Chronic Lymphocytic Leukemia Cells Co-Opt CD200, CD270, CD274 and CD276 to Induce Impaired Actin Polarization At the T Cell Immune Synapse

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 802-802
Author(s):  
Alan G. Ramsay ◽  
Andrew J. Clear ◽  
Alexander Davenport ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Direct Contact ◽  
Actin Polymerization ◽  
Flow Cytometric Analysis ◽  
Cd8 T Cell ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric ◽  
Cell Synapse

Abstract Abstract 802 We have previously demonstrated that impaired formation of the T cell immunological synapse in response to autologous (auto) antigen-presenting cells (APCs) is a global immunosuppressive mechanism in chronic lymphocytic leukemia (CLL) (J Clin Invest. 2008;118(7):2427-2437). Polymerization of F-actin beneath the area of the T cell:APC contact site generates a structural support for signaling molecules to assemble and regulate appropriate CD4+ T cell activation and cytolytic CD8+ T cell (CTL) effector function. Importantly, direct contact interaction with tumor cells was shown to induce defective actin polarization at the synapse in previously healthy allogeneic (allo) peripheral blood (PB) T cells. Here we have extended our functional screening coculture assays and show that CD200, CD270 (TNF receptor, TNFR-superfamily 14, SF14), CD274 (programmed death ligand 1, PD-L1), and CD276 (B7-H3) are co-opted by primary CLL cells (n=25) to induce impaired actin polymerization at the CD3+ T cell synapse. Antibody neutralizaton of these CLL ligands significantly increased allo T cell synapse actin polymerization with APCs compared to isotype control treated cells (P<.01). Counteracting the combined activity of all four inhibitory proteins on CLL cells showed the largest increase in F-actin synapse polymerization. Importantly, we further demonstrate that direct contact coculture with CLL cells further augmented F-actin polymerization defects in auto PB patient T cells (isolated from low white blood cell count CLL patients), that was prevented by the prior blockade of these CLL inhibitory ligands (P<.01). Next we analyzed the in situ expression of inhibitory ligands and receptors by immunohistochemistry using a CLL lymphoid tissue microarray (TMA). Significantly higher expression of CD200+ CD270+ CD274+ CD276+ CD20+ CLL cells, and CD272+ (B and T lymphocyte attenuator, BTLA) CD279+ (PD-1) CD3+ T cells were detected compared to healthy counterpart cells from reactive control lymph node samples (P<.0001). Notably, higher expression of CD200+ CD274+ CLL cells correlated with poor disease outcome (P<.01). Flow cytometric analysis of peripheral blood patient cells showed that these inhibitory ligands were up-regulated on circulating CLL cells and also their receptors on auto T cells compared to age-matched healthy donor cells (P<.05). Next we investigated the impact of lenalidomide on CLL immunosuppressive signaling interactions with T cells. Both pretreatment of CLL cells with lenalidomide prior to primary coculture and direct addition of drug significantly increased (P <.01) subsequent allo T cell F-actin synapse polarization compared to control treated experiments. Flow cytometric analysis identified that lenalidomide downregulated the expression of these CLL inhibitory ligands and cognate receptors on allo T cells during intercellular contact interactions (P <.01), but not when age-matched healthy B cells were used. We next investigated the effect on cytolytic synapse function and demonstrated that allo CD8+ T cell killing function was significantly enhanced (P <.05) following combinational antibody blockade of CLL inhibitory ligands or lenalidomide treatment compared to control treated leukemic cells. Importantly, lenalidomide treatment blocked further augmented synapse impairment in auto T cells from CLL patients following coculture with CLL tumor cells. As members of the Rho family of GTPases, including RhoA, Rac1 and Cdc42 have been described as key regulators of actin polymerization, we measured their activated GTP-bound state in T cells following direct-contact interaction with CLL tumor cells. We demonstrate decreased active RhoA and Rac1 levels in TCR-stimulated allo T cells on coculture with CLL cells compared with primary coculture with healthy B cells (P <.05). In contrast, combinational antibody blockade of the CLL inhibitory ligands or lenalidomide treatment increased T cell Rho GTPase activity including Cdc42 (P <.05). In conclusion, our findings identify a new mechanism of cancer immunoescape in which CLL tumor cells co-opt multiple inhibitory B7-related molecules that can mediate global immunosuppressive actin defects in both auto and allo T cells. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Functional Screening Studies Identify Combinational Activity of PD-L1 and CD200 In Mediating Dysfunctional T Cell Immunological Synapse Formation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 696-696
Author(s):  
Alan G. Ramsay ◽  
Andrew James Clear ◽  
Alexander Davenport ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Suppression ◽  
Neutralizing Antibodies ◽  
Synapse Formation ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Immune Synapse ◽  
Cell Synapse

