Ag-specific type 1 CD8 effector cells enhance methotrexate-mediated antitumor responses by modulating differentiated T cell localization, activation and chemokine production in established breast cancer

Clinical Immunology (2008) 128, 205–218

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Ag-specific type 1 CD8 effector cells enhance methotrexate-mediated antitumor responses by modulating differentiated T cell localization, activation and chemokine production in established breast cancer☆ Mark J. Dobrzanski a,⁎, Joyce B. Reome b , James C. Hylind b , Kathleen A. Rewers-Felkins a , Khaliquzzaman Abulsamad a , Shawna L. Adams a a b

Texas Tech University School Of Medicine, Department of Internal Medicine, Amarillo, Texas 79106, USA Trudeau Institute, Saranac Lake, NY 12983, USA

Received 22 October 2007; accepted with revision 26 March 2008 Available online 3 June 2008 KEYWORDS Host antitumor responses; Chemokines/cytokines; Chemotherapy; Adoptive T cell transfer; Endogenous T effector cells; Type 1 antitumor responses; Tc1/Th1 T cell subpopulations

Abstract The chemotherapeutic agent methotrexate is widely used in the treatment of breast cancer. Although its mechanism-of-action has been defined, less is known about its interaction with T cell-mediated antitumor responses. Type 1 CD8 T cell-mediated immune responses (Tc1) are cytolytic, produce IFN-gamma and are associated with effective antitumor responses. Using a murine transgenic TCR tumor model, we show that single-dose treatment with methotrexate enhanced CD8-mediated type 1 antitumor responses when administered 3 days prior to Tc1 effector cell transfer. Co-treatment with methotrexate not only enhanced donor Tc1 cell accumulation and persistence at sites of primary tumor growth, but also promoted elevated levels of activated donor TIL cells. This markedly enhanced the appearance of endogenous differentiated (CD44High) CD8 tumor-infiltrating cells when compared to that of corresponding groups receiving either MTX or Tc1 cell transfer alone. Such cells were acutely activated as defined by co-expression of surface markers associated with TCR engagement (CD69) and T cell activation (CD25) at both early (days 1–8) and late (days 12–20) stages following treatment.

Abbreviations: TIL, Tumor-Infiltrating T Cells; TSA-HA, HA-expressing TS/A breast adenocarcinoma; Tc1/Th1, CD8+ or CD4+ Tcells producing type 1 cytokines; MTX, Methotrexate. ☆ This work was supported by grants through the United States Army Medical Research and Development Command DAMD 17-01-1-0429 (to M.J.D.) and The Harrington Cancer Research Foundation (to M.J.D.). ⁎ Corresponding author. Texas Tech University School Of Medicine, Department of Internal Medicine,1400 Wallace Boulevard, Rm 214, Amarillo, Texas 79106, USA. E-mail address: [email protected] (M.J. Dobrzanski). 1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.03.518

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M.J. Dobrzanski et al. Conversely, such animals showed an early decrease in CD4+/CD44High/CD25+/CD69+ T cells that correlated with delays in tumor growth in vivo. Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IP-10 in vivo. This suggested that Tc1 cell transfer, in combination with chemotherapy, can enhance antitumor responses by modulating immunoregulatory T cells involved in homeostasis and immune tolerance within the tumor environment. These studies offer insight into mechanisms that enhance T cell-based immunotherapy in cancer. © 2008 Elsevier Inc. All rights reserved.

Introduction Breast cancer is the second leading cause of cancer-related deaths among women in North America. Conventional chemotherapeutic agents, such as methotrexate (MTX), are antimetabolite drugs widely used in the treatment of various primary and metastatic breast malignancies [1–5]. They inhibit cancer cell growth and initiate tumor cell apoptosis by targeting distinct enzymatic reactions that influence, in part, the tumor environment, cell sensitivity, survival and treatment efficacy. Although such chemotherapeutic-based strategies improve response rates and survival among patients with breast cancer, their interaction and effects on various tumor Ag-specific T cell subpopulations and their antitumor immune responses remain relatively undefined. The application of cellular immunotherapeutic principles to the treatment of breast cancer is a relatively new undertaking [6,7]. Adoptive T cell immunotherapy, which involves the ex vivo expansion and activation of select tumor Ag-specific T cells and their subsequent re-administration into cancer patients, is one such approach that has been shown to induce promising, yet limited, antitumor responses to certain types of established malignancies [7–11]. The resistance of solid tumors to these and other cellular immunotherapeutic strategies has been attributed, in part, to the phenotypic and/or intrinsic properties of tumor-infiltrating T cell populations (TIL), the immunoregulatory microenvironments of the tumor, the presence or absence of pertinent cytokines and/or regulatory cells induced by endogenous homeostatic immune responses to progressively growing tumor [12–23]. These barriers have prompted for the investigation and use of other therapies in combination with T cell immunotherapy to enhance response rates among cancer patients. Aside from their cytolytic potentials, type 1 CD8+ effector T cells (Tc1) express up-regulated levels of CD44 and CD25 and characteristically secrete IFN-γ following Ag encounter [24–28]. Using a previously described murine TCR-transgenic tumor immunotherapy model, we have shown that adoptively transferred Ag-specific Tc1 cells localize at the tumor site and effectively delay tumor growth and progression, in part, by different mechanisms involving donor cell-derived cytokines and recruitment of select endogenous Teffector/memory cells [29–31]. In the current study, we show that single-dose treatments with MTX or Tc1 effector cell transfer alone can induce marginally significant effects on tumor regression among mice with established breast malignancies. However, in mice treated with the former, 3 days prior to Tc1 effector cell transfer, we show a significantly enhanced effect in Ag-specific type 1 CD8-mediated antitumor responses that resulted in substantial delays in tumor growth and progression. More interestingly, our results show that co-therapy with MTX (i) not

