Notch Inhibition in Alloreactive CD4+ and CD8+ T Cells Blocks Graft-Versus-Host Disease by Inducing a Hyporesponsive Program with Features of T Cell Anergy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 340-340
Author(s):  
Ashley R Sandy ◽  
Jooho Chung ◽  
Ivy T Tran ◽  
Gloria T Shan ◽  
Ann Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ifn Gamma ◽  
Cell Compartments ◽  
Alloreactive T Cells ◽  
Notch Inhibition

Abstract Abstract 340 Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo-BMT). We previously identified Notch signaling as an essential regulator of allogeneic CD4+ T cell responses mediating GVHD after allo-BMT. Alloreactive CD4+ T cells expressing the pan-Notch inhibitor DNMAML induced markedly less severe GVHD as compared to wild-type T cells, leading to improved survival of the recipients. Notch-deprived T cells had preserved in vivo expansion and cytotoxicity. However, alloreactive DNMAML CD4+ T cells produced markedly decreased amounts of multiple proinflammatory cytokines, including TNF-alpha, IFN-gamma, and IL-2. This was associated with increased expansion of Foxp3+ CD4+ T regulatory cells. Thus, Notch signaling is an attractive new therapeutic target to control GVHD without eliminating the anti-cancer activity of allo-BMT. To elucidate the mechanisms of Notch action in GVHD, we studied the effects of Notch inhibition in alloreactive CD4+ and CD8+ T cells using minor and major histocompatibility antigen-mismatched models of allo-BMT. In the B6 anti-BALB/b minor antigen-mismatched model, recipients of B6 T cells were protected from lethal acute GVHD upon DNMAML expression in the CD4+, CD8+ or both T cell compartments. In the B6 anti-BALB/c MHC-mismatched model, DNMAML CD4+ or CD8+ T cells transplanted alone or in combination induced significantly less GVHD and resulted in improved survival compared to wild-type T cells. Upon ex vivo restimulation with anti-CD3/CD28 antibodies, both CD4+ and CD8+ DNMAML alloreactive T cells had markedly decreased production of IFN-gamma. These findings suggest that Notch signaling has parallel functions in CD4+ and CD8+ T cells. We then studied expression of Tbx21 (encoding T-bet) and Eomes, the key transcription factors regulating Ifng transcription in CD4+ Th1 and CD8+ T cells, respectively. DNMAML alloreactive T cells had preserved amounts of Tbx21 mRNA and T-bet protein, and increased levels of Eomes transcripts and protein. These data differ from past reports indicating that Notch signaling controls T cell differentiation through direct regulation of Tbx21 and Eomes expression. Ex vivo restimulation of DNMAML CD4+ and CD8+ T cells with PMA (diacylglycerol analog) and ionomycin (calcium ionophore) rescued IFN-gamma production by both T cell compartments and partially restored IL-2 production by CD4+ T cells, suggesting abnormal signaling downstream of the T cell receptor. After anti-CD3/CD28 restimulation, DNMAML alloreactive T cells showed markedly decreased phosphorylation of Mek1 and Erk1/2, indicating defective Ras/MAPK activation. PMA was sufficient to rescue Erk1/2 activation. NFkB activity was also significantly impaired in alloreactive DNMAML T cells as assessed with a NFkB-luciferase reporter transgene. Abnormal responsiveness was acquired in vivo during alloreactive T cell priming, since naïve DNMAML T cells had preserved Ras/MAPK activation. Moreover, alloreactive Notch-deprived T cells had elevated levels of intracellular cAMP and increased expression of the anergy-associated genes, Dgka and Egr3. Thus, alloreactive DNMAML T cells had features reminiscent of T cell anergy. Given that in vivo proliferation in irradiated recipients and cytotoxicity of DNMAML alloreactive T cells were largely preserved, our data suggest a “split anergy” phenotype with differential effects on distinct T cell effector functions. Altogether, our results reveal a parallel role for Notch signaling in both the CD4+ and CD8+ T cell compartments that differ from all previous reports of Notch action in mature T cells. Understanding the role of Notch signaling in alloreactive T cells is essential for harnessing the therapeutic potential of Notch inhibition in GVHD. Disclosures: No relevant conflicts of interest to declare.

