E␮-TCL1 mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction Gullu Gorguna,1, Alan G. Ramsayb,1, Tobias A. W. Holderrieda,1, David Zahriehc, Rifca Le Dieub, Fenglong Liuc, John Quackenbushc, Carlo M. Croced, and John G. Gribbenb,2 aDepartments of Medical Oncology and cBiostatistics and Computational Biology, The Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; dDepartment of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210; and bInstitute of Cancer, Barts and London School of Medicine, University of London, London EC1M 6BQ, United Kingdom Contributed by Carlo M. Croce, February 21, 2009 (sent for review November 26, 2008) Preclinical animal models have largely ignored the immune-sup- impairment of T-cell function and alteration of gene expression pressive mechanisms that are important in human cancers. The in CD4 and CD8 T cells to that observed in human patients with identification and use of such models should allow better predic- this disease. The causal relationship between leukemia and the tions of successful human responses to immunotherapy. As a induction of T-cell changes is demonstrated in vivo by the finding model for changes induced in nonmalignant cells by cancer, we that infusion of CLL B cells into nonleukemia-bearing E␮-TCL1 examined T-cell function in the chronic lymphocytic leukemia (CLL) mice rapidly induces these changes, demonstrating a causal E␮-TCL1 transgenic mouse model. With development of leukemia, relationship. This model allows dissection of the molecular E␮-TCL1 transgenic mice developed functional T-cell defects and changes induced in CD4 and CD8 T cells by interaction with alteration of gene and protein expression closely resembling leukemia cells, and further supports the concept that cancer changes seen in CLL human patients. Furthermore, infusion of CLL results in complex abnormalities in the immune microenviron- cells into young E␮-TCL1 mice induced defects comparable to those ment. The similarities with human CLL, including reversible seen in mice with developed leukemia, demonstrating a causal immunological synapse dysfunction with an immunomodulating relationship between leukemia and the T-cell defects. Altered drug (1), validates the use of E␮-TCL1 mice as a model for pathways involved genes regulating actin remodeling, and T cells further analyses of ways to prevent and reverse cancer-induced exhibited dysfunctional immunological synapse formation and immune dysfunction. T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of Results this animal model of CLL and define a versatile model to investigate Dysfunctional T-Cell Function in E␮-TCL1 Mice That Have Developed both the molecular mechanisms of cancer-induced immune sup- Leukemia. To test the hypothesis that development of leukemia pression and immunotherapeutic repair strategies. would induce changes in the nonmalignant cells of the immune system, we examined changes in T-cell function in E␮-TCL1 ancer cells induce changes in the tumor microenvironment. transgenic mice (6, 7) as they developed leukemia from 12- CThe mechanisms whereby this occurs are poorly understood, months of age [see supporting information (SI) Fig. S1]. Trans- but include direct cancer cell-T cell interactions, production of genic expression of TCL1 in B cells has no demonstrable effect immune-suppressive factors by cancer cells, and induction of on T cells, because before development of leukemia (tested at regulatory T-cell subsets (1, 2). Understanding the elusive 3–4 months) transgenic mice had normal T-cell numbers, CD4/ mechanisms of tumor-driven immune evasion will aid the re- CD8 ratio, cytokine production on mitogen, and antigenic finement of existing cancer immunotherapy strategies and iden- stimulation (data not shown), and as described below, similar tify novel treatments. To date, preclinical animal models that gene-expression profiles to WT mice. closely model human cancer and, in particular, include exami- However, once the mice developed CLL they had decreased nation of the immune-suppressive mechanisms used by cancer in vivo antigen-specific T-cell activation, suppressed T-cell mi- cells have been under-characterized. The identification and use togenic proliferation, and impaired induction of idiotype- of such models should allow better predictions of successful specific CD8 T cells capable of killing CLL cells (Fig. 1) human responses to immunotherapy. compared to age-matched control WT mice or E␮-TCL1 trans- Chronic lymphocytic leukemia (CLL) is an ideal model cancer genic mice without CLL. Leukemic mice had dysfunctional to study immune T cells that have been exposed to circulating T-cell lymphokine production (Th2-preponderant) with in- tumor B cells and is associated with immune dysfunction. CLL creased IL1, IL4, and IL6 but decreased