only enhanced donor Tc1 effector cell accumulation and localization at sites of primary tumor growth, but also promoted elevated levels of activated donor Tc1 effector cells co-expressing CD25 within the tumor environment, (ii) diminished “early” infiltration and localization levels of differentiated endogenous CD4/CD44High TIL cell subpopulations co-expressing CD25 and CD69, (iii) promoted a progressive increase in corresponding endogenous CD8 TIL cell subpopulations at both early (days 1–8) and late (days 12– 20) stages following treatment, and (iv) promoted and facilitated the accumulation and persistence of endogenous T cells expressing up-regulated levels of the chemokine IP-10. Although these results correlated with diminished tumor growth rates among mice with established mammary tumors in vivo, such treatments resulted in only partial responses and did not result in long term survival. We discuss the potential role of cytoablative co-treatment in Ag-specific CD8-mediated type 1 antitumor responses and their potential regulatory and homeostatic effects on both donor and endogenous TIL cell subpopulations. Lastly, we further provide a potential mechanism for IP-10-producing Tcells during the effector phase of the type 1 antitumor response in progressive disease.

Materials and methods Animals Female Thy 1.1 BALB/c mice (H-2d), 6 to 10 weeks of age, were obtained from Jackson Laboratories (Bar Harbor, ME). The Thy 1.2 Clone-4 TCR-transgenic mouse strain, on the BALB/c background (H-2d), was originally obtained from Dr. Linda Sherman (The Scripps Research Institute, La Jolla, CA). These mice express a transgenic TCR vβ8.2/vα10 specific for the IYSTVASSL peptide of hemagglutinin (HA) in the context of MHC class I, H2-Kd [32]. Animals were maintained and treated according to animal care committee guidelines of the National Institutes of Health, Trudeau Institute and Texas Tech University Health Science Center.

TS/A-HA tumor cell line The TS/A-HA tumor cell line was generated and characterized as previously described and is an aggressive, poorly immunogenic murine mammary adenocarcinoma cell line of BALB/c origin [30]. The parent TS/A tumor cell line expressing transfected HA (TS/A-HA) was generated using LipofectAMINE reagent (Invitrogen, Carlbad, CA) following manufacturer's instructions. Briefly, the HA gene from the Mount Sinai strain of the PR8 influenza virus was sub-cloned into the β-actin expression vector obtained from Dr. Bernadette Scott (Queen

Tc1 effector cell transfer and chemotheraphy in breast cancer Elisabeth II Medical Center, Nedlands, Australia). Transfected cells were selected by culture in medium containing the neomycin analogue geneticin (Life Technologies, Gaithersburg, MD) at a final concentration of 500 μg/ml. The level of HAsurface expression on transfected cells was measured by FACS analysis, using the biotinylated HA-specific mAb H36-4-5.2 that was kindly provided by Dr. Walter Gerhard (The Wistar Institute, Philadelphia, PA). Clones expressing HA were sorted using a Becton Dickinson FACS Calibur (San Jose, CA) and individual clones were generated by limiting dilution. Subconfluent monolayers of either parent TS/A or TS/A-HA, in log growth phase, were harvested with addition of 0.25% trypsin (Gibco) in HBSS and washed three times in DMEM media containing 10% fetal calf serum (HyClone Inc). The TS/ A-HA tumor cell line progressively grows in vivo without evidence of spontaneous regression when injected subcutaneously (sc) into mammary fat pads (right anterior quadrant of chest) of normal syngeneic BALB/c mice. Such a model provides a defined orthotopic mammary tumor model that correlates with both “early” local–regional and/or “late” systemic stages of progressive human breast cancer [30,33].