Notch Signaling in Graft-Versus-Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-14-SCI-14
Author(s):  
Ivan Maillard
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Graft Versus Host Disease ◽  
Graft Versus Host ◽  
Cell Responses ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Notch Inhibition

Abstract SCI-14 Notch is a highly conserved signaling pathway with multiple functions in health and disease. In the hematopoietic system, Notch was first described for its essential role at early stages of T cell development in the thymus and for its involvement in T cell acute lymphoblastic leukemia. In addition, Notch is being increasingly recognized as a potent regulator of antigen-driven mature T cell responses with context-dependent effects. These effects are influenced by the regulated expression of Jagged and Delta-like Notch ligands in antigen-presenting cells under the control of Toll-like receptors and other innate signals. We are investigating the importance of the Notch pathway in T cell alloimmunity in the setting of allogeneic bone marrow or peripheral blood stem cell transplantation (allo-BMT). After allo-BMT, alloimmune T cell responses mediate beneficial graft-versus-tumor (GVT) activity as well as detrimental graft-versus-host disease (GVHD). Using the pan-Notch inhibitor DNMAML and other genetic models of Notch inhibition, we have discovered an essential function for Notch signaling in donor-derived alloreactive T cells. In multiple mouse models of allo-BMT, infusion of Notch-deficient T cells as compared to wild-type T cells led to dramatically reduced GVHD severity and lethality. This effect was apparent for both CD4+ and CD8+ T cells and did not involve global immunosuppression, since Notch-deficient T cells proliferated normally and expanded in host lymphohematopoietic organs. However, Notch-deficient donor T cells failed to accumulate in the gut, a key GVHD target organ, and produced markedly decreased amounts of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. In parallel, Notch inhibition was associated with increased accumulation of FoxP3+CD4+ T cells. Decreased cytokine production could not be explained by a classical T helper differentiation defect. In contrast to their defective induction of GVHD, Notch-deficient T cells remained capable of mediating cytotoxic and anti-tumor responses both in vitro and in vivo, leading to preserved GVT activity even against large numbers of tumor cells. We are currently investigating the molecular and cellular mechanisms of Notch action in alloreactive T cells. We are also exploring the role of individual Notch receptors and ligands at the alloimmune synapse in vivo. Altogether, our findings identify Notch inhibition in donor T cells as a novel strategy to induce beneficial immunomodulation rather than global immunosuppression after allo-BMT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selective anergy of V beta 8+,CD4+ T cells in Staphylococcus enterotoxin B-primed mice.

1990 ◽  
Vol 172 (4) ◽  
pp. 1065-1070 ◽  
Author(s):  
Y Kawabe ◽  
A Ochi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Anergy ◽  
Specific Cytotoxicity ◽  
Enterotoxin B ◽  
Staphylococcus Enterotoxin B

The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.

In Vivo Blockade of Individual Notch Ligands and Receptors Provides a New Targeted Therapeutic Approach In Graft-Versus-Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 819-819
Author(s):  
Ivy T Tran ◽  
Ashley R Sandy ◽  
Alexis Carulli ◽  
Gloria T Shan ◽  
Vedran Radojcic ◽  
...  
Keyword(s):  
T Cells ◽  
Notch Signaling ◽  
Gamma Secretase ◽  
Notch Receptors ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Notch Inhibition ◽  
Total Body ◽  
Notch Ligands