Abstract Abstract 696 The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1738-1738
Author(s):  
Benedetta Apollonio ◽  
Mariam Fanous ◽  
Mohamed-Reda Benmebarek ◽  
Stephen Devereux ◽  
Patrick Hagner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Lymphocytic Leukemia ◽  
Immune Synapse ◽  
Cell Synapse

Abstract Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse. Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01). In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. Disclosures Hagner: Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

scholarly journals Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer

Blood ◽  
2012 ◽  
Vol 120 (7) ◽  
pp. 1412-1421 ◽  
Author(s):  
Alan G. Ramsay ◽  
Andrew J. Clear ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Immune Evasion ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Small Gtpases ◽  
Lymphocytic Leukemia ◽  
Functional Screening ◽  
Immunologic Synapse

Abstract Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand–induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell–inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.

scholarly journals T-Cell Large Granular Lymphocytic Leukemia with Extremely Rare Immunophenotype (CD4/CD8 Double-Positive) Followed by Multiple Myeloma Diagnosis

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Dina Soliman ◽  
Sherin Sallam ◽  
Susanna Akiki ◽  
Deena Mudawi ◽  
Feryal Ibrahim
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Viral Infections ◽  
Cytotoxic T Cells ◽  
Flow Cytometric Analysis ◽  
Lymphocytic Leukemia ◽  
Double Positive ◽  
Large Granular Lymphocytic Leukemia ◽  
First Case

T-cell large granular lymphocytic leukemia is characterized by clonal expansion of a CD3+/CD57+ subpopulation, which are typically CD8+ positive cytotoxic T- cells, and can only be diagnosed if there is a persistent, greater than 6 months, elevation of LGL in the blood (usually 2–20 × 109/L), in the absence of an identifiable cause. T-LGLL has been associated with reactive conditions such as autoimmune diseases and viral infections and has also been reported in association with hematologic and non-hematologic malignancies. We report a case of asymptomatic CD4/CD8 double-positive T-LGLL. Flow cytometry on peripheral blood revealed a subpopulation of CD4/CD8 double-positive T cells expressing CD57 and cTIA. Clonality was established by flow cytometric analysis of T-cell receptor V(â) region repertoire which showed that >70% of the cells failed to express any of the tested V(â) regions. Clonality was further confirmed by PCR with the detection of clonal TCR beta and TCR gamma gene rearrangements. Six months later, she presented with persistent lower back pain and diagnosed with IgG kappa multiple myeloma. CD4/CD8 double-positive T-large granular leukemia is the first case reported in the literature. This rare phenotype is either underreported or a truly rare clinical entity. More studies are warranted to characterize the pathogenesis and clinical characteristics of this group of patients and to further assess the relationship between multiple myeloma and T-LGLL as a cause-and-effect relationship or simply related to the time at which diagnosis has been made.

