scholarly journals Mertk Inhibition Promotes Anti-Leukemia Immunity By Reversing T Cell Suppression Via the PD-1 Axis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4019-4019
Author(s):  
Alisa Lee-Sherick ◽  
Kristen Jacobsen ◽  
Curtis J. Henry ◽  
Rebecca Parker ◽  
Xiaodong Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Suppression ◽  
Tnf Α ◽  
Equity Ownership ◽  
Cell Suppression

Abstract The efferocytic receptor MerTK is expressed on monocytes/macrophages and facilitates tolerogenic T cell suppressive clearance of apoptotic cellular debris. Given the rapid cell turn-over in leukemia, we hypothesized that ongoing efferocytosis is leukemia-permissive and inhibition of MerTK on monocytes/macrophages will reverse T cell suppression leading to enhanced anti-leukemia immunity. To test this hypothesis, we inoculated mice with syngeneic murine BCR-ABL p185 acute lymphoblastic leukemia (ALL) cells, which do not express MerTK. The effects of MerTK inhibition were evaluated in wild-type (WT) mice treated daily with MRX-2843, a MerTK small molecule inhibitor, and in mice with a MerTK knockout mutation (Mertk-/-). Median survival was prolonged in WT mice treated with MRX-2843 (40 days) compared to vehicle treatment (20 days, p<0.01). In littermate Mertk-/- mice, all but one mouse survived without evidence of leukemia 150 days after inoculation (p<0.0001). To explore the T cell suppressive effects of MerTK inhibition on CD11b+ monocytes/macrophages, we used flow cytometry to study ex vivo mixed cell cultures using Mertk-/- or WT murine splenocytes +/- ALL cells treated +/- MRX-2843. Co-culture with ALL cells for 24 (36.0%), 48 (34.9%) and 72 (36.6%) hours resulted in a significant increase in expression of co-inhibitory ligands PD-L1 and PD-L2 on CD11b+ cells compared to cultures without ALL cells (2.03%, 3.2% and 7.5%, p<0.0001), or cultures with ALL cells treated with MRX-2843 (8.3%, 15.3% and 9.5%, p<0.0001). Similarly, PD-L1/PD-L2 co-expression on CD11b+ cells from Mertk-/- mice was not significantly altered in the presence (16.0%) or absence (7.0%) of ALL. Furthermore, there was no significant change in PD-L1/PD-L2 expression when Mertk-/- co-cultures were treated with MRX-2843 (12.6%). These findings were recapitulated in vivo. There was no change in PD-L1/PD-L2 expression on CD11b+ cells from Mertk-/- mice inoculated with ALL (4.8% spleen; 0.8% marrow) compared to uninoculated Mertk-/- mice (7.2% spleen; 1.2% marrow) or uninoculated WT littermates (2.0% spleen; 0.7% bone marrow), whereas CD11b+ cells from WT mice inoculated with ALL demonstrated a significant upregulation of PD-L1/PD-L2 (24.1% spleen, p<0.0001; and 4.2% bone marrow, p<0.001), which was downregulated upon treatment with MRX-2843 (1.9% spleen, p<0.01; 1.2% bone marrow, p<0.05) In these experiments, expression of PD-1, the co-inhibitory receptor which binds PD-L1/PD-L2, on T cells was also assessed. In WT mice inoculated with ALL, expression of PD-1 on T cells from spleens (74.2% CD4+; 49.2% CD8+) and bone marrow (65.9% CD4+; 73.0% CD8+) was significantly increased compared to spleens (10.0% CD4+; 4.1% CD8+) and bone marrow (36.4% CD4+; 8.5% CD8+) from uninoculated control mice (p<0.0001). However, expression of PD-1 was not significantly different in spleens (28.3% CD4+; 7.2% CD8+) and bone marrow (66.2% CD4+; 29.2% CD8+) from Mertk-/- mice inoculated with ALL compared with spleens (20.0% CD4+; 4.7% CD8+) and bone marrow (62.2% CD4+; 12.4% CD8+) from uninoculated Mertk-/- mice. To determine whether changes in PD-1 expression were indicative of T cell activation or exhaustion, T cells treated with α-CD3 beads were added to splenocyte and ALL cell co-cultures for 24 hours and intracellular IFN-ɣ and TNF-α levels were assessed using flow cytometry. The fraction of T cells producing IFN-ɣ/TNF-α significantly increased in co-cultures treated with MRX-2843 (44.2% CD4+; 51.1% CD8+) compared to vehicle-treated co-cultures (27.4% CD4+, p<0.0001; 36.8% CD8+, p<0.0001). Similarly, IFN-ɣ/TNF-α production increased in co-cultures with Mertk-/- splenocytes and ALL cells (48.3% CD4+, p<0.0001; 51.2% CD8+, p<0.0001) compared to co-cultures with WT splenocytes and activation was not further augmented by treatment with MRX-2843 (48.9% CD4+, 51.5% CD8+). Similar results were obtained when T cells from Mertk-/-mice were used, demonstrating that the effects of T cell activation were dependent on the presence of MerTK on CD11b+ cells, but not T cells. In conclusion, inhibition of MerTK on CD11b+ monocytes/macrophages reversed T cell suppression in response to leukemia via the PD-1 axis. MerTK inhibitors are in early phase clinical trials and these data demonstrate a potential immunotherapeutic use for MerTK inhibitors in the treatment of ALL. Disclosures Wang: Meryx: Equity Ownership, Patents & Royalties. Frye:Meryx: Equity Ownership, Patents & Royalties. Earp:Meryx: Equity Ownership. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership.