Abstract Abstract 819 Notch signaling is a cell-cell communication pathway with multiple functions in health and disease. Notch ligands of the Delta-like (Dll1, 3, 4) or Jagged (Jagged1, 2) family interact with one of four mammalian Notch receptors (Notch1-4), leading to proteolytic activation of the receptors by gamma-secretase. We have discovered a critical role for Notch signaling in the differentiation of pathogenic host-reactive T cells during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Expression of the pan-Notch inhibitor DNMAML in donor T cells led to markedly reduced GVHD severity, without causing global immunosuppression (Blood 2011, 117(1): 299–308). These findings identify Notch signaling in alloreactive T cells as an attractive therapeutic target after allo-BMT. To explore preclinical strategies of Notch blockade in GVHD, we first assessed the effects of systemic pan-Notch inhibition with gamma-secretase inhibitors. In the B6 anti-BALB/c MHC-mismatched model of allo-BMT, administration of the gamma-secretase inhibitor dibenzazepine was as efficient as genetic strategies at blocking Notch target gene expression and production of inflammatory cytokines in donor T cells (IFN-γ, TNF-α, IL-2). However, dibenzazepine induced severe gastrointestinal toxicity after total body irradiation due to inhibition of both Notch1 and Notch2 in the gut epithelium. To avoid these side effects, we hypothesized that targeting individual Notch receptors or ligands could provide safe therapeutic Notch blockade after allo-BMT. Among the four mammalian Notch receptors (Notch1-4), donor alloreactive T cells expressed Notch1 and Notch2. Host dendritic cells expressed Notch ligands of the Jagged and Delta-like (Dll) families, with markedly increased Dll4 but not Jagged1/2 transcripts after total body irradiation. This suggested that blockade of Notch1 and/or Notch2 in T cells or Delta-like Notch ligands in dendritic cells could abrogate GVHD. To explore this possibility, we used specific monoclonal antibodies to neutralize Notch receptors and ligands in vivo after allo-BMT (Nature 2006, 444(7122):1083–7; Nature 2010, 464(7291): 1052–7). Combined blockade of Notch1 and Notch2 in vivo reduced the production of key inflammatory cytokines by alloreactive CD4+ and CD8+ T cells to a similar extent as DNMAML-mediated pan-Notch inhibition. Inhibition of Notch1 alone led to a large decrease in cytokine secretion, indicating that Notch1 is a dominant non-redundant Notch receptor in alloreactive T cells. Consistently, transplantation of Notch1-deficient but not Notch2-deficient B6 T cells allowed for decreased GVHD and improved survival in BALB/c recipients, similarly to global Notch inhibition by DNMAML. We then studied the consequences of inhibiting Dll1, Dll4 or both Dll1/Dll4 Notch ligands during acute GVHD. Combined Dll1/Dll4 blockade was as potent as DNMAML expression in decreasing cytokine production by alloreactive T cells, demonstrating that Delta-like and not Jagged ligands are the key Notch agonists at the alloimmune synapse. Dll4 inhibition was superior to Dll1 blockade in reducing cytokine production, abrogating GVHD, and prolonging recipient survival. Importantly, combined Dll1/Dll4 inhibition provided long-term protection against GVHD morbidity and mortality, while avoiding severe gastrointestinal side effects from Notch inhibition. Protection was observed even upon transient Dll1/Dll4 blockade during 1–2 weeks after transplantation. Altogether, our data suggest that Notch1 and Dll4 preferentially interact during alloreactive T cell priming and identify novel strategies to safely and efficiently target individual elements of the Notch pathway after allo-BMT. Humanized antibodies against Notch receptors and ligands were designed to block both mouse and human proteins, thus our preclinical work could lead to new strategies for GVHD control in human patients. Disclosures: Shelton: Genentech Inc.: Employment. Yan:Genentech Inc.: Employment. Siebel:Genentech Inc.: Employment.

scholarly journals T cell memory. Long-term persistence of virus-specific cytotoxic T cells.

1989 ◽  
Vol 169 (6) ◽  
pp. 1993-2005 ◽  
Author(s):  
B D Jamieson ◽  
R Ahmed
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Biological Properties ◽  
Antigenic Specificity ◽  
Ctl Response

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.

scholarly journals Delta-like Ligands Expressed By Stromal Cells in Secondary Lymphoid Organs Deliver an Early Pulse of Notch Signaling and Drive T Cell Pathogenicity in Acute Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 841-841
Author(s):  
Jooho Chung ◽  
Christen L. Ebens ◽  
Vedran Radojcic ◽  
Ute Koch ◽  
Ann Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Stromal Cells ◽  
Acute Gvhd ◽  
Hematopoietic System ◽  
Alloreactive T Cells ◽  
Equity Ownership