Generation of HA-specific Tc1 effector T cell subpopulations To obtain CD8 effector T cells to HA peptide, single cell suspensions from spleens and lymph nodes of HA-BALB/c mice were washed twice in HBSS and resuspended in DMEM — 10% FCS. CD8 enriched T cells were obtained by passing lymphoid cell suspensions through nylon wool columns and treating with anti-CD4 (RL172.4), anti-heat-stable antigen (J11D), anti-MHC class II (D3.137, M5114, CA4) mAbs, and complement. Small resting CD8 T cells were harvested from percoll gradients (Sigma) and resuspended to appropriate cell concentrations in culture media. Naive CD8 cells were typically N 90% pure as demonstrated by immunofluorescent Ab staining. Antigen presenting cells (APC) were enriched from spleens of normal BALB/c mice by anti-Thy 1.2 (HO13.14 and F7D5), anti-CD4 (RL172.4) and anti-CD8 (3.155) mAbs and complement. T cell-depleted APCs were pulsed with HA peptide (11 mM) for 30 min at 37 °C and treated with mitomycin C (50 μg/ml, Sigma) for an additional 30 min at 37 °C. For Tc1 effector cell generation, naive CD8 T cells from HABALB/c transgenic mice (2 × 105 cells/ml) were stimulated with mitomycin C-treated HA peptide-pulsed APCs (6 × 105 cells/ml) in the presence of IL-2 (20 U/ml, X63.IL-2 supernatants), IL-12 (2 ng/ml, kindly provided by Dr. Stanley Wolf (Genetics Institute/Wyeth, Cambridge, MA), and anti-IL-4 mAb (200 U/ml, X63.Ag.IL-4 supernatants). Effector Tcell cultures were incubated for 4 days with additional IL-2 (20 U/ml) added to the cultures on day 2 to promote CD8 cell expansion. As shown in our earlier studies [29,30,34], Tc1 cells demonstrated potent tumor Ag-specific CTL activity to HA peptideexpressing tumor cell targets in vitro. Moreover, Tc1 effector cells produced substantial amounts of IFN-γ with no detectable levels of IL-4, IL-5, or GM-CSF. Flow cytometric analysis showed that Tc1 cells expressed typical cell surface Ag markers associated with effector cell phenotype. Effector cell populations were CD8+CD4− and expressed up-regulated levels of both CD44 and CD25 and down-regulated levels of CD62L.

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Adoptive T cell transfer model Syngeneic female Thy 1.1 BALB/c mice were injected subcutaneously with 0.2 ml of a single cell suspension containing 2×105 TS/A-HA tumor cells in the right anterior mammary region. Ten days following tumor challenge, mice were treated with various doses of Thy 1.2 HA Ag-specific Tc1 effector cells (i.v.) and tumor growth monitored. Briefly, tumor growth was measured every 3 days using vernier calipers and volumes (mm3) obtained by multiplying the measured length by the measured width by the calculated mean of these measured values. Data are presented as either the mean tumor volume or as the tumor growth rate which is the absolute value of the slope of the line as determined by regression analysis (95% confidence intervals). The ratio of the growth rates among groups of mice receiving different treatments to corresponding groups of untreated mice×100 is depicted as the relative tumor growth rate.

Drug treatment and chemotherapy Ready-to-use injectable solutions of MTX (Lederle Pharmaceutical Division of American Cyanamid Company, Pearl River, NY) in normal saline were prepared and provided by the hospital pharmacy. Groups of mice with established TS/A-HA mammary tumors (n = 6–10) were treated one time with MTX 7 days after tumor challenge. Drug treatment at doses ranging from 500 to 0.5 mg/kg body weight, was administered i.p. in 0.5 ml volumes of normal saline. For adoptive Tcell cotherapy studies, mice were treated i.v. with 1 × 107 tumorreactive CD8 T cells 3 days after drug treatment with singledose MTX (i.e. 10 days post tumor challenge). Tumor growth was measured every 3 days using vernier calipers and tumor volumes and growth rates were calculated as described above. Body weights of mice were measured twice weekly to monitor treatment-related toxicity. Untreated tumor-bearing animals served as controls.

Flow cytometric analysis Single cell suspensions of tumor were obtained by mechanical dispersion through nylon mesh screens in DMEM — 5% FCS and washed three times in fluorescent antibody buffer (FAB) consisting of 1% bovine serum albumin and 0.02% sodium azide in 0.01 M phosphate buffered saline, pH 7.2. Immune cell populations were phenotyped by their expression of surface markers using either direct or indirect immunofluorescence staining techniques [29,30]. Lymphocytes (106), pretreated with FcR block, were incubated for 20 min on ice with 100 μl of FAB containing 1 μg of various mAbs conjugated to either Biotin, PE, FITC, or Cy-Chrome. For biotinylated mAbs, strept-avidin allophycocyanin or strept-avidin cychrome was used as a second step reagent. The mAbs used include anti-CD90.1 (Thy 1.1; Pharmingen), anti-CD90.2 (Thy 1.2; Pharmingen), anti-CD8 (Caltag Laboratories, Bulingame, CA.) or anti-CD4 (Pharmingen), anti-CD44 (eBioscience, San Diego, CA; clone IM7), anti-CD3 (Pharmingen), anti-CD25 (eBioscience), and anti-CD69 (eBioscience). Stained cell preparations were than washed three times in FAB, and analyzed by multiparameter flow cytometry using a Becton Dickinson FACS Calibur (San Jose, CA). Surface

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M.J. Dobrzanski et al. donor cells and cultured with either nothing or plate-bound anti-CD3 for 5 h at 37 °C. Total RNA from either unstimulated or plate-bound anti-CD3 cell cultures from Tc1 effector celltreated or untreated tumor-bearing mice were prepared by tissue homogenation in TRIzol reagent (GIBCO). mRNA levels were quantitated using the RiboQuant Multiprobe Ribonuclease Protection Assay (RPA) system (Pharmingen) with the mCK-5 chemokine mRNA detection probe sets. Bands were detected using the Molecular Imager FX with the Quantity One Software analysis program (BIO-Rad Labs, Hercules, CA) and normalized against the L32 housekeeping gene as Relative Units.