IL2, IFN-␥, and IL-12␤ is the most common adult leukemia in North America and production. These mice also had impaired proliferation and IL-2 Europe and is currently incurable. Immunotherapeutic strategies production following cross-linking of CD3/CD28 (data not are believed to represent a promising treatment modality for this shown). These data identify defective T-cell function with the disease if immune suppression can be controlled (3). Extensive research has been carried out using CLL human patient cells, Author contributions: G.G., A.G.R., T.A.W.H., R.L.D., C.M.C., and J.G.G. designed research; including the characterization of altered gene and protein- G.G., A.G.R., T.A.W.H., R.L.D., and J.G.G. performed research; C.M.C. contributed new expression profiles (4) and suppressed functional responses in reagents/analytic tools; D.Z., F.L., and J.Q. analyzed data; and G.G., A.G.R., T.A.W.H., R.L.D., patient T cells compared to healthy-donor T cells (1, 5). and J.G.G. wrote the paper. We sought to determine whether development of leukemia in The authors declare no conflict of interest. the well-established E␮-TCL1 transgenic murine model of CLL Data deposition: The data reported in this paper have been deposited in the Gene (6) would induce changes in nonmalignant T cells. To examine Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE8836). this, we examined changes in gene-expression profile, protein 1G.G., A.G.R., and T.A.W.H. contributed equally to this work. expression, and function in T cells from E␮-TCL1 transgenic 2To whom correspondence should be addressed. E-mail: [email protected]. mice as they developed CLL. We demonstrate that development This article contains supporting information online at www.pnas.org/cgi/content/full/ of CLL in these transgenic mice is associated with similar 0901166106/DCSupplemental. 6250–6255 ͉ PNAS ͉ April 14, 2009 ͉ vol. 106 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901166106 Downloaded by guest on September 28, 2021 A B C Wild type Eµ-Tcl1 mice without CLL 60 Wild type Eμ-Tcl1 without CLL 10000 Eµ-Tcl1 mice with CLL Eμ-Tcl1 without CLL 20 Eμ-Tcl1 with CLL 50 Eμ-Tcl1 with CLL 3 8000 40 6000 30 10 p=0.0003 % lysis CPM x10 CPM 4000 20 2000 p=0.05 10 (CPM) 0 0 [3H]-Thymidine incorporation 0 0.5 1.0 Unstimulated PMA-Ionomycin 3 10 30 Peptide ng/ml Effector:Target Ratio Fig. 1. Development of CLL in E␮-TCL1 mice induces suppressed T-cell function. (A) Decreased in vivo gp33 antigen-specific T-cell activation in mice with CLL. WT mice and mice with and without CLL were immunized with gp33 peptide. On day 8, spleen cells from immunized mice (n ϭ 3 in each group) were stimulated with indicated concentrations of KAVYNFATM peptide in triplicate for 3 days before [3H]-Thymidine incorporation was measured. (B) Decreased response to mitogen activators in mononuclear cells from E␮TCL1 mice with CLL compared to those without CLL. Mononuclear cells were stimulated with 100-ng/ml PMA and 4-␮g/ml ionomycin for 3 days. Proliferation was measured using [3H]-Thymidine. (C) Decreased idiotype-specific cytotoxicity against CLL cells by CD8 T cells. CD8 cells were stimulated in vitro with dendritic cells (DCs) pulsed with Ig heavy chain (IgVH)-derived peptide GSAINYAPSI weekly for 4 weeks. Killing of CLL cells expressing this IgVH by the resulting T-cell lines was assessed at the effector:target ratios shown from wild type and E␮-TCL1 mice with and without CLL. (A–C) Results are the mean Ϯ SD and are representative of at least 3 independent experiments. development of CLL by using this mouse model that is not a regulatory subset. Changes in expression of chemokines and result of aging or the expression of the transgene alone. chemokine receptors in CLL mice were similar to those in CLL patients (4). Molecular Basis for Defective T-Cell Function in the E␮-TCL1 Mouse Model. To understand the molecular basis for the observed Comparison of T-Cell Defects Between E␮-TCL1 Mouse Model and functional defects and to compare changes seen in mice and Human Patients. An important question was whether the changes patients with CLL (4), we performed gene-expression profiling. in gene-expression profiles observed in the mice with CLL were Unsupervised analysis of highly purified CD4 and CD8 T cells similar to those observed in human patients with this disease. To in CLL mice demonstrated altered gene-expression profiles examine this, analysis of gene-expression changes in T cells in compared to WT mice or to young E␮-TCL1 mice without CLL mice compared with those in patients (4) was performed disease (Fig. S2). These findings again support the hypothesis using RESOURCERER (8, 9), a database for annotating and that the altered T-cell gene-expression is not a result of aging or linking microarray resources within and across species.