Abstract Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands results in near-complete protection from acute GVHD in mouse models of allogeneic bone marrow transplantation. Notch-deprived alloreactive T cells proliferate and accumulate in vivo, but produce dramatically reduced levels of the proinflammatory cytokines IFNγ, TNFα and interleukin-2 (IL-2) (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., J Clin Invest 2013). In this study, we sought to: 1) determine the kinetic requirements for Notch signaling in the pathogenesis of acute GVHD; 2) identify the essential cellular compartment that delivers Dll1 and/or Dll4 ligands to incoming alloreactive T cells. In the B6 anti-BALB/c major histocompatibility complex-mismatched model, a single dose of Dll1 and Dll4 blocking antibodies at the time of transplantation abolished alloreactive T cell production of IFNγ, TNFα, and IL-2, increased regulatory T cell numbers (as assessed at day 10), and conferred long-term protection from GVHD. Conversely, delaying antibody administration by only two days after transplantation resulted in persistent T cell cytokine production, no changes in regulatory T cell numbers, and loss of long-term protection from GVHD. These findings identify a critical early window of Notch activity that promotes the pathogenesis of acute GVHD. To identify the dominant cellular source of Dll1 and Dll4, we assessed the impact of Cre-mediated Dll1 and Dll4 inactivation within host hematopoietic, donor hematopoietic, or host non-hematopoietic tissues. Bone marrow chimeras that lacked Dll1 and Dll4 solely within the host hematopoietic system were generated from poly(I:C)-induced Mx1-Cre;Dll1fl/fl;Dll4fl/fl donor mice. Both donor chimerism and Cre-mediated excision efficiency were >97%. Unlike systemic Dll1/4 blockade, Dll1 and Dll4 inactivation within the host hematopoietic system failed to decrease GVHD mortality or severity. Likewise, Mx1-Cre-mediated deletion of Dll1 and Dll4 within the donor hematopoietic system had minimal effects on T cell proinflammatory cytokines. In contrast, Ccl19-Cre-mediated Dll1 and Dll4 inactivation within host stromal cells profoundly impaired donor T cell production of IFNγ, TNFα, and IL-2, and resulted in long-term protection from GVHD. Lineage tracing in Ccl19-Cre x ROSA26-YFP mice revealed Cre activity within a small subset of CD45-negative lymph node and spleen stromal cells, but not in professional hematopoietic antigen-presenting cells. These data suggest that a specialized subset of non-hematopoietic stromal cells delivers an early pulse of Notch signaling to alloreactive T cells during acute GVHD. To our knowledge, these results provide the first in vivo evidence for non-motile secondary lymphoid-resident stromal cells as critical drivers of T cell-mediated immune pathology, with a central role for Notch signaling in this process. Transient interference with Notch ligand function or with their expression by the stromal cell niche in the peri-transplant period could serve as a novel therapeutic strategy for GVHD. Disclosures Yan: Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Gene-Modified CD8+ T Cells Undergo Functional Polarization to Effector and Central Memory Cells in Response to Antigen Exposure.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1531-1531
Author(s):  
Paul Neeson ◽  
Amanda Shin ◽  
Tsin Tai ◽  
Karen Chen ◽  
Joanne Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Effector Memory ◽  
Ifn Gamma ◽  
Lewis Antigen ◽  
Negative Cell Line ◽  
Ley Antigen

Abstract Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:- phenotype (CD45RA, CCR7, CD28, CD27 and perforin); homeostatic cytokine (IL-15/IL-7) proliferation; response to Lewis antigen contact including cell proliferation and cytokine secretion. We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116. Figure 1. LeY T cells have an effector memory phenotype and respond to Lewis antigen expressing cell lines by secreting IFN-gamma. Following the transduction culture, the CD8+ LeY-T cells (A) expressed high levels of perforin and an EM phenotype. In (B), LeY T or empty vector control T cells were co-cultured with tumour cells overnight and intracellular cytokine secretion assay performed. The LeY CD8+ T cells responded to Lewis antigen expressing OVCAR cells by secreting IFN-gamma, whereas no response was observed with the negative cell line HCT-116.