Figure 1 Systemic administration of single-dose MTX or Tc1 cell transfer effectively delays mammary tumor growth in mice with established breast tumors. Mice (n = 6–10/gp) were injected subcutaneously in the mammary fat pad region with 2 × 105 TS/A-HA tumor cells. Seven days later, various concentrations of HA Ag-specific-Tc1 effector cells (A) were adoptively transferred (i.v.) into tumor-bearing mice and the mean tumor volumes were determined as described in Materials and methods. In B, groups of tumor-bearing mice were treated i.p. with various concentrations of methotrexate (MTX) and the tumor volumes were determined. Untreated tumor-bearing mice served as controls. Numbers in parenthesis represent the range of acute survival following drug treatment. Data are expressed as the mean +/− the SEM of three independent experiments.

marker analysis was performed using Cell Quest Software and the percent positive and absolute cell numbers were determined.

Analysis of endogenous T cell-derived chemokine mRNA expression in primary mammary tumor tissue Freshly-generated Tc1 donor cells (Thy 1.2) or TIL cells from treated or untreated tumor-bearing recipient mice (Thy 1.1) were harvested and single cell suspensions from primary tumors were obtained at different time points following tumor challenge. T cells were enriched by negative selection using mAb and complement. Endogenous T cell suspensions were further treated with anti-Thy 1.2 mAb to eliminate

Figure 2 Co-therapy with single-dose MTX enhances Agspecific Tc1 effector cell-mediated antitumor responses in mice with established breast tumors. Mice (n = 6–10 mice/gp) were injected sc in the mammary fat pad region with 2 × 105 TS/ A-HA tumor cells. Seven days later, groups of tumor-bearing mice were treated (i.p.) with MTX (50 mg/kg body weight) or nothing. Three days later, specified groups of untreated or drugtreated mice received 1 × 107 Tc1 effector cells (i.v.) and tumor volumes (A) and growth rates (B) were determined as described in Materials and methods. The relative tumor growth rate is determined by regression analysis (95% confidence intervals). The absolute value of the slope of the regression line is the tumor growth rate. The ratio of the tumor growth rates among groups of mice receiving different treatments to corresponding groups of untreated mice × 100 is depicted on the x-axis. Untreated and Tc1 effector cell-treated tumor-bearing mice served as controls. Results are expressed as the mean +/− the SEM of 4 independent experiments. ⁎, p b 0.01 for treated animals versus non-treated animals; ⁎⁎, p b 0.05 for animals receiving combinatorial therapy versus corresponding groups of animals receiving single-treatment therapy.

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Figure 3 Effects of MTX on donor Tc1 effector cell localization, persistence and acute activation at sites of primary tumor growth among animals with established mammary tumors. TS/A-HA tumor-bearing mice (Thy 1.1) were either untreated or treated with MTX (50 mg/kg body weight) 7 days following tumor challenge. Three days later, specified groups of mice were treated with 1 × 107 Tc1 effector cells (Thy 1.2) as described in Figure 2. At specified time points following Tc1 cell transfer, tumors from groups of mice were harvested and single cell suspensions were labeled with anti-Thy 1.2 and CD8 mAbs. Lymphocytes, distinguished by their forward and side scatter, were analyzed by multiparameter flow cytometry. The absolute cell numbers of donor T cells were calculated as the percentage of positive staining cells × the total number of mononuclear cells per tissue. Results are expressed as the mean ± SEM of 3–5 mice per group/time point in three independent experiments. In B, at early (days 1–8) and late (days 12–20) stages following Tc1 cell transfer, mammary tumors were harvested and labeled with anti-Thy 1.2, anti-CD8, or anti-CD44 and the percentage of T cells coexpressing CD25 surface Ag were assessed by multicolor flow cytometry. Gates were set on donor Thy 1.2/CD8/CD44High T cell populations and cells co-expressing CD25 surface Ag were determined. Data shown are from a representative experiment showing the percentage of tumor or spleen-derived Thy 1.2/CD8/CD44High T cells co-expressing CD25 surface Ag. Gates for all figures were adjusted to appropriate isotype controls to distinguish regions of nonspecific and specific mAb staining. Results are representative of three independent experiments. ⁎, p b 0.01 for animals receiving both MTX and Tc1 cell transfer versus corresponding groups of animals receiving Tc1 cell transfer alone.

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Statistical analysis For differences in tumor size and growth between experimental and control groups, the Mann–Whitney Rank Sum Test was used. For T cell subpopulation comparison studies and growth rate analysis among experimental and control groups, we use the Student's t-test provided in the PRISM Graph Pad statistical software package. Statistical significance was defined as a p value less than 0.05 for all analysis.

