
Klaus-Dieter Preuss ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
Lorenz Thurner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Bispecific Antibodies ◽  
B Cell Lymphomas ◽  
Lymphoma Cells

Abstract Background Autoantigens are suspected to play an important role in the pathogenesis of different types of B cell neoplasia. Suggestive of this hypothesis is the restricted usage of a stereotyped IgHv repertoire in CLL, MCL and DLBCL. Further evidence supporting this notion is the identification of specific autoantigens as the target of the B-cell receptor from malignant lymphomas and myelomas, such as paratarg-7 as antigenic target for 15% of paraproteins of MGUS and MM patients. ARS2 was previously identified as the autoantigenic target for the B-cell receptor of approximately 25% of DLBCLs of the ABC type, here termed ARS2 reactive lymphomas. We had recently shown that the B-cell receptor antigens can be used to target B-cell lymphoma cells in vitro and in vivo in an approach designated as BARs (B-cell receptor antigens for reverse targeting), the first therapeutic strategy in oncology with absolute and exclusive specificity for the malignant clone. To test whether BARs can substitute the B-cell binding antibody (e.g. CD19) in T-cell engaging bispecific antibodies, we designed a bispecific CD3/BAR product consisting of a recombinant single chain fragment (scFv) against CD3 linked to ARS2 (CD3-ARS2). One arm of this construct should engage the T cell co-receptor CD3 of human T cells, and the other site should bind to the B cell receptor of ARS2 reactive lymphomas thus specifically redirecting and activating T cells against lymphoma cells. Material and methods VL and VH genes of the CD3 - OKT3 hybridoma and the DNA sequence of the 33 amino acids containing the B-cell receptor binding epitope of ARS2 were cloned into a pcDNA 3.1 vector by standard cloning techniques. VH and VL were linked by a glycine-serine linker, as was VL to the ARS2 epitope resulting in VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2 peptide chain. The final cloning product was transfected in HEK cells for production of the bispecific construct. Binding capacity to lymphoma cell lines (OCI-Ly3, U2932, HBL-1) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of CD3-ARS2 after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay. Results The CD3 - ARS2 bispecific construct bound to CD3 on T cells and the B-cell receptor of ARS2 reactive lymphoma cells. CD3/ARS2 induced rapid cytotoxicity exclusively in ARS2 reactive lymphoma cell lines at concentrations as low as 250 ng/ml with an effector - target ratio of 10:1. Specific T-cell mediated cytotoxicity reached 40% after 4 hours. Lymphoma cell lines with BCRs of a specificity other than ARS2 were not affected. Conclusion The CD3/BAR construct is a novel therapeutic principle for the treatment of B-cell lymphomas, suggesting that BARs might also be useful as part of CAR-T cells. Compared to CD3/CD19 bispecific antibodies the CD3/BARs are exclusively cytotoxic against the malignant clone and spare normal B-cells. This should considerably reduce the acute toxicity of T-cell engaging bispecific constructs and circumvent long-term B cell depletion. Experiments comparing the cytotoxic capacity of CD3/BARs with CD3/CD19 bispecific antibodies are underway, as are analyses evaluating possible synergisms of these constructs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immune Functions in Mice Lacking Clnk, an SLP-76-Related Adaptor Expressed in a Subset of Immune Cells

2004 ◽  
Vol 24 (13) ◽  
pp. 6067-6075 ◽  
Author(s):  
Oliver Utting ◽  
Bradley J. Sedgmen ◽  
Tania H. Watts ◽  
Xiaoshu Shi ◽  
Robert Rottapel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mast Cells ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Immune Cells ◽  
Leukemia Cell ◽  
Cell Development ◽  
Immune Functions

ABSTRACT The SLP-76 family of immune cell-specific adaptors is composed of three distinct members named SLP-76, Blnk, and Clnk. They have been implicated in the signaling pathways coupled to immunoreceptors such as the antigen receptors and Fc receptors. Previous studies using gene-targeted mice and deficient cell lines showed that SLP-76 plays a central role in T-cell development and activation. Moreover, it is essential for normal mast cell and platelet activation. In contrast, Blnk is necessary for B-cell development and activation. While the precise function of Clnk is not known, it was reported that Clnk is selectively expressed in mast cells, natural killer (NK) cells, and previously activated T-cells. Moreover, ectopic expression of Clnk was shown to rescue T-cell receptor-mediated signal transduction in an SLP-76-deficient T-cell line, suggesting that, like its relatives, Clnk is involved in the positive regulation of immunoreceptor signaling. Stimulatory effects of Clnk on immunoreceptor signaling were also reported to occur in transfected B-cell and basophil leukemia cell lines. Herein, we attempted to address the physiological role of Clnk in immune cells by the generation of Clnk-deficient mice. The results of our studies demonstrated that Clnk is dispensable for normal differentiation and function of T cells, mast cells, and NK cells. Hence, unlike its relatives, Clnk is not essential for normal immune functions.

Inhibitory MHC class I receptors on NK cells and T cells

1996 ◽  
Vol 17 (2) ◽  
pp. 86-91 ◽  
Author(s):  
Lewis L. Lanier ◽  
Joseph H. Phillips
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Class I
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