Critical and Opposing Effects of T Cell-Derived TNFα and Interferon-γ on IL-17 Induction Assessed in Vitro and in Vivo Following Allogeneic Bone Marrow Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3482-3482
Author(s):  
Minghui Li ◽  
Kai Sun ◽  
Mark Hubbard ◽  
Doug Redelman ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Allogeneic Bmt

Abstract IL-17-producing CD4 T cells (Th17) are a recently identified T helper subset that plays a role in mediating host defense to extracellular bacteria infections and is involved in the pathogenesis of many autoimmune diseases. In vitro induction of IL-17 in murine CD4+ T cells has been shown to be dependent on the presence of the proinflammatory cytokines TGF-β and IL-6 whereas IFNγ can suppress the development of Th17 cells. In the current study, we examined the roles of TNFα and IFNγ on IL-17 production by purified T cells in vitro and in vivo after allogeneic bone marrow transplantation (BMT). We present findings that expression of TNFα by the T cell itself is necessary for optimal development of Th17 under in vitro polarizing conditions. A novel role for T cell-derived TNFα in Th17 induction was observed when in vitro polarization of Tnf−/−CD4+ T cells resulted in marked reductions in IL-17+CD4+ T cells compared to Tnf+/+CD4+ T cells. In marked contrast, T cell-derived IFNγ markedly inhibited Th17 development as more IL-17+CD4+ T cells were found in Ifnγ−/−CD4+ T cells than in Ifnγ+/+CD4+ T cells, and of particular interest was the dramatic increase in IL-17+CD8+ cells from Ifnγ−/− mice. To determine if T cell-derived TNFα or IFNγ can regulate Th17 development in vivo we examined the differentiation of alloreactive donor T cells following allogeneic BMT. We have found that donor-derived Th17 cells can be found in lymphoid tissues and GVHD-affected organs after allogeneic BMT. However, transfer of Tnf−/− CD4+ T cells after allogeneic BMT resulted in marked reductions in Th17 cells in the spleen (18×103 vs 7×103, P<0.05). In agreement with the in vitro data and in contrast to what was observed with transfer of Tnf−/− CD4+ T cells, transfer of donor Ifnγ−/− T cells resulted in marked increases in not only IL-17+CD4+ but also IL-17+CD8+ T cells infiltrating the liver (7×103 vs 14×103, P<0.05; 4×104 vs 12.5×104, P<0.05). These results suggest that the donor T cell-derived TNFα and IFNγ opposingly regulate IL-17 induction of both CD4+ and CD8+ T cells in vitro and after allogeneic BMT which correlates with GVHD pathology.

Artificial APC-Based Generation of IL-21 Secreting CD4+ T Cells That Can Provide Help to CD8+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 466-466
Author(s):  
Makito Tanaka ◽  
Marcus Butler ◽  
Sascha Ansén ◽  
Osamu Imataki ◽  
Alla Berezovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Binding Assay ◽  
Cell Responses ◽  
Large Numbers ◽  
A Cell

