Mesenchymal Stromal Cells Suppress T-and B-Cells via Galectin-9 in a Donor Dependent Manner

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1248-1248
Author(s):  
Christopher Ungerer ◽  
Patricia Quade-Lyssy ◽  
Reinhard Henschler ◽  
Erhard Seifried ◽  
Heinfried Radeke ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Immune Suppression ◽  
Immune Cells ◽  
Stromal Cells ◽  
Coagulation Factor ◽  
T And B Cells ◽  
Ifn Γ

Abstract Abstract 1248 Therapeutic approaches using multipotent mesenchymal stromal cells (MSCs) are advancing in regenerative medicine, transplantation and autoimmune diseases. Until now the way of action for MSC-mediated immune suppression is still controversial and relies most probably on a multifactorial mechanism. MSCs have been demonstrated to produce the suppressive molecules hepatocyte growth factor (HGF), tumor growth factor-β (TGF-β), prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO). Furthermore, it has been described that immunosuppression by MSCs is enhanced via stimulation with interferon-γ (IFN-γ). Recently, galectin-1, a β-galactoside binding lectin with immune modulatory properties, has been added to the group of immune modulatory molecules that are responsible for MSC-mediated immune suppression. Here, we identified galectin-9 (Gal-9) as a new molecule involved in MSC-mediated immune modulation. First, we isolated MSCs from bone marrow of randomly selected donors and performed several in vitro experiments regarding their immune modulatory potential (e.g proliferation and IgG production). Interestingly, Gal-9 was the only investigated protein, which was strongly upregulated in MSCs upon activation with IFN-γ. We moreover demonstrate that Gal-9 is a major mediator of the anti-proliferative effect of MSCs on T-cells. Although a B-cell suppressive function of Gal-9 has previously not been reported, we were surprised to detect the same inhibitory effect on isolated B-cells. Proliferation of immune cells was triggered upon either stimulation with either PHA and LPS, or CD40L and PHA. Activation of MSCs with IFN-γ resulted in a major decrease of proliferation of both T-cells and B-cells. In addition, Gal-9 and activated MSCs contribute to the suppression of VZV triggered immunoglobulin release as well. Again activation of MSCs with IFN-γ decreased the IgG release, whereas blocking Gal-9 with lactose, a well characterized inhibitor of Gal-9 function, reversed the effect almost completely. Further, we determined that Gal-9 expression levels (mRNA and protein) distinguish between MSC cultures from different donors after activation. Among donors, we could differentiate between individuals with high Gal-9 levels and higher immune modulatory potential and such with low Gal-9 expression and lower immune modulatory potential. Compared to untreated MSCs we demonstrated a three- to fifty-fold rise in Gal-9 levels after prior activation with IFN-γ. In addition, we demonstrated the upregulation of Gal-9 in MSCs by cell-cell contacts with either T-or B-cells. The upregulation was additionally at least two fold increased by previeously activating MSCs with IFN-γ. Because our group is interested in the therapy of hemophilia A and because of the unxpected suppressive effect of Gal-9 on B-cells and B-cell function, we next tested the effect of MSCs and Gal-9 on the induction of inhibitory antibodies to coagulation factor VIII (FVIII). Mice were immunized with human coagulation factor VIII (FVIII) in the presence or absence of either human MSCs, anti-murine Gal-9 or human Gal-9. As predicted, MSCs suppressed and anti-Gal-9 antibodies anhanced antibody formation. However in contrary to the expected, human Gal-9 co-treatment enhanced the anti-FVIII antibody response. A set of additional experiments revealed, that human Gal-9 suppresses murine regulatory T-cells in vivo. Further, in contrast to human immune cells, murine-derived T- and B-cells did not respond to human recombinant Gal-9 in vitro, but human IFN-γ activated MSCs were able to suppress proliferation of murine immune cells. Because of only 60% homology of murine and human Gal-9 we assume that the murine model cannot predict the function of human Gal-9 and that MSC-mediated immune modulatory functions are exerted via alternative pathways in this setting. Experiments with murine Gal-9 to demonstrate the in vivo function of Gal-9 are ongoing. In conclusion, Gal-9 is novel mediator of MSC immunomodulatory functions and affectsmultiple immune cell types including B-cells. Gal-9 is differentially expressed in MSCs from different donors and may therefore serve as a predictive indicator for clinical MSC functionality. Disclosures: No relevant conflicts of interest to declare.