T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5174-5174
Author(s):  
Olga Y. Azhipa ◽  
Scott D. Rowley ◽  
Michele L. Donato ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cell Responses ◽  
Flow Cytometric ◽  
Complexity Index

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

The Plant-Derived Agent Silvestrol Has B-Cell Selective Activity In Vitro in Chronic Lymphocytic Leukemia Patient Cells and In Vivo in the Tcl-1 Mouse Model of CLL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3123-3123 ◽  
Author(s):  
David M. Lucas ◽  
Ryan B. Edwards ◽  
Michael D. De Lay ◽  
Derek A. West ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Tumor Cells ◽  
Lymphocytic Leukemia ◽  
Pathological Examination ◽  
Cell Percentage ◽  
Leukocyte Counts

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options, especially for high-risk populations such as the del(17p13) patient subset. Currently available therapies for CLL, even if effective, can have significant detrimental effects on remaining T cells, leaving patients at risk of potentially lethal opportunistic infections. New agents with unique mechanisms of action, independence of key resistance pathways, and selectivity for tumor cells are crucial to make an impact on patient survival. Silvestrol, a structurally unique compound isolated from the plant genus Aglaia, exhibited potent activity against several tumor cell lines and moderate in vivo activity in the P388 mouse leukemia model (J. Org. Chem. 2004, 69:3350; ibid. 69:6156). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. The LC50 (concentration lethal to 50% of cells relative to untreated control) of silvestrol was 6.5 nM at 72 hours by MTT assay. We performed assays to determine CLL patient cell viability at 72 hours with or without drug washout at various times. In these studies, silvestrol showed up to 50% killing at 72 hours with only a four hour exposure, and reached maximum efficacy with a 24 hour exposure. Silvestrol was similarly effective against cells from CLL patients with or without del(17p13). Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. In MTT assays using isolated CD3+ or CD19+ cells, and in whole blood from healthy volunteers and CLL patients, silvestrol demonstrated substantially more cytotoxicity toward B cells than T cells. We then tested silvestrol using Tcl-1 transgenic mice, which are initially normal but develop a slow-progressing B cell leukemia very similar to human CLL. Lymphocytes obtained from spleens of Tcl-1 mice with leukemia were incubated ex vivo with 80 nM silvestrol and analyzed by flow cytometry. Silvestrol produced an 88% reduction in the B cell percentage after 24 hours with no negative effect on the T cell percentage (8% increase), in contrast to 1 μM fludarabine, which affected both B cell (22% reduction) and T cell (14% reduction) subsets. Non-leukemic mice of the Tcl-1 background strain were treated with 1.0, 1.5 and 2.5 mg/kg/day silvestrol for 5 days to determine a tolerable dose. Three of five mice treated with 2.5 mg/kg/day died at the beginning of the second week of treatment. However, none of the animals treated at 1.0 or 1.5 mg/kg showed signs of toxicity or weight loss even after two full weeks of treatment and were normal at pathological examination. Tcl-1 mice with evidence of leukemia as determined by elevated leukocyte counts and enlarged spleens were then treated with silvestrol at 1.5 mg/kg/day × 5 days for two weeks. Treated mice experienced decreased overall leukocyte counts relative to vehicle controls. Furthermore, CD19+ cell numbers and percentages diminished substantially while the T cells were only mildly affected. Additional leukemic Tcl-1 mice are currently being treated and studies are underway examining the mechanism of action of silvestrol in CLL cells.

Generation of CD8 T Cell-Mediated Protective Immunity against Tumor Escapees

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2623-2623 ◽  
Author(s):  
Bindu Varghese ◽  
Behnaz Taidi ◽  
Adam Widman ◽  
James Do ◽  
R. Levy
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Cd8 T Cell