Results Single-dose treatment with Tc1 effector cell transfer or MTX alone delays mammary tumor growth and progression in mice with established breast tumors To initially address the potential therapeutic role of Agspecific Tc1 effector cell subpopulations in established breast malignancies, we utilized our TCR-transgenic T cell/ murine breast tumor model. Following sc TS/A-HA tumor challenge (2 × 105), graded numbers of in vitro-generated effector T cell subpopulations were intravenously transferred 7 days following tumor challenge and therapeutic efficacy was evaluated by measuring tumor volumes and growth rates. As shown in Figure 1A, mice receiving doses of 5–100 × 105 of HA-Tc1 effector cells showed similarly significant (p b 0.05) decreases in tumor growth and progression when compared with that of untreated tumor-bearing control animals. Whereas, transfer of 10-fold less Tc1 effector cells, at numbers as low as 0.5 × 105, resulted in no therapeutic effect. This suggested that Tc1 effector cell responses not only participated in T cell-mediated delays in tumor growth but also appeared to reach a therapeutic threshold that resulted in partial responses. In parallel studies, groups of tumor-bearing mice were treated systemically (i.p.) with various doses of MTX 7 days following tumor challenge. As shown in Figure 1B, singledose treatment with MTX alone, at either 5 or 50 mg/kg body weight was tolerated and significantly (p b 0.05) delayed mammary tumor growth in mice with established tumor when compared to that of corresponding untreated control mice. Administration of either higher (500 mg/kg body weight) or lower (0.5 mg/kg body weight) drug doses

211 was ineffective with a substantially lowered range in treatment survival in the former. Moreover, both spleen and tumor cell death to MTX at various drug doses ranging from 500 to 0.005 mg/ml were dose-dependent and titratable to doses as low as 0.5 mg/ml in vitro (data not shown). This suggested that (i) lymphocytes and TSA-HA tumor cell viability were sensitive to MTX treatment in vitro and (ii) at drug concentrations of 50 mg/kg body weight, MTX was both tolerated by tumor-bearing animals and significantly effective in delaying tumor cell growth and progression in vivo.

Co-therapy with MTX augments CD8-mediated type 1 antitumor responses in animals with established breast cancer Since single-dose treatment with cyto-reductive drug or Tc1 effector cell transfer showed significant, yet limited, effects on delaying tumor cell growth and progression in vivo, we next investigated their potential combinatorial effects on adoptively transferred Tc1 effector cells in vivo. Seven days following TS/A-HA tumor challenge, mice were treated i.p. with MTX at drug doses (50 mg/kg body weight) found to be both well tolerated and effective in delaying TS/A-HA tumor cell growth in vivo. Three days later, groups of mice received 1 × 107 Tc1 effector cells intravenously and tumor growth rates and volumes were determined. As shown in Figures 2A and B, treatment with MTX alone effectively delayed tumor growth when compared to groups of untreated control mice. Moreover, in corresponding groups of mice receiving MTX and Tc1 effector cell transfer, both tumor growth rates and volumes were markedly reduced when compared to that of animals receiving either chemotherapy alone, effector cell therapy alone or nothing. This suggested that pre-treatment of tumor-bearing mice with the breast cancer agent MTX can markedly enhance the therapeutic efficacy of Tc1 effector cell-mediated immune responses.

Co-therapy with MTX enhances localization and persistence of adoptively transferred Ag-specific Tc1 effector cells at sites of primary tumor growth Since therapeutic efficacy by adoptively transferred T cells is largely dependent, in part, on the accumulation and

Figure 4 Co-therapy with MTX and Tc1 effector cell transfer enhances endogenous CD8 T cell localization, differentiation and acute activation at sites of primary tumor growth in vivo. Tumors from recipient (Thy 1.1) tumor-bearing mice treated with either MTX alone or MTX and Tc1 effector cell (Thy 1.2) transfer were obtained as described in Figure 3. Single cell suspensions were labeled with antiThy 1.1, anti-CD8, anti-CD44, anti-CD69 or anti-CD25 surface markers. Gates were set on Thy 1.1/CD8/CD44High T cells co-expressing surface markers associated with acute TCR engagement (CD69) and/or early stage T cell activation (CD25) and assessed by multicolor flow cytometry. Numbers indicate the percentage of differentiated (Thy 1.1/CD8/CD44High) tumor-infiltrating endogenous CD8 T cells co-expressing elevated levels of CD25 and CD69 surface Ag. Data are representative of four independent experiments. Untreated and corresponding groups of Tc1 effector cell-treated tumor-bearing mice served as controls. In B, gates were set on Thy 1.1/CD8 T cells and the absolute cell numbers of endogenous CD8 cells co-expressing CD44High were assessed. The absolute cell numbers were calculated as the percentage of positive staining cells × the total number of mononuclear cells per tissue. In parallel studies, gates were set on Thy 1.1/CD8/CD44High T cells and the absolute cell numbers of differentiated T cells co-expressing the surface activation markers CD69 (C) and CD25 (D) were determined. Results are expressed as the mean +/− the SEM of 3–5 mice per group/time point. Untreated and corresponding Tc1 effector cell-treated tumor-bearing mice served as controls. ⁎, p b 0.01 for treated animals versus non-treated animals; ⁎⁎, p b 0.005 for animals receiving combinatorial therapy versus corresponding groups of animals receiving singletreatment therapy.