In Vivo Activated CD103+CD4+ Regulatory T Cells Ameliorate Ongoing Chronic GVHD.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 350-350
Author(s):  
Dongchang Zhao ◽  
Chunyan Zhang ◽  
Tangsheng Yi ◽  
Chia-Lei Lin ◽  
Ivan Todorov ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Chronic Gvhd ◽  
Treg Cells ◽  
Dependent Manner ◽  
T And B Cells ◽  
Ifn Γ

Abstract The αEβ7 integrin CD103 is an excellent marker for identifying in vivo activated CD4+ regulatory T (Treg) cells. CD103− naive Treg cells from donors are effective in prevention but ineffective in treatment of graft versus host disease (GVHD). It is unknown whether in vivo activated donor CD103+ Treg cells can effectively treat ongoing GVHD. We have recently reported a new chronic GVHD model, in which donor DBA/2 (H-2d) spleen cells were transferred to the irradiated BALB/c (H-2d) recipients. The recipients showed chronic GVHD-like syndrome with high-levels of serum autoantibodies, proteinuria, and hair-loss, and the disease induction required both donor CD4+ T and B cells in a cell dose dependent manner (Blood, 2006). In the current studies, we observed that the percentage of CD103+ cells among donor CD4+ T cells in the recipients without disease was up to 45% and was more than two fold higher than that of the recipients with disease. The CD103+CD4+ T cells were more than 97% FoxP3+, which was similar to natural CD25hi Treg cells, but the former expressed markedly higher levels of CCR5 as compared to the latter. The CD103+ Treg cells showed markedly stronger suppression capacity as compared to freshly isolated as well as in vitro anti-CD3-activated CD25hi natural Treg cells. When injected into ongoing chronic GVHD recipients with severe proteinuria, CD103+ Treg cells (1x106) reversed proteinuria and tissue damages in 92% (11/12) of the recipients, and the recipients survived for more than 100 days. In contrast, the in vitro activated natural Treg cells reversed disease in only 25% (2/8) of the recipients, and the freshly isolated CD25hi natural Treg cells reversed none (0/12), and the treated recipients died within 45 days, which was similar to control PBS treated recipients. Furthermore, we found that infusion of the CD103+ Treg cells significantly reduced CD138+ antibody-secreting plasma cells in spleen and reduced serum levels of autoantibodies; and that infusion of the CD103+ Treg cells also significantly reduced CD44loCD62LhiSCA-1hi post-mitotic CD4+ T memory stem cells in spleen as well as pathogenic IFN-γ+CD4+ T cells in liver and nephritogenic IFN-γ+IL-10+CD4+ T cells in kidney tissues. Our results indicate that, different from CD103− naive natural Treg cells that prevent GVHD via suppressing donor T cell activation and expansion, CD103+ Treg cells ameliorate ongoing chronic GVHD via reducing activated pathogenic T and B cells.

scholarly journals In VitroMeasles Virus Infection of Human Lymphocyte Subsets Demonstrates High Susceptibility and Permissiveness of both Naive and Memory B Cells

2018 ◽  
Vol 92 (8) ◽  
pp. e00131-18 ◽  
Author(s):  
Brigitta M. Laksono ◽  
Christina Grosserichter-Wagener ◽  
Rory D. de Vries ◽  
Simone A. G. Langeveld ◽  
Maarten D. Brem ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Immune Suppression ◽  
Peripheral Blood ◽  
Measles Virus ◽  
Memory B Cells ◽  
T And B Cells ◽  
B Cell Subsets