scholarly journals Both Maturation and Survival of Human Dendritic Cells are Impaired in the Presence of Anergic/Suppressor T Cells

2003 ◽  
Vol 10 (1) ◽  
pp. 61-65 ◽  
Author(s):  
L. Frasca ◽  
C. Scottà ◽  
G. Lombardi ◽  
E. Piccolella
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Costimulatory Molecule ◽  
T Cell Suppression ◽  
Cell Suppression ◽  
Anergic T Cells ◽  
Human Dendritic Cells

T cell suppression is a well established phenomenon, but the mechanisms involved are still a matter of debate. Mouse anergic T cells were shown to suppress responder T cell activation by inhibiting the antigen presenting function of DC. In the present work we studied the effects of co-culturing human anergic CD4+T cells with autologous dendritic cells (DC) at different stages of maturation. Either DC maturation or survival, depending on whether immature or mature DC where used as APC, was impaired in the presence of anergic cells. Indeed, MHC and costimulatory molecule up-regulation was inhibited in immature DC, whereas apoptotic phenomena were favored in mature DC and consequently in responder T cells. Defective ligation of CD40 by CD40L (CD154) was responsible for CD95-mediated and spontaneous apoptosis of DC as well as for a failure of their maturation process. These findings indicate that lack of activation of CD40 on DC by CD40L-defective anergic cells might be the primary event involved in T cell suppression and support the role of CD40 signaling in regulating both activation and survival of DC.

scholarly journals CCL19 enhances CD8+ T-cell responses and accelerates HBV clearance

2021 ◽  
Author(s):  
Yan Yan ◽  
Wei Zhao ◽  
Wei Liu ◽  
Yan Li ◽  
Xu Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Crucial Role ◽  
Cell Activation ◽  
Hbv Infection ◽  
Host Immune System ◽  
Tnf Α ◽  
Ifn Γ ◽  
High Level

