scholarly journals 702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A730-A730
Author(s):  
Wenqing Jiang ◽  
Zhengyi Wang ◽  
Zhen Sheng ◽  
Jaeho Jung ◽  
Taylor Guo
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Gene Expression Analysis ◽  
Cell Activation ◽  
Clinical Development ◽  
Cynomolgus Monkeys ◽  
Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

scholarly journals Immunologic ignorance of vascular endothelial cells expressing minor histocompatibility antigen

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4588-4595 ◽  
Author(s):  
Beatrice Bolinger ◽  
Philippe Krebs ◽  
Yinghua Tian ◽  
Daniel Engeler ◽  
Elke Scandella ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Histocompatibility Antigen ◽  
Target Cells ◽  
Minor Histocompatibility Antigen

Abstract Endothelial cells (ECs) presenting minor histocompatibility antigen (mhAg) are major target cells for alloreactive effector CD8+ T cells during chronic transplant rejection and graft-versus-host disease (GVHD). The contribution of ECs to T-cell activation, however, is still a controversial issue. In this study, we have assessed the antigen-presenting capacity of ECs in vivo using a transgenic mouse model with beta-galactosidase (β-gal) expression confined to the vascular endothelium (Tie2-LacZ mice). In a GVHD-like setting with adoptive transfer of β-gal–specific T-cell receptor–transgenic T cells, β-gal expression by ECs was not sufficient to either activate or tolerize CD8+ T cells. Likewise, transplantation of fully vascularized heart or liver grafts from Tie2-LacZ mice into nontransgenic recipients did not suffice to activate β-gal–specific CD8+ T cells, indicating that CD8+ T-cell responses against mhAg cannot be initiated by ECs. Moreover, we could show that spontaneous activation of β-gal–specific CD8+ T cells in Tie2-LacZ mice was exclusively dependent on CD11c+ dendritic cells (DCs), demonstrating that mhAgs presented by ECs remain immunologically ignored unless presentation by DCs is granted.

scholarly journals Human Immunodeficiency Virus-Specific Circulating CD8 T Lymphocytes Have Down-Modulated CD3ζ and CD28, Key Signaling Molecules for T-Cell Activation

2000 ◽  
Vol 74 (16) ◽  
pp. 7320-7330 ◽  
Author(s):  
Linda A. Trimble ◽  
Premlata Shankar ◽  
Mark Patterson ◽  
Johanna P. Daily ◽  
Judy Lieberman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Cd8 T Lymphocytes ◽  
Immunodeficiency Virus

ABSTRACT Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3ζ, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3ζ down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3ζ-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3ζ−. CD8 T cells with down-modulated CD3ζ also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR+ CD62L−). After T-cell activation, CD3ζ-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor α-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3ζ is not reexpressed even after IL-2 exposure.

The immunological impact of the RAF inhibitor BMS908662: Preclinical and early clinical experience in combination with CTLA-4 blockade.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2521-2521 ◽  
Author(s):  
Margaret Callahan ◽  
Gregg Masters ◽  
Jessica Katz ◽  
Valerie Russell ◽  
Ruth Ann Roman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mapk Pathway ◽  
Preclinical Studies ◽  
Anti Tumor Activity