ABSTRACTMeasles is characterized by a transient immune suppression, leading to an increased risk of opportunistic infections. Measles virus (MV) infection of immune cells is mediated by the cellular receptor CD150, expressed by subsets of lymphocytes, dendritic cells, macrophages, and thymocytes. Previous studies showed that human and nonhuman primate memory T cells express higher levels of CD150 than naive cells and are more susceptible to MV infection. However, limited information is available about the CD150 expression and relative susceptibility to MV infection of B-cell subsets. In this study, we assessed the susceptibility and permissiveness of naive and memory T- and B-cell subsets from human peripheral blood or tonsils toin vitroMV infection. Our study demonstrates that naive and memory B cells express CD150, but at lower frequencies than memory T cells. Nevertheless, both naive and memory B cells proved to be highly permissive to MV infection. Furthermore, we assessed the susceptibility and permissiveness of various functionally distinct T and B cells, such as helper T (TH) cell subsets and IgG- and IgA-positive memory B cells, in peripheral blood and tonsils. We demonstrated that TH1TH17 cells and plasma and germinal center B cells were the subsets most susceptible and permissive to MV infection. Our study suggests that both naive and memory B cells, along with several other antigen-experienced lymphocytes, are important target cells of MV infection. Depletion of these cells potentially contributes to the pathogenesis of measles immune suppression.IMPORTANCEMeasles is associated with immune suppression and is often complicated by bacterial pneumonia, otitis media, or gastroenteritis. Measles virus infects antigen-presenting cells and T and B cells, and depletion of these cells may contribute to lymphopenia and immune suppression. Measles has been associated with follicular exhaustion in lymphoid tissues in humans and nonhuman primates, emphasizing the importance of MV infection of B cellsin vivo. However, information on the relative susceptibility of B-cell subsets is scarce. Here, we compared the susceptibility and permissiveness toin vitroMV infection of human naive and memory T- and B-cell subsets isolated from peripheral blood or tonsils. Our results demonstrate that both naive and memory B cells are more permissive to MV infection than T cells. The highest infection levels were detected in plasma cells and germinal center B cells, suggesting that infection and depletion of these populations contribute to reduced host resistance.

Mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell properties

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2632-2638 ◽  
Author(s):  
Moïra François ◽  
Raphaëlle Romieu-Mourez ◽  
Sophie Stock-Martineau ◽  
Marie-Noëlle Boivin ◽  
Jonathan L. Bramson ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Stromal Cells ◽  
Antigen Processing ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Recent studies involving bone marrow mesenchymal stromal cells (MSCs) demonstrated that interferon (IFN)–γ stimulation induces major histocompatibility complex (MHC) class II–mediated antigen presentation in MSCs both in vitro and in vivo. Concordantly, we investigated the ability of MSCs to present extracellular antigen through their MHC class I molecules, a process known as cross-presentation. Using an in vitro antigen presentation assay, we demonstrated that murine MSCs can cross-present soluble ovalbumin (OVA) to naive CD8+ T cells from OT-I mice. Cross-presentation by MSC was proteasome dependent and partly dependent on transporter associated with antigen-processing molecules. Pretreatment of MSC with IFN-γ increased cross-presentation by up-regulating antigen processing and presentation. However, although the transcription of the transporter associated with antigen processing-1 molecules and the immunoproteasome subunit LMP2 induced by IFN-γ was inhibited by transforming growth factor-β, the overall cross-presentation capacity of MSCs remained unchanged after transforming growth factor-β treatment. These observations were validated in vivo by performing an immune reconstitution assay in β2-microglobulin−/− mice and show that OVA cross-presentation by MSCs induces the proliferation of naive OVA-specific CD8+ T cells. In conclusion, we demonstrate that MSCs can cross-present exogenous antigen and induce an effective CD8+ T-cell immune response, a property that could be exploited as a therapeutic cell-based immune biopharmaceutic for the treatment of cancer or infectious diseases.

Adoptive Therapy with Human CD19 (hCD19)-Targeted T Cells Further Modified to Express IL-12 Overcome the Need for Prior Lymphodepletion to Eradicate Established hCD19+ Tumors and Induce B Cell Aplasias In Immune Competent hCD19+ Transgenic Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2097-2097
Author(s):  
Erik Hayman ◽  
James Lee ◽  
Hollie J. Pegram ◽  
Renier Brentjens
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Constitutive Expression ◽  
Significant Advance ◽  
B Cell Malignancies ◽  
Ifn Γ