Abstract Background Chemokine (C–C motif) ligand 19 (CCL19) is a leukocyte chemoattractant that plays a crucial role in cell trafficking and leukocyte activation. Dysfunctional CD8+ T cells play a crucial role in persistent HBV infection. However, whether HBV can be cleared by CCL19-activated immunity remains unclear. Methods We assessed the effects of CCL19 on the activation of PBMCs in patients with HBV infection. We also examined how CCL19 influences HBV clearance and modulates HBV-responsive T cells in a mouse model of chronic hepatitis B (CHB). In addition, C–C chemokine-receptor type 7 (CCR7) knockdown mice were used to elucidate the underlying mechanism of CCL19/CCR7 axis-induced immune activation. Results From in vitro experiments, we found that CCL19 enhanced the frequencies of Ag-responsive IFN-γ+ CD8+ T cells from patients by approximately twofold, while CCR7 knockdown (LV-shCCR7) and LY294002 partially suppressed IFN-γ secretion. In mice, CCL19 overexpression led to rapid clearance of intrahepatic HBV likely through increased intrahepatic CD8+ T-cell proportion, decreased frequency of PD-1+ CD8+ T cells in blood and compromised suppression of hepatic APCs, with lymphocytes producing a significantly high level of Ag-responsive TNF-α and IFN-γ from CD8+ T cells. In both CCL19 over expressing and CCR7 knockdown (AAV-shCCR7) CHB mice, the frequency of CD8+ T-cell activation-induced cell death (AICD) increased, and a high level of Ag-responsive TNF-α and low levels of CD8+ regulatory T (Treg) cells were observed. Conclusions Findings in this study provide insights into how CCL19/CCR7 axis modulates the host immune system, which may promote the development of immunotherapeutic strategies for HBV treatment by overcoming T-cell tolerance.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

scholarly journals Akt Fine-tunes NF-κB-dependent Gene Expression during T Cell Activation

2011 ◽  
Vol 286 (41) ◽  
pp. 36076-36085 ◽  
Author(s):  
Jing Cheng ◽  
Binh Phong ◽  
David C. Wilson ◽  
Raphael Hirsch ◽  
Lawrence P. Kane
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Profiling ◽  
Leukocyte Activation ◽  
Mrna Transcription ◽  
Tnf Α ◽  
Fine Tune

Activation of the NF-κB signaling pathway is critical for leukocyte activation and development. Although previous studies suggested a role for the Akt kinase in coupling the T cell antigen receptor and CD28 to NF-κB activation in T cells, the nature of the role of Akt in this pathway is still unclear. Using a targeted gene profiling approach, we found that a subset of NF-κB-dependent genes required Akt for optimal up-regulation during T cell activation. The selective effects of Akt were manifest at the level of mRNA transcription and p65/RelA binding to upstream promoters and appear to be due to altered formation of the Carma1-Bcl10 complex. The proinflammatory cytokine TNF-α was found to be particularly sensitive to Akt inhibition or knockdown, including in primary human blood T cells and a murine model of rheumatoid arthritis. Our findings are consistent with a hierarchy in the expression of NF-κB-dependent genes, controlled by the strength and/or duration of NF-κB signaling. More broadly, our results suggest that defining the more graded effects of signaling, such as those demonstrated here for Akt and the NF-κB pathway, is important to understanding how cells can fine-tune signaling responses for optimal sensitivity and specificity.

scholarly journals Rapid Flow Cytometry Method for Quantitation of LFA-1-Adhesive T Cells

2006 ◽  
Vol 13 (3) ◽  
pp. 403-408 ◽  
Author(s):  
Brian Crucian ◽  
Mayra Nelman-Gonzalez ◽  
Clarence Sams
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Peripheral Blood Mononuclear ◽  
Simple Method ◽  
Rapid Flow

ABSTRACT Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an “adhesive” state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-μm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19− CD16− CD45RO+ CD62L+ CD27+ CD57−. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1141-1141
Author(s):  
Elena E. Solomou ◽  
Valeria Visconte ◽  
Federica Gibellini ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Failure ◽  
Protein Levels ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p&lt;0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p&lt;0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene’s regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