Abstract Abstract 466 CD8+ T cells are thought to be major players in T cell immunity because of their potent direct effector function. However, many studies have demonstrated that CD4+ T cells also play a critical role by providing help which optimizes CD8+ T cell responses. In vivo experiments using murine models have suggested that common cytokine receptor γ-chain cytokines such as IL-2, IL-15 and IL-21 are mediators of this CD4+ T cell help. Previously, we generated K562-based artificial APC (aAPC) by transducing HLA-A2, CD80, and CD83. This aAPC can generate large numbers of antigen-specific CD8+ CTL with a central/effector memory phenotype and potent effector function. These CTL are surprisingly long-lived and can be maintained in vitro without any feeder cells or cloning. We are currently conducting a clinical trial where large numbers of anti-tumor CD8+ CTL generated ex vivo using this aAPC and IL-2/IL-15 are adoptively transferred to patients with advanced cancer. Early results have demonstrated that adoptively transferred anti-tumor CTL can expand and persist as memory T cells for longer than 6 months without lymphodepletion or cytokine administration. Furthermore, some patients have demonstrated objective clinical responses. These in vivo results suggest that K562-based aAPC might serve as a clinically important APC to generate large numbers of antigen-specific T cells for adoptive therapy. Based upon these observations, we have generated a K562-derived aAPC that can expand antigen-specific CD4+ T cells capable of providing help to CD8+ T cells. One challenge with the study of human HLA class II-restricted antigen-specific CD4+ T cells lies in the fact that there is no DR allele with a frequency greater than 25% in any race or ethnic extraction. To overcome this issue, we targeted HLA-DP0401 (DP4), which is positive in 64% of Caucasians and is the most frequent HLA allele in many other ethnic groups. aAPC was generated by sequentially transducing DPA1*0103, DPB1*0401, CD80 and CD83 to HLA class I-, class II-, CD54+, CD58+ K562. Using this aAPC and 57 overlapping peptides encompassing the full-length protein, we identified three DP4-restricted immunogenic epitopes derived from CMV pp65. One of the 3 epitopes, peptide #23 (aa 221-240) appeared to be an immunodominant epitope, since specific CD4+ T cells were expanded from all donors tested. A cell-based in vitro competitive binding assay confirmed that #23 binds DP4 molecules. #23-specific CD4+ T cells generated using aAPC and low dose IL-2/IL-15 were long-lived, up to 4 months in vitro without any feeder cells or cloning, and were able to recognize APC exogenously pulsed with pp65 protein. ELISPOT showed that #23-specific CD4+ T cells were able to secrete IL-2, IL-4, IFN-γbut not IL-10 in an antigen-specific manner. Interestingly, intracellular cytokine staining revealed that a fraction of IFN-γsecreting CD4+ T cells concurrently produced IL-4. Most importantly, using an aAPC expressing HLA-A2, DP4, CD80, and CD83, we were able to demonstrate that pp65-specific CD4+ T cells can provide help to pp65-specific CD8+ T cells in an antigen-specific way. Survivin is an attractive target antigen for tumor immunotherapy, since it is expressed by many tumor types and is indispensable for tumor growth. We have also successfully generated DP4-restricted Survivin-specific CD4+ T cells using this aAPC. Using a cell-based in vitro binding assay, 5 Survivin-derived peptides with high binding capacity to DP4 molecules were identified. Among these 5 peptides, peptide #90 (aa 90-104) bound DP4 most potently. aAPC pulsed with #90 was able to induce antigen-specific CD4+ T cell responses from cancer patients. These CD4+ T cells were also long-lived, up to 3 months in vitro and secreted IL-2, IL-4, and IFN-γbut not IL-10. Interestingly, IL-21 was also produced upon antigen-specific stimulation. It should be noted that our K562-based aAPC did not expand Foxp3+ regulatory T cells under the experimental conditions tested. Taken all together, we have established a K562-based aAPC to generate large numbers of HLA-DP4-restricted antigen-specific CD4+ T cells that possess longevity and functional competence. Based upon our previous success in clinical translation of K562-based aAPC for CD8+ T cells and the high prevalence of HLA-DP4, generating a clinical grade version of this aAPC for CD4+ T cells is of high priority. Disclosures: No relevant conflicts of interest to declare.

Redirected CD4+ T Cells towards HLA Class I-Restricted WT1 Epitope Successfully Display Multifactorial Helper Function for the Enhancement of Antileukemia Efficacy Mediated by Redirected T-Cell Based Immunocelltherapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3153-3153
Author(s):  
Yukihiro Miyazaki ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Sachiko Okamoto ◽  
Hiroaki Asai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Proliferation And Differentiation ◽  
Ifn Γ

Abstract Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

Pharmacological Inhibition of Hsp90 Destabilizes the Histone Methyltransferase Ezh2 in Alloreactive T Cells and Reduces Graft-Versus-Host Disease While Retaining Anti-Leukemic Effects in Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 234-234
Author(s):  
Qingrong Huang ◽  
Shan He ◽  
Yuanyuan Tian ◽  
Changhong Li ◽  
Yuting Gu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Activated T Cells ◽  
Pharmacological Inhibition ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Genetic Deletion ◽  
And Function