Abstract Abstract 2097 Most patients with B cell malignancies, including acute and chronic B cell leukemias as well as most B cell lymphomas, are either incurable or will die from their disease. For this reason novel approaches are needed to treat these patients. To this end, we have previously generated genes encoding artificial T cell receptors, termed chimeric antigen receptors (CARs), specific to the CD19 antigen expressed on normal B cells as well as most B cell malignancies. Human T cells retrovirally modified to express hCD19 targeted CARs lyse hCD19+ tumor cells in vitro and eradicate established systemic hCD19+ tumors in SCID-Beige mice. In order to better assess the efficacy of CD19-targeted CAR modified T cells in a more clinically relevant setting, we have generated a novel syngeneic immune competent hCD19 tumor model utilizing transgenic C57BL6 mice (C57BL6 mCD19-/- hCD19+/−) which exclusively express hCD19 on normal B cells bearing systemic established syngeneic EL4(hCD19) thymoma tumors. Initial studies of EL4(hCD19) bearing C57BL6 mCD19-/- hCD19+/− mice treated with syngeneic T cells expressing the hCD19-targeted CAR, termed 19mz, failed both to eradicate tumor and to induce predicted B cell aplasias. In contrast, following initial lymphodepleting cyclophosphamide chemotherapy, mice treated with 19mz modified T cells both effectively eradicated established tumors as well as induced long-term persistent B cell aplasias. Studies on the effects of cyclophosphamide lymphodepletion in this model revealed elevations of both serum IL-12 and IFN-γ and a decrease in circulating CD4+ Foxp3+ regulatory T cells. In light of these findings we subsequently further modified 19mz+ T cells to express murine IL-12. Constitutive expression of IL-12 by 19mz+ T cells significantly enhanced both cytotoxicity and IFN-γ production as assessed by in vitro assays. Strikingly, we found that 19mz/IL-12 modified T cells no longer required prior lymphodepletion to either induce B cell aplasias or to successfully eradicate established EL4(hCD19) tumors in C57BL6 mCD19-/- hCD19+/− mice. Further in vivo mechanistic studies of these 19mz/IL-12 modified T cells demonstrated a requisite role of autocrine IL-12 mediated signaling of modified T cells through the IL12 receptor expressed on modified T cells, as well as a significant role for IL-12 induced IFN-γ production in mediating the observed in vivo B cell aplasias and anti-tumor effects seen in the absence of prior cyclophosphamide lymphodepletion. Significantly, all mice infused with IL-12 secreting tumor targeted T cells tolerated therapy well with no obvious side effects as assessed by direct observation. These data highlight an exciting and potentially significant advance in the emerging field of adoptive immunotherapy of cancer utilizing genetically modified autologous T cells. Further these studies provide the initial rationale for the clinical application of autologous CD19 CAR targeted IL-12 secreting T cells in the treatment of patients with advanced chemotherapy refractory B cell malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche

2021 ◽  
Vol 9 (3) ◽  
pp. e001803
Author(s):  
Louise M E Müller ◽  
Gemma Migneco ◽  
Gina B Scott ◽  
Jenny Down ◽  
Sancha King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Immune Responses ◽  
Stromal Cells ◽  
Tumor Burden ◽  
Effector Memory ◽  
In Vivo Model ◽  
Bm Niche

BackgroundMultiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported.MethodsThis study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment.ResultsUsing the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes.ConclusionThese data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.

scholarly journals Immunological Reaction in TNF-α-Mediated Osteoclast Formation and Bone ResorptionIn VitroandIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Hideki Kitaura ◽  
Keisuke Kimura ◽  
Masahiko Ishida ◽  
Haruka Kohara ◽  
Masako Yoshimatsu ◽  
...  
Keyword(s):  
T Cells ◽  
Bone Metabolism ◽  
Stromal Cells ◽  
Fas Ligand ◽  
Bone Marrow Cells ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Bone Diseases