Targeting The T Cell Component Of The Tumour Microenvironment In Chronic Lymphocytic Leukaemia; A Potential Therapeutic Strategy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4147-4147
Author(s):  
Kirsty M Cuthill ◽  
Andrea Gail Sherman Buggins ◽  
Pj Chana ◽  
Stephen Devereux
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Lymphocytic Leukaemia ◽  
Nuclear Translocation ◽  
Tumour Microenvironment

Abstract It has recently become clear that B cell receptor (BCR) activation plays an important role in the pathogenesis of chronic lymphocytic leukaemia (CLL); a fact that is underlined by the marked efficacy of drugs that inhibit components of this pathway. Although the underlying mechanisms remain unclear, CLL BCRs have been shown to recognize a variety of autoantigens and there is evidence of ongoing activation of a number of downstream signaling molecules including Syk, Erk, Akt and the NFkB and NFAT family of transcription factors. In addition to BCR activation, it is thought that signals from other cells in the tumour microenvironment such as T cells, the vascular endothelium and other stromal cells may also play a role in promoting the growth of the disease. In the present study we chose to revisit the effects of ciclosporin (CsA), a calcineurin antagonist with effects on antigen receptor signaling, in CLL. When this agent is used to treat the autoimmune complications of CLL, concurrent responses in the underlying disease have been noted in about 20% of patients, although the underlying mechanism has not been thoroughly investigated. Since CsA primarily inhibits T cell activation we hypothesized that its effects in CLL might be due to a reduction in T cell mediated co-stimulation in the lymph nodes. We therefore investigated the effect of CsA on the activation of CLL B and T cells using conventional and multispectral imaging flow cytometry to measure the expression of activation markers and the nuclear translocation of NFAT and NFKB family transcription factors. Cells were collected from eight unselected patients with a confirmed diagnosis of CLL for each study. T and B cells were purified by negative immunomagnetic selection and activated by incubation with phorbol ester and ionomycin (PMA/I) or CD40L transfected fibroblasts in the presence of absence of CsA. The activation of CD4+ T cells and CD19+ CLL cells was assessed by staining for CD69/interferon gamma (IFNΥ) and CD69/CD25 respectively. Nuclear translocation of NFATc2 and NFKB p65 was measured by image flow cytometry (Amnis Imagestream). Leukaemia and Lymphoma Research provided the funding for this study. NFkB(p65) translocation at 30 minutes was inhibited by a mean of 22.5% (p=0.0003) in activated CLL CD4+ T cells treated with CsA compared to those treated with vehicle control (VC). Similarly, in the presence of CsA, NFAT-c2 translocation was inhibited by a mean of 24.3% (p=0.008) at 10 minutes in CLL CD4+ T cells compared to those treated with VC. NFkB(p65) translocation was not inhibited (mean of differences=0.63%, p=0.645) and NFAT-c2 translocation was minimally inhibited (mean of differences = -4%, p = 0.007) in activated CLL B Cells treated with CsA. The proportion of activated CLL CD4+ T cells expressing both CD69 and IFNΥ was reduced by 13.2% (p=0.003) in the presence of CsA whereas there was no inhibition of CD25(-1.5, p=0.16) and CD69(-1.4, p=0.5) expression in activated CLL B cells treated with CsA. In summary, CsA had a profound effect on CD4+ T cell activation in patients with CLL, as demonstrated by the reduction in NFkB (p65), NFAT-c2 nuclear translocation and CD69/IFNΥ expressing cells. In contrast, there was a minimal effect on NFAT-c2 translocation in activated CLL B cells and no impact on NFkB (p65) translocation or the expression of CD25 and CD69. These findings suggest that the previously documented activity of CsA in CLL is not due to a direct effect on the tumour but is instead indirect and mediated through inhibition of other microenvironment derived signals such as those provided by activated CD4+ T cells. Since it is likely that these co-stimulatory effects act in concert other signals, such as those induced by BCR activation, reexamination of CsA and similar agents in CLL would thus seem warranted. Disclosures: No relevant conflicts of interest to declare.