Abstract Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD involves complex interactions of immune cells, induction of host-reactive donor effector T cells, and donor T cell-mediated injury to normal tissues. Epigenetic changes have been implicated in T cell-mediated GVHD. We previously described that genetic deletion of Ezh2, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), reduced GVHD in mice but preserved graft-versus-leukemia (GVL) responses. Several selective inhibitors of Ezh2 have been recently discovered (e.g. GSK126, UNC1999 and EPZ6438), which specifically reduce the levels of H3K27me3 but not EZH2 protein. Unexpectedly, our preliminary studies showed that administration of GSK126 failed to prevent GVHD in mice. This stands in contrast to our findings that genetic deletion of T cell Ezh2 leads to GVHD inhibition, and suggest that Ezh2 may regulate GVHD through a mechanism independent of H3K27me3. Identifying an optimal method to target T cell Ezh2 for controlling GVHD remains an unmet need. Using experimental mouse models, we demonstrate that functional heat shock protein (Hsp)90 is critical for maintaining Ezh2 protein stability and function in activated T cells. Pharmacological inhibition of Hsp90 destablizes Ezh2 protein in alloreactive T cells, reduces GVHD but preserves GVL effects in mice. To determinethe molecule(s) that is critical for maintaining Ezh2 protein stablility in T cells, we performed mass spectrum (MS) analysis and identified 25 Ezh2-interacting proteins that showed higher intensities than others in T cell receptor (TCR)-activated CD8+ T cells. Among them, we found a group of proteins associated with protein folding and degradation, including Hsp90. Hsp90 is a molecular chaperone required for the stability and function of several key signaling intermediates (e.g., AKT, Raf1 and ERK1/2). Using reciprocal co-immunoprecipitation assay, we confirmed that Ezh2 and Hsp90 directly interacted with each other in TCR-activated CD8+ T cells. Pharmacological inhibition of Hsp90 using its specific inhibitor AUY922, which is currently in phase II clinical trials for cancer therapy, effectively reduced Ezh2 protein without decreasing H3K27me3 24 hours after treatment. This effect was accompanied by decreased proliferation and survival of TCR-activated T cells in vitro. Retroviral overexpression of Ezh2 in T cells markedly improved their proliferation in the presence of AUY922, suggesting that reducing Ezh2 by Hsp90 inhibition is an important mechanism that reduces proliferation and survival of activated CD8+ T cells. Building on these observations, we examined the impact of inhibiting Hsp90 on GVHD by administering AUY922 to B6 mice receiving MHC-identical minor histocompatibility antigen-mismatched C3H.SW mouse CD8+ T cells and T cell-depleted bone marrow (BM). While about 80% of control B6 recipients died from severe GVHD, 80% of AUY922-treated B6 recipients survived without clinical signs of severe GVHD by 84 days after transplantation. In vivo AUY922 administration reduced the survival and expansion of alloreactive T cells, and decreased the fequency of alloreactive T effector cells producing IFN-g and TNF-a. To rule out the model-specific effect of AUY922, we used a haplo-identical B6 into BDF1 mouse model of GVHD. Using CFSE-labeled donor T cells, we first validated that in vivo administration of AUY922 to unirradiated BDF1 mice receiving parent B6 T cells selectively reduced the expansion of alloantigen-reactive donor T cells, but did not impair the expansion and survival of donor T cells that did not respond to alloantigens. In lethally irradiated BDF1 mice receiving B6 T cells and BM, AUY922 administration reduces lethal GVHD, with approximately 50% of them surviving long-time. Importantly, AUY922 treatment preserved GVL activity of donor T cells, leading to significantly improved survival of BDF1 recipients challenged with A20 leukemic cells (Fig.1). Taken together, our findings identified a previously unrecognized molecular mechanism by which Ezh2 and Hsp90 are integrated to regulate alloreactive T cell responses and GVHD. Targeting the Ezh2-Hsp90 complex using AUY922 represents a novel and clinically relevant approach to reduce GVHD while preserving GVL effects, thereby improving the efficacy of allo-HSCT. Disclosures No relevant conflicts of interest to declare.

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