Tumor necrosis factor-α(TNF-α) is a cytokine produced by monocytes, macrophages, and T cells and is induced by pathogens, endotoxins, or related substances. TNF-αmay play a key role in bone metabolism and is important in inflammatory bone diseases such as rheumatoid arthritis. Cells directly involved in osteoclastogenesis include macrophages, which are osteoclast precursor cells, osteoblasts, or stromal cells. These cells express receptor activator of NF-κB ligand (RANKL) to induce osteoclastogenesis, and T cells, which secrete RANKL, promote osteoclastogenesis during inflammation. Elucidating the detailed effects of TNF-αon bone metabolism may enable the identification of therapeutic targets that can efficiently suppress bone destruction in inflammatory bone diseases. TNF-αis considered to act by directly increasing RANK expression in macrophages and by increasing RANKL in stromal cells. Inflammatory cytokines such as interleukin- (IL-) 12, IL-18, and interferon-γ(IFN-γ) strongly inhibit osteoclast formation. IL-12, IL-18, and IFN-γinduce apoptosis in bone marrow cells treated with TNF-α  in vitro, and osteoclastogenesis is inhibited by the interactions of TNF-α-induced Fas and Fas ligand induced by IL-12, IL-18, and IFN-γ. This review describes and discusses the role of cells concerned with osteoclast formation and immunological reactions in TNF-α-mediated osteoclastogenesisin vitroandin vivo.

scholarly journals P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A3.2-A4
Author(s):  
J Grün ◽  
I Piseddu ◽  
C Perleberg ◽  
N Röhrle ◽  
S Endres ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
List Type ◽  
Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

scholarly journals 323 Immunogenic syngeneic model MC38-OVA for the preclinical evaluation of immune evasion and checkpoint blockade

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A348-A348
Author(s):  
Jessie Wang ◽  
Kaixia Lian ◽  
Jia Zheng ◽  
Chenpan Nie ◽  
Annie An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Immune Evasion ◽  
Syngeneic Mouse ◽  
Ifn Γ ◽  
Draining Lymph Nodes ◽  
Syngeneic Tumors

BackgroundThe development of immuno-oncology (I/O) therapeutics has revolutionized the cancer treatment landscape. Despite this achievement, the mechanism behind limited responses is poorly understood. Tumor immune evasion has been reported to arise through the loss of tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways, which are crucial to CD8+ T cell-mediated killing. Syngeneic mouse models have been widely used as they have an intact immune system, are easily accessible, and have a vast array of historical data for comparison. However, limited syngeneic models respond to immune checkpoint inhibitors, possibly due to low intrinsic immunogenicity. The expression of ovalbumin (OVA) has previously shown to sufficiently alter the susceptibility of syngeneic tumors to host T cell-mediated responses. In this study, the newly developed OVA-expressing MC38 syngeneic line was characterized for tumor immunity, checkpoint blockade response and response durability.MethodsMurine colon cancer MC38 cells were transduced by lentiviral vector with chicken OVA coding cDNA. A single clone was selected, and OVA expression was confirmed by western blot. The MC38-OVA cells were subcutaneously implanted into immunocompetent mice to evaluate the tumorigenicity and in vivo response to anti-PD-1 antibody treatment. Blood was collected 2 days post final dose of anti-PD-1 treatment for phenotypic analysis by FACS. Spleen and tumor draining lymph nodes were collected at termination for FACS analysis of IFN-γ+ T cells and OVA specific CD8+ T cells. Adoptive transfer was evaluated by challenge studies in both MC38-OVA and MC38 tumor-bearing mice with T cells derived from MC38-OVA mice, anti-PD-1 cured mice and OT-I mice. In vitro killing assays were performed to evaluate the function of adoptive CD3+ T cells transfer.ResultsOVA-expressing MC38 presented complete regression under anti-PD-1 treatment in vivo. T cell expansion was observed after anti-PD-1 treatment in peripheral blood with increased IFN-γ+ T cells in both tumor-draining lymph nodes and spleen. Additionally, anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited MC38-OVA and MC38 tumor growth following adoptive transfer. CD3+ T cells from MC38-OVA-bearing mice and OT-I mice showed anti-tumor immunity in vivo. In vitro killing assay demonstrated increased immunity.ConclusionsSyngeneic mouse tumor models are preferred preclinical models for I/O research, despite limited intrinsic immunogenicity. OVA expression in syngeneic tumors largely increased T cell-mediated immunity to enhance antigen-specific T cell responses during tumorigenesis, providing novel immunogenic models for preclinical immunotherapy evaluation.

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