Overcoming Immunoescape Mechanisms of BCL1 Leukemia and Induction of CD8 + T-Cell–Mediated BCL1-Specific Resistance in Mice Cured by Targeted Polymer-Bound Doxorubicin Marek Kovar, 1 Jakub Tomala, 1 Helena Chmelova, 1 Lubomir Kovar, 1 Tomas Mrkvan, 1 Radka Joskova, 1 Zuzana Zakostelska, 1 Tomas Etrych, 2 Jiri Strohalm, 2 Karel Ulbrich, 2 Milada Sirova, 1 and Blanka Rihova 1 1 Department of Immunology and Gnotobiology, Institute of Microbiology and 2 Laboratory of Biomedical Polymers, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic Abstract BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4 + and CD8 + T cells are required for establishment of the resistance, but only CD8 + T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory mole- cules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expan- sion of CD4 + CD25 + Foxp3 + regulatory T (T reg ) cells. Although it has been shown that depletion of T reg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of T reg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded T reg cells can indeed promote tumor progression by using mice with selectively expanded T reg cells before inoculation of BCL1 leu- kemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of T reg ) can signif- icantly improve the therapeutic outcome of chemotherapy. [Cancer Res 2008;68(23):9875–83] Introduction In this study, we used a conjugate of a synthetic, water-soluble, and biocompatible copolymer based on N -(2-hydroxypropyl)me- thacrylamide (HPMA) with doxorubicin bound via a Gly-Phe(D,L)- Leu-Gly spacer (1, 2) and containing either human polyclonal (i.e., nonspecific) antibody or B1 monoclonal antibody (mAb). B1 mAb specifically binds with high affinity to an idiotype of surface IgM on BCL1 leukemia (3). Thus, we used a conjugate containing potent cytostatic drug specifically targeted to BCL1 cells (2), which enabled us to effectively treat mice bearing the leukemia while causing only minimal damage to the immune system (1, 4). Because genetic instability is a hallmark of all malignant cells (5), tumors accumulate enormous number of mutations, and although that only small proportion of these occur in open reading frames of genes expressed by tumor cells, it is inevitable that any given cancer cell will express at least few new antigenic determinants that could be recognized by the immune system (6). Numerous innate and adaptive immune effector cells participate in the recognition and destruction of cancer cells, a process that is known as cancer immunosurveillance (5–7). Cancer cells can escape innate and adaptive immune either by immunosubver- sion or by immunoselection (8–12). Immunosubversion is a pro- cess of active suppression of the immune response by tumor cells. Tumors use numerous different mechanisms (further referenced as immunoescape mechanisms) of immunosubversion (8, 13–16), including down-regulation of MHC class I expression (9, 10) and up-regulation of expression of CD95L (16), indoleamine-2,3-dioxy- genase (14, 15), and arginase-1 or transforming growth factor-h and interleukin (IL)-10 (17). Moreover, a significant expansion of regulatory T (T reg ) cells, which are capable of inhibiting both CD4 + and CD8 + T-cell responses, was observed during progression of many types of tumors both in mice and humans (18). It was reported previously that conjugates based on poly(HPMA) containing doxorubicin are capable to completely cure tumor- bearing mice and that some of these mice show tumor-specific resistance (1, 4). The goal of our study was to investigate this resistance; specifically, we aimed to determine which subset of immunocompetent cells is necessary for the establishment and which for the maintenance of the resistance. Another aim was to determine which immunoescape mechanisms can be used by BCL1 cells to avoid rejection by immune system. We identified several mechanisms used by BCL1 leukemia and we also showed that their abrogation can significantly improve the therapeutic outcome of chemotherapy. Moreover, we provided the first direct evidence that mice with expanded T reg cells show faster tumor progression than mice with normal counts of T reg cells. Materials and Methods Mice. Female BALB/c mice were obtained from the breeding colony at the Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i. Mice were used at 9 to 15 wk of age. All experiments were approved by the Animal Welfare Committee at the Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. Cell lines. The murine B-cell leukemia BCL1, B16F10 melanoma, and RAW264.7 cell lines were purchased from the American Type Culture Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Marek Kovar, Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4-Krc 14220, Prague, Czech Republic. Phone: 420-241-062- 365; Fax: 420-241-721-143; E-mail: makovar@biomed.cas.cz. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1979 www.aacrjournals.org 9875 Cancer Res 2008; 68: (23). December 1, 2008 Research Article Research. on October 28, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from