Characterisation of Human CD83 Expression on Immune Cells and Their Targeting with CD83 Antibodies to Prevent Graft Versus Host Disease in Allogeneic Haematopoietic Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3081-3081
Author(s):  
Derek NJ Hart ◽  
Xinsheng Ju ◽  
Zehra Elgundi ◽  
Nirupama Verma ◽  
Pablo Silveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Splice Variants ◽  
Scid Mice ◽  
Human Pbmc ◽  
Graft Versus Host ◽  
Equity Ownership

Abstract Introduction: CD83 is an important marker of activated dendritic cells (DC) but it is also expressed on other immune cells. Polyclonal anti-CD83 antibody depletes activated DC and prevents human peripheral blood mononuclear cell (PBMC) induced xenogeneic graft versus host disease (GVHD) in immunosuppressed SCID mice (J Exp Med 2009;206;387). We therefore generated a potential therapeutic human anti-CD83 mAb (3C12C), which had similar efficacy and T cell sparing effects in the same model (Leukemia 2015; in press). To investigate the specific immunosuppressive effect of 3C12C further, we undertook a comprehensive analysis of CD83 expression and its glycosylation pattern on various immune cell populations and tested the effect of 3C12C on T cell function using preclinical models, including a human CD83 (hCD83) knock in (KI) mouse. Methods: A panel of mouse and recombinant mAbs to hCD83 were used to analyse its expression by flow cytometry on resting and activated healthy donor PBMC. The expression of potential CD83 splice variants was examined by PCR. T cell expression was examined by flow cytometry and confocal microscopy after PHA, CD3/CD28 beads and allogeneic mixed leukocyte reaction (alloMLR) culture. Control human IgG1 (trastuzumab) and 3C12C mAbs were tested (0.125mg d-1) in a xenogeneic model of GVHD utilizing human PBMC transplanted into total body irradiation and anti-NK conditioned SCID mice. The genetically engineered hCD83 KI mouse was shown to be immune-competent and used to test the effect of 3C12C on LPS activated DC and T cells. Results: There were distinct CD83 splice variants (full length CD83, splicing variant CD83a, CD83b and CD83c) in different immune cells. CD83 glycosylation status also differed with high glycosylation required for surface expression on activated DC, whereas its expression on activated B cells and monocytes was resistant to de-glycosylation. Increases in CD83 expression on T cells occurred early with different kinetics, underlining the distinct signal pathway involved. The 3C12C mAb reduced T cell proliferation in the alloMLR but did not affect cytomegalovirus (CMV) or influenza (Flu) specific CD8+T cell numbers. Treatment with 3C12C prevented GVHD in human PBMC transplanted SCID mice, which otherwise developed histological GVHD between d8-13. Human DC were activated by d2 and expressed the CMRF-44 activation marker plus CD83, CD80 and CD86. Treatment with 3C12C mAb eliminated CD83+ CMRF44+ DC early post-transplant and reduced T cell activation. Further studies, established CMV and Flu specific T cells were retained and responded to antigen by IFNg production. Furthermore, Treg numbers were preserved. The 3C12C mAb depleted LPS activated DC in hCD83 KI mice in experiments performed prior to commencing transplant studies. Conclusion: These findings suggest that the potential therapeutic human anti-CD83 mAb induced significant immune suppression, by depletion of activated DC and consequential modulation of T cell activation. The reduction in allo/xeno activated T cells may result in part from a direct effect of anti-CD83 on early T cell responses. This apparently selective immunosuppressive effect preserves anti-viral T cell immunity and Treg pathways, suggesting that 3C12C merits further investigation as a novel agent for GVHD prophylaxis. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