2521 Background: Two new approaches to treat advanced melanoma have transformed the standard of care: the CTLA-4 blocking antibody, ipilimumab, and the targeted inhibitor of mutated BRAF, vemurafenib. These agents are mechanistically unique and combination therapy is a promising next step. We evaluated the combination of BMS908662 (662), a pan RAF inhibitor, with CTLA-4 blockade in preclinical studies and report first-in-human clinical experience with this combination. Methods: 1) We tested the impact of 662 on T cells in vitro, using cultured human T cells, and in vivo, using OT-1 transgenic mice. T cell activation and MAPK pathway signaling were assessed in parallel. 2) Preclinical studies measuring the anti-tumor activity of combination therapy were performed in CT-26 and SA1N tumor models. 3) Three pts with BRAF mutant stage IV melanoma were treated at MSKCC on CA206005, an IRB-approved protocol, receiving ipilimumab (3 mg/kg) and 662 (25 mg bid) (NCT01245556). Two pts consented to an IRB-approved protocol permitting immune monitoring. Results: 1) In vitro studies demonstrate that 662 can potentiate T cell activation after stimulation. This corresponds with increased MAPK pathway signaling, consistent with paradoxical activation of the MAPK pathway in wild type cells, a class effect of RAF inhibitors. In vivo, enhanced expansion of OT-1 cells after ovalbumin challenge was seen in mice treated with 662. T cell expansion was greatest in mice treated with a combination of CTLA-4 blockade and 662 (p<0.05). 2) Both preclinical models demonstrate superior anti-tumor activity with combination therapy compared to monotherapy (p<0.05). 3) All pts treated on protocol CA206005 tolerated combination therapy. New keratoacanthomas and SCCs, likely related to 662, were identified. One pt has an ongoing response at 10 mos (-85%), one had stable disease for 24 wks (-19%) and a third had disease progression. Enhanced MAPK signaling in PBMCs after treatment with 662 was detected ex vivo. Conclusions: RAF inhibitors may potentiate T cell activation in vitro and in vivo, offering one explanation for the enhanced anti-tumor activity seen in combination with CTLA-4 blockade in pre-clinical models.

scholarly journals 697 Tumor-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A739-A739
Author(s):  
Michael Hedvat ◽  
Veronica Zeng ◽  
Juan Diaz ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cancer Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bispecific Antibodies ◽  
Anti Tumor Activity

BackgroundT cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Solid tumors often lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling could be beneficial in solid tumors. We designed tumor-associated-antigen (TAA) x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of TAA and TCR engagement. Clinical application of this class of antibodies has potential to enhance activity of either anti-PD(L)1 antibodies or TAA x CD3 T cell engagers.MethodsWe designed a stability and affinity optimized anti-CD28 antibody that can be paired with TAA of choice to engage CD28 monovalently using Xencor’s XmAb 2+1 and 1+1 platforms. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo anti-tumor and T cell proliferative activity of B7H3 x CD28 bispecific antibodies were determined in tumor-bearing huPBMC-NSG mice treated simultaneously with TAA x CD3 bispecific antibody. In vivo activity of PDL1 x CD28 antibodies was determined with hCD28 KI mice inoculated with MC38 tumors expressing hPDL1-antigen. Finally, safety and tolerability of B7H3 x CD28 and PDL1 x CD28 was determined in cynomolgus monkeys.ResultsB7H3 x CD28 and PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of target antigen. B7H3 x CD28, alone or in combination with anti-PD1 antibody, enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. PDL1 x CD28 also enhanced CMV+ cell expansion but did not synergize with anti-PD1 antibody treatment. B7H3 x CD28 significantly enhanced in vivo anti-tumor activity of TAA x CD3 antibodies while also promoting greater T cell expansion. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 antibody inhibited tumor growth greater than an anti-PDL1 antibody alone. B7H3 x CD28 and PDL1 x CD28 were well tolerated in cynomolgus monkeys.ConclusionsB7H3 x CD28 and PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development.

561 DuoBody®-PD-L1×4–1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4–1BB co-stimulation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Alexander Muik ◽  
Isil Altintas ◽  
Rachelle Kosoff ◽  
Friederike Gieseke ◽  
Kristina Schödel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cytokine Production ◽  
Immune Cell ◽  
Anti Tumor Activity

BackgroundCheckpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T-cell- and NK-cell-mediated anti-tumor immunity and has been shown to synergize with CPI in preclinical models. DuoBody-PD­L1×4-1BB is a first-in-class, Fc-silenced, bispecific next-generation checkpoint immunotherapy that activates T cells through PD-L1 blockade and simultaneous PD-L1-dependent 4-1BB co-stimulation. Here we present preclinical evidence for the mechanism of action of DuoBody-PD-L1×4-1BB, and proof-of-concept using mouse-reactive mbsAb-PD-L1×4-1BB in vivo.MethodsRNA sequencing analyses was performed on primary human CD8+ T cells that were co-cultured with PD-L1+ monocytes in the presence of anti-CD3/anti-CD28 and test compounds. T-cell proliferation and cytokine production were analyzed in primary human T-cell and mixed lymphocyte reaction (MLR) assays in vitro, and using patient-derived tumor-infiltrating lymphocytes (TILs). Cytotoxic activity was assessed in co-cultures of CLDN6+PD-L1+ MDA-MB-231 tumor cells and CLDN6-TCR+CD8+ T cells. Anti-tumor activity of mbsAb-PD-L1×4-1BB was tested in vivo using the CT26 mouse tumor model. Immunophenotyping of the tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs) and peripheral blood was performed by flow cytometry.ResultsDuoBody-PD-L1×4-1BB significantly induced expression of genes associated with immune cell proliferation, migration and cytokine production in activated CD8+ T cells, which were not altered by CPI. DuoBody-PD-L1×4-1BB dose-dependently enhanced expansion of human TILs ex vivo. DuoBody-PD-L1×4-1BB dose-dependently enhanced T-cell proliferation and pro-inflammatory cytokine production in vitro (e.g. IFNγ and TNFα; in polyclonal and antigen-specific T-cell proliferation assays and MLR), which was dependent on crosslinking to PD-L1+ cells and superior to CPI or the combination of Fc-silenced PD-L1- and 4-1BB-specific antibodies. DuoBody-PD-L1x4-1BB induced upregulation of degranulation marker CD107a and granzyme B in CD8+ T cells, resulting in antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro, superior to CPI. In mice bearing subcutaneous CT26 tumors, a model that was insensitive to PD-L1 blockade, mbsAb-PD-L1×4-1BB elicited tumor rejection in the majority of the mice at active dose levels and significantly improved survival. Dose-dependent anti-tumor activity was associated with expansion of tumor antigen-specific T cells in the blood and enhanced immune-cell activation in tdLNs and TME.ConclusionsCombining PD-L1 blockade with conditional 4-1BB co-stimulation using bispecific antibodies induced T-cell activation, expansion, and cytotoxic activity in vitro and potent anti-tumor activity in vivo superior to CPI. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT03917381).Ethics ApprovalAll mice studies were performed by BioNTech SE at its research facilities in Germany, and the mice were housed in accordance with German federal and state policies on animal research. All experiments were approved by the regulatory authorities for animal welfare in Germany. The use of tumor tissue resections was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

scholarly journals CD8+ T Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T Cell Differentiation

2021 ◽  
Author(s):  
Juan Fernandez-Garcia ◽  
Fabien Franco ◽  
Sweta Parik ◽  
Antonino A Pane ◽  
Dorien Broekaert ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Differentiation

Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.

scholarly journals Role of Lymphotoxin α in T-Cell Responses during an Acute Viral Infection

2002 ◽  
Vol 76 (8) ◽  
pp. 3943-3951 ◽  
Author(s):  
M. Suresh ◽  
Gibson Lanier ◽  
Mary Katherine Large ◽  
Jason K. Whitmire ◽  
John D. Altman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
T Cell Responses ◽  
Cell Responses ◽  
Lymphotoxin Α

ABSTRACT The importance of lymphotoxin α (LTα) in lymphoid organogenesis is well established. Although LTα has been implicated in the pathogenesis of T-cell-mediated immunopathologies, the requirement for LTα in T-cell activation and effector function in vivo is not well understood. To determine the role of LTα in T-cell activation in vivo, we compared the generation of antigen-specific T-cell responses between wild type (+/+) and LTα-deficient (LTα−/−) mice during an acute infection with lymphocytic choriomeningitis virus (LCMV). Our studies showed that LCMV-infected LTα−/− mice had a profound impairment in the activation and expansion of virus-specific CD8 T cells in the spleen, as determined by cytotoxicity assays, intracellular staining for gamma interferon, and staining with major histocompatibility complex class I tetramers. Further, the nonlymphoid organs of LTα−/− mice also contained substantially lower number of LCMV-specific CD8 T cells than those of +/+ mice. Greatly reduced virus-specific CD8 T-cell responses in LTα−/− mice led to a defect in LCMV clearance from the tissues. In comparison to that in +/+ mice, the activation of LCMV-specific CD4 T cells was also significantly attenuated in LTα−/− mice. Adoptive transfer experiments were conducted to determine if abnormal lymphoid architecture in LTα−/− mice caused the impairment in the activation of LCMV-specific T-cell responses. Upon adoptive transfer into +/+ mice, the activation and expansion of LCMV-specific LTα−/− T cells were restored to levels comparable to those of +/+ T cells. In a reciprocal cell transfer experiment, activation of +/+ T cells was significantly reduced upon transfer into LTα−/− mice. These results showed that impairment in the activation of LCMV-specific T cells in LTα−/− mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LTα−/− T cells.