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Tc1 effector cell transfer and chemotheraphy in breast cancer persistence of responsive transferred donor T cells at the site of tumor growth, we next attempted to assess the localization kinetics and activation state of tumor-infiltrating donor cells following co-treatment with MTX in vivo. Syngeneic mice (n = 6–10 mice/gp) were injected sc in the mammary fat pad region with 2 × 105 TS/A-HA tumor cells. Seven days later when tumors were established, groups of mice were either treated (i.p.) with MTX alone (50 mg/kg body weight) or nothing. Three days later, specified groups of mice received 1 × 107 Tc1 effector cells as described above. Using Thy 1 congenic mice to distinguish between donor (Thy 1.2) and recipient (Thy 1.1) CD8 T cell populations, TS/A-HA mammary tumors were harvested at various time intervals following donor cell transfer and Tc1 cells were enumerated by multicolor flow cytometric analysis. As shown in Figure 3A, donor Tc1 cells (Thy 1.2/ CD8), among tumor-bearing mice receiving Tc1 therapy alone, progressively accumulated at the tumor site as early as day 2 post T cell transfer. However, in Tc1-treated mice receiving MTX 3 days earlier, donor cell numbers not only accumulated by day 2 but also increased nearly 3-fold by day 4 and remained elevated for up to 16 days post Tc1 treatment. Moreover, such tumor-infiltrating Tc1 cells remained CD44High and co-expressed elevated proportions (b 98%) of the acute cell surface activation marker CD25 at both early (days 1–8) and late (days 12–20) stages following co-therapy (Fig. 3B-top panel). Interestingly, differentiated donor cells in corresponding groups of tumor-bearing mice receiving Tc1 cell transfer alone showed diminished levels of CD25 expression at sites of tumor growth. Although mice receiving Tc1 donor cells alone maintained elevated levels of the differentiation surface Ag CD44, the proportion of CD25 co-expression among these cells at both early and late time points at sites distal (spleen) to primary tumor growth was substantially diminished when compared to that of corresponding groups of tumor-bearing mice receiving both MTX and Tc1 cotherapy (Fig. 3B). This suggested that co-treatment with MTX can enhance differentiated Tc1 effector cell localization, persistence and acute activation at sites both proximal and distal to mammary tumor cell growth in vivo.

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Co-therapy with Tc1 effector cell transfer and MTX enhances endogenous differentiated CD8 T cell localization, persistence and activation at sites of primary breast tumor growth in vivo Since prior treatment with MTX enhanced donor Tc1 effector cell localization, persistence and antitumor responses, we next investigated such combinatorial treatments and their effects on endogenous CD8 effector cell localization and phenotype at sites of primary tumor growth. Using Thy 1 congenic mice and multiparameter flow cytometry, endogenous tumor-infiltrating CD8 T cells (Thy 1.1/CD8) were quantitatively and qualitatively assessed for cell surface markers associated with effector/memory cell differentiation (CD44High) in vivo [29,30]. As shown in Figures 4A and B, the frequency and cell number of endogenous differentiated T cells (Thy 1.1/CD8/ CD44High) among groups of mice receiving Tc1 cell transfer alone were significantly elevated at early stages (days 1–8) following treatment when compared to that of corresponding cells in mice either untreated or treated with MTX alone. However, the frequencies and numbers of such cells in mice receiving both MTX and Tc1 cell transfer were comparatively greater at corresponding time points. Similar results were obtained in corresponding groups of mice at later time points (days 12–20) receiving such therapies (Figs. 4A and B). In parallel studies, we further assessed endogenous effector/memory CD8 T cell subpopulations (Thy 1.1/CD8/ CD44High) for surface marker expression associated with acute TCR engagement (CD69) and/or early stage T cell activation (CD25) in vivo [30,35]. Aside from mice receiving MTX alone, all groups of mice showed that nearly all tumor-infiltrating Thy 1.1/CD8/CD44High cells co-expressed both CD25 and CD69 at both early and late stages following treatment in vivo (Fig. 4A). Moreover, tumor-bearing mice receiving MTX prior to Tc1 cell transfer showed markedly elevated levels in both frequency and cell numbers when compared to groups of mice receiving either MTX alone, Tc1 cell transfer alone or no treatment at corresponding time points following treatment (Figs. 4A–D). This suggested that single-dose MTX in combination with adoptively transferred Tc1 effector cells can effectively facilitate and/or enhance the persistence and acute activation of endogenous effector/memory CD8 T cell subpopulations