Presence of Vasoactive Intestinal Polypeptide Signaling in Plasmacytoid Dendritic Cells Improves Survival in a Murine Allo-BMT Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2154-2154
Author(s):  
Jing-Xia Li ◽  
Jian-Ming Li ◽  
Edmund K Waller
Keyword(s):  
Bone Marrow ◽  
Weight Loss ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Expansion ◽  
Cell Activation ◽  
Post Transplant ◽  
Cytokine Analysis

Abstract Introduction: Pre-clinical murine experiments and clinical data from allogeneic bone marrow transplantation (allo-BMT) have shown that increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft results in better clinical outcomes with less severe graft-versus-host disease (GvHD) and improved survival. The mechanism by which donor pDC modulate GvHD is unknown. Knowing that vasoactive intestinal polypeptide (VIP) is an immunosuppressive peptide , we reasoned that VIP signaling might play a role in regulating T-cell activation and expansion, and the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-BMT. We have tested the hypothesis that VIP synthesis by donor pDCs can modulate T cell alloreactivity. Methods: To explore the mechanisms by which pDC and VIP signaling regulate T cell activation in murine allo-BMT, we prepared B6-background donor cell grafts and transplanted them into lethally irradiated B10.BR recipients. In experiment 1, recipients were transplanted with grafts containing the combination of 5 x 103 VIP-GFP hematopoietic stem cells (HSC) and 3 x 106 VIP-wild type (VIP-WT) or VIP-knock out (VIP-KO) splenocytes. At day 7, splenocytes were isolated for flow cytometric analysis looking for GFP signal, which represents VIP-promotor activity. Experiment 2 used combinations of 5 x 103 VIP-WT HSC, 1 x 106 luciferase+ T cells, and 50 x 103 VIP-WT or VIP-KO pDC from B6 as donor grafts. Recipients were monitored for survival and GvHD based on fur texture, posture, activity, skin integrity and weight loss. T cell expansion was measured by bioluminescent imaging (BLI). Serum cytokines from bleeds at day 3 and day 8 post-transplant were analyzed using a Luminex 38 plex panel. Some recipients were euthanized on day 3 for intracellular cytokine analysis of splenic T cells. Results: In experiment 1, 7 days post-transplant, analysis of splenocytes from all mice showed increased activity of the VIP gene promoter in donor pDC that were derived from HSC, compared to other cell types. The VIP promoter signal was also stronger in donor HSC-derived pDCs, if originally transplanted with VIP-KO splenocytes. In experiment 2 over 70% of mice receiving HSC+T+VIP-WT pDC in the BM graft survived to day 100 post-transplant, while those getting VIP-KO pDC instead only had 30% survival (Fig 1A). All surviving recipients were fully engrafted by day 30. BLI revealed greater total T-cell proliferation (measured as radiance) in recipients of VIP-KO pDC (Fig 1B). Furthermore, recipients of VIP-KO pDC had more severe acute GvHD, with increased weight loss and GvHD clinical scores (Fig 1C, 1D). Some recipients were euthanized and their serum were collected for cytokine analysis on day 8 post-transplant, which showed up-regulation of pro-inflammatory or chemotactic cytokines MCP1, IL-1, IL-12, IL-17 in T cells co-transplanted with VIP-KO pDC compared to WT pDC. Conclusion: The present findings show that: 1) VIP is produced by donor pDC early after allo-BMT; 2) absence of VIP production by donor pDC leads to increased T-cell expansion in a murine allo-BMT model. Thus the pDC-T cell VIP signaling pathway is a critical element in controlling donor T cell alloreactivity after allo-BMT. Future studies will include VIP qPCR to confirm VIP production in donor pDC post-transplant, and determine the mechanism by which VIP production by pDC regulates T cell activity and modulates GvHD. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2525-2525
Author(s):  
Paul M Tyler ◽  
Mariah M Servos ◽  
Boris Klebanov ◽  
Trinayan Kashyap ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
T Cell Development ◽  
Immune Homeostasis ◽  
Dosing Regimen ◽  
Employment Equity ◽  
Equity Ownership

Abstract Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016). Disclosures Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.

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