A Mouse Model for Immunotherapeutic Reversal of Leukemia-Induced T Cell Dysfunction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 30-30
Author(s):  
Alan G. Ramsay ◽  
Gullu Gorgun ◽  
Tobias A.W. Holderried ◽  
David Zahrieh ◽  
Fenglong Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Immune Synapse ◽  
Immunological Recognition

Abstract Understanding the elusive mechanisms of tumor-driven immune evasion will aid the refinement of existing cancer immunotherapy strategies and identify novel treatments. To date, pre-clinical animal models that closely model human cancer, including the immune suppressive mechanisms utilized by cancer cells, have been under-characterized. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in non-malignant cells by cancer, we examined T cell function in Eμ-TCL1 transgenic mice as they developed leukemia from 12-months of age. Transgenic expression of TCL1 in B cells had no demonstrable effect on T cells, however, mice with leukemia had decreased in vivo antigen specific T cell activation, suppressed T cell mitogenic proliferation and impaired induction of idiotype specific CD8 T cells capable of killing CLL cells compared to control WT mice (age-matched throughout study) or Eμ-TCL1 transgenic mice without CLL. Leukemic mice also had dysfunctional T cell lymphokine production (Th2-preponderant). To understand the molecular basis for the observed functional defects and to compare changes seen in mice and patients with CLL we performed gene expression profiling. Analysis of highly purified CD4 and CD8 T cells in CLL mice demonstrated altered gene expression profiles compared to WT mice or to young Eμ-TCL1 mice without disease. Of note, infusion of CLL cells into young Eμ-TCL1 mice induced gene expression changes comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T cell defects. Analysis of gene expression changes in T cells in CLL mice compared with those in patients was performed using RESOURCERER, a database for annotating and linking microarray resources within and across species and identified 50 overlapping genes in CD4 T cells and 45 overlapping genes in CD8 T cells. The majority of differentially expressed genes in CD4 T cells from both mice and patients with CLL were involved in cell proliferation and activation pathways with increase in Lck. Multiple defects within the actin cytoskeletal formation pathways were identified in both CD4 and CD8 T cells including Cdc42. Integrity of the T cell cytoskeleton is essential to regulate the dynamic signaling required for T cell activation and effector function in response to immunological recognition of antigen-presenting cells (APCs). T cell conjugates from mice with leukemia had suppressed antigen-dependent F-actin accumulation and early T cell signaling at the immune synapse with CLL cells (APCs) compared to WT mice conjugates. Moreover, we have demonstrated that infusion of CLL cells into young mice induces this T cell defect, demonstrating an in vivo immunomodulating mechanism utilized by tumor cells. Treatment of both CLL cells and autologous T cells from leukemic mice with lenalidomide (0.5 μM for 24 h) enhanced the formation of the F-actin immune synapse and recruitment of tyrosine-phosphorylated proteins irrespective of the presence of exogenous antigen. Of note, the capacity to repair immunological recognition with this agent was associated with increased recruitment of the cytoskeletal signaling molecules Lck and Cdc42 to the immunological synapse, regardless of whether the gene was increased or decreased on gene expression profiling. These results demonstrate that leukemia cells induce changes in multiple T cell pathways regulating antigen recognition and effector function. The similarities with human CLL including reversible immunological synapse dysfunction with an immunomodulating drug validates the use of Eμ-TCL1 mice as a model for further analyses of ways to prevent and reverse cancer-induced immune dysfunction. The use of this model to understand and reverse the molecular changes in T cells induced by leukemia will likely have broad applications to maximize immune responses in patients.

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