Figure 5 Combinatorial treatment of tumor-bearing mice with MTX and Tc1 effector cell transfer influences the localization and activation of differentiated CD4 effector T cell subpopulations at sites of primary tumor growth in vivo. Tumors from recipient (Thy 1.1) tumor-bearing mice treated with either MTX alone or MTX and Tc1 effector cell (Thy 1.2) transfer were obtained as described in Figure 4. Single cell suspensions were labeled with anti-Thy 1.1, anti-CD4, anti-CD44, anti-CD69 or anti-CD25 surface markers. Gates were set on Thy 1.1/CD4/CD44High T cells co-expressing CD25 and CD69 surface markers and assessed by multicolor flow cytometry. Numbers indicate the percentage of differentiated (Thy 1.1/ CD4/CD44High) tumor-infiltrating endogenous CD4 T cells co-expressing elevated levels of CD25 and CD69 surface Ag. Data are representative of four independent experiments. Untreated and corresponding groups of Tc1 effector cell-treated tumorbearing mice served as controls. In B, gates were set on Thy 1.1/CD4 T cells and the absolute cell numbers of endogenous CD4 cells co-expressing CD44High were assessed. The absolute cell numbers were calculated as the percentage of positive staining cells × the total number of mononuclear cells per tissue. In parallel studies, gates were set on Thy 1.1/CD4/CD44High T cells and the absolute cell numbers of differentiated T cells co-expressing the surface activation markers CD25 (C) and CD69 (D) were determined. Results are expressed as the mean +/− the SEM of 3–5 mice per group/time point. Untreated and corresponding Tc1 effector cell-treated tumor-bearing mice served as controls. ⁎, p b 0.01 for treated animals versus nontreated animals; ⁎⁎, p b 0.05 for animals receiving combinatorial therapy versus corresponding groups of animals receiving single-treatment therapy.

214 that potentially affect, in part, both early and late stages of Ag-specific CD8-mediated type 1 antitumor responses at sites of primary tumor growth.

Co-therapy with MTX modulates the localization kinetics and activation of differentiated tumorinfiltrating CD4 effector T cell subpopulations that influence adoptively transferred Ag-specific Tc1mediated antitumor responses in vivo Since CD4 T cells and their subpopulations may have different sensitivities to various chemotherapeutic drugs that may affect their functional and immunoregulatory potentials [36–40], we next investigated the localization kinetics, persistence and activation of differentiated CD4 T cells following combinatorial treatment with MTX in vivo. As shown in Figures 5A and B, mice receiving MTX alone showed a significant decrease in the cell number and frequency of differentiated Thy 1.1/CD4/CD44High T cells at both early (days 1–8) and late (days 12–20) stages following treatment when compared to that of corresponding groups receiving MTX and Tc1 cell transfer or Tc1 cell transfer alone. Moreover, groups of mice receiving combinatorial therapy showed a significant decrease in corresponding T cell numbers when compared to that of mice receiving Tc1 cell transfer alone. In parallel studies, we further evaluated select subpopulations of differentiated CD4 T cells as defined by effector cell surface markers associated with TCR-mediated activation. As shown in Figures 5A and C, groups of mice receiving either combinatorial treatment, Tc1 cell transfer alone or nothing showed that nearly all (N 92%) differentiated CD4/CD44High T cells co-expressed CD25 surface Ag at both early and late time points following treatment. However, in mice receiving MTX alone, the proportion and cell numbers of tumorinfiltrating CD4/CD44High/CD25+ T cells were markedly lower at early time points when compared to that of corresponding treated mice. Interestingly, at later time points, the frequency of such cells in MTX-treated animals appeared to recover with higher proportions of CD4/CD44High Tcells co-expressing CD25. Similar results were obtained with endogenous Thy 1.1/CD4/ CD44High T cells co-expressing CD69 in these same animals (Figs. 5A and D). These results further correlated with the observation that co-therapy with MTX and Tc1 cell transfer induced lower tumor growth rates than either single treatment alone (Fig. 5). Collectively, this suggested that prior cotreatment with MTX can modulate both the initial/early localization and persistence of “select” differentiated CD4 effector T cell subpopulations in vivo and further influence/ enhance adoptively transferred Ag-specific Tc1-mediated antitumor responses within the primary tumor environment.

Co-therapy with adoptively transferred tumorreactive Tc1 effector cells and MTX enhances endogenous T cell-derived IP-10 expression within the tumor environment that correlates with both endogenous T cell localization and delayed mammary tumor growth Interferon inducible protein 10 (IP-10) has been shown to be expressed by T cells that aid in enhancing local type 1-like T cell-mediated immune responses at sites of inflammation