Abstract Introduction: Anti-idiotype antibodies against B cell lymphoma have shown remarkable success in causing tumor regression in the clinic. In addition to their known ability to mediate ADCC, anti-idiotype antibodies have also been shown to directly inhibit the proliferation of tumor cells by sending negative growth signals via the target idiotype. However, further studies to investigate this mechanism have been hindered by the failure of patient tumor cells to grow ex vivo. Methods and Results: In order to study this phenomenon further, we developed an antibody against the idiotype on an A20 mouse B lymphoma cell line. A radioactive thymidine incorporation assay showed decreased A20 cell proliferation in the presence of the anti-id antibody ex vivo. In vivo, when mice were treated intraperitoneally (i.p.) with 100 μg of antibody 3 hours post-tumor inoculation (1×106 A20 subcutaneously (s.c.)), tumor growth was delayed for greater than 40 days after which the tumor began to grow once again. Further analysis of these escaping tumor cells by flow cytometry showed that that the tumor cells escaped the antibody-mediated immune response by down-regulating expression of idiotype and IgG on their surfaces although the cells retained idiotype expression intracellularly. This down-regulation of surface idiotype rendered the tumor cells resistant to both ADCC and signaling-induced cell death. The addition of an immunostimulatory bacterial mimic (CpG-DNA; 100 μg × 5 intratumoral (i.t.) injections; Days 2, 3 4, 6 & 8) to antibody therapy (Day 0; 100 μg i.p.) cured large established tumors (Day 0 = 1 cm2) and prevented the occurrence of tumor escapees (p&lt;0.0001). Antibody plus CpG combination therapy in tumor-bearing mice deficient for CD8+ T cells demonstrated the critical role of CD8+ T cells in A20 tumor eradication (p&lt;0.005). Depletion of CD4+ T cells was found to have no significant impact on the therapy. We also found that when mice were inoculated with two tumors and treated with anti-idiotype antibody (i.p.) followed by intratumoral CpG in just one tumor (Day 0=1 cm2; anti-idiotype antibody 100 μg Day 0; 100 μg CpG Days 2, 3, 4, 6 & 8), untreated tumors regressed just as well as CpG-treated tumors indicating a systemic anti-tumor immune response was generated. Conclusion: Anti-idiotype therapy, although effective in delaying tumor growth, frequently generates antigen-loss variants. However, we found that when anti-idiotype antibodies were combined with CpG, even large established tumors were cured due to systemic CD8+ T cell-dependent tumor immunity. Rather than simply mediating ADCC against a single tumor antigen, which requires the constant infusion of antibody to hamper tumor growth, we hypothesize a cytotoxic T-cell response against many tumor antigens was also generated. Such a diverse T-cell repertoire can prevent the emergence of tumor escapees and collectively provide long-lasting tumor protection. These pre-clinical results suggest that anti-tumor antibodies combined with CpG warrant further study in patients with B cell lymphoma.

Defective T Cell Migration in Chronic Lymphocytic Leukemia Is Repaired by Lenalidomide

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3117-3117
Author(s):  
Alan G. Ramsay ◽  
Lena Svensson ◽  
Nancy Hogg ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Direct Contact ◽  
Immune Surveillance ◽  
Lymphocytic Leukemia ◽  
T Cell Migration

Abstract We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p&lt;0.01). We further postulated that direct contact of CLL tumor cells with healthy T cells would induce this migratory defect. Healthy CD4 or CD8 T cells were cocultured with either allogeneic CLL B cells or allogeneic healthy B cells and subsequently used in migration assays. Co-culture with CLL cells resulted in significantly reduced T cell migration compared with co-culture with healthy B cells (~ 44% reduction in migration, n=6, p&lt;0.01). Evidence that direct contact was required to induce this migratory defect was shown when no effect was observed when cell-cell adhesion was prevented by pretreatment of CLL cells with anti-ICAM-1 blocking antibody prior to primary co-culture with healthy T cells. This cancer-induced migratory defect was repaired when CLL T cells were pretreated with the immunomodulatory drug Lenalidomide (1μM for 1hr). Treatment with this agent enhanced the migratory potential of CLL T cells to a speed comparable to untreated and treated healthy T cells. The finding that lenalidomide can restore rapid migration in patient T cells provides evidence that this agent may increase immune surveillance in CLL patients.

Prevention of Acute GvHD During MHC Haploidentical BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2455-2455
Author(s):  
Weston Miller ◽  
Caleb E. Wheeler ◽  
Angela Panoskaltsis-Mortari ◽  
Allan D Kirk ◽  
Christian P Larsen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rhesus Macaque ◽  
Cd8 T Cells ◽  
Flow Cytometric Analysis ◽  
Costimulation Blockade ◽  
Flow Cytometric ◽  
Gvhd Prophylaxis ◽  
High Level ◽  
Cd127 Expression

Abstract Abstract 2455 Poster Board II-432 Introduction: While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods: Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results: Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD. In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions: We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures: No relevant conflicts of interest to declare.

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