M.J. Dobrzanski et al. [41–47]. Since we have previously shown that adoptively transferred Ag-specific Tc1-mediated antitumor responses were notably dependent on IFN-γ and profoundly facilitated the local accumulation of phenotypically distinct endogenous T cell subpopulations within the tumor environment [29– 31], we next assessed the expression kinetics of endogenous T cell-derived IP-10 following co-therapy with Tc1 cell transfer and MTX. Endogenous T cells (Thy 1.1) were further enriched by treatment with anti-heat-stable antigen (J11D), anti-MHC class II (D3.137, M5114, CA4), and anti-Thy 1.2 (HO13.14 and F7D5) mAbs and complement for elimination of adoptively transferred donor cells and other non-T immune cell populations from either untreated or treated tumorbearing mice. Recovered cells were re-stimulated with plate-bound anti-CD3 mAb, and IP-10 chemokine mRNA was detected by RNase protection assays and normalized against the L32 housekeeping gene as Relative Units for comparative analysis. As shown in Figure 6, groups of untreated TS/A-HA tumor-bearing mice showed an early elevation in IP-10 gene expression that precipitously declined with tumor progression. In contrast, Tc1-treated tumor-bearing mice showed a similarly early and more prolonged elevation of such T cellderived chemokine in vivo. However, in Tc1-treated mice receiving MTX 3 days earlier, levels of IP-10 production were increased and remained elevated at both early and late stages following therapy. Animals receiving MTX alone

Figure 6 Endogenous T cell-derived IP-10 chemokine gene expression profiles in mammary tumors following co-therapy with Tc1 cell transfer and MTX. Mammary tumors from mice treated with either MTX alone or MTX and Tc1 (Thy 1.2) cell transfer (n =2/time point/experiment) were harvested at various time intervals and single cell suspensions were obtained as described in Figure 3. Endogenous T cells (Thy 1.1) were further enriched by treatment with anti-heat-stable antigen (J11D), anti-MHC class II (D3.137, M5114, CA4), and anti-Thy 1.2 (HO13.14 and F7D5) mAbs and complement for elimination of adoptively transferred donor cells and other non-T immune cells. Resulting T cell populations were cultured with either nothing or plate-bound anti-CD3 for 5 h at 37 °C. Tcell cultures were harvested and total RNA was prepared as described in Materials and methods. IP-10 chemokine mRNA was detected by RNase protection assays and normalized against the L32 housekeeping gene as Relative Units for comparative analysis. Untreated and corresponding groups of Tc1 effector cell-treated tumor-bearing mice served as controls. Results are expressed as the mean+/−the SEM of values derived from three to four mice per group per designated time point in two independent experiments. ⁎, p b 0.01 for treated animals versus non-treated animals; ⁎⁎, pb 0.05 for animals receiving combinatorial therapy versus corresponding groups of animals receiving single-treatment therapy.

Tc1 effector cell transfer and chemotheraphy in breast cancer showed a diminished level of T cell-derived IP-10 production at both corresponding early (days 1–8 post Tc1 cell transfer) and late (days 12–20 post Tc1 cell transfer) stages when compared to that of other treated tumor-bearing animals. Moreover, IP-10 production among corresponding non-stimulated control cultures (no anti-CD3 restimulation) was negligible (data not shown). This suggests that MTX administration prior to adoptively transferred Tc1 effector cell transfer effectively promoted, maintained and facilitated the accumulation of endogenous T cells expressing upregulated levels of IP-10.

Discussion Using a previously described murine transgenic T cell-tumor immunotherapy model, we show that single-dose treatment with MTX or Tc1 effector cell transfer alone can induce significant, although limited, tumor regression among mice with established breast tumors. However, in mice treated with MTX, 3 days prior to Tc1 effector cell transfer, we show a markedly enhanced effect in Ag-specific type 1 CD8mediated antitumor responses that resulted in substantially enhanced delays in tumor growth and progression. Our results showed that prior treatment of tumor-bearing animals with single-dose MTX resulted in not only enhanced donor Tc1 effector cell accumulation at sites of primary tumor growth, but also promoted elevated levels of donor cells co-expressing the cell surface activation marker CD25. Tumor regression following chemotherapy has been shown to result in an elevation of both Ag and pro-inflammatory factors released from dead or dying tumor cells that can profoundly influence T cell recruitment, local cell activation and function [36–40]. Although such drug-mediated cell death mechanisms are beyond the scope of this study, others have shown that cytoablative drug treatments can result in substantial tumor de-bulking and enhancement of lymphoid space within the tumor environment. Subsequently, this would enable adoptively transferred Tc1 cells the opportunity to localize, expand and functionally orchestrate type 1-like immune responses at sites of tumor growth [36–40,48]. Alternatively, differences in the nature of MTX-mediated tumor cell death may also influence ensuing antitumor immune responses that may also affect local T cell activation and function in vivo [36,49]. For example, non-apoptotic death pathways which include necrosis, autophagy and mitotic devastation are differentiated by particular combinations of morphological and biochemical changes that may influence T cell-mediated responses. Alternatively, pre-treatment with cytoablative drugs may increase tumor cell sensitivity through apoptosis that may down-regulate tumor cell survival factors or enhance T cellmediated killing by up-regulating expression of tumor-associated death receptors, such as CD95 that elevate Fas-dependent CTL lysis [36,49–51]. In either instance, our data show that prior treatment with MTX augmented adoptively transferred Ag-specific Tc1 effector cell localization, persistence and activation at the sites of primary tumor growth that appeared to result in the emergence and enhancement of a more favorable environment for adoptively transferred CD8 type 1-like immune responses in vivo. Since select T cells and their subpopulations have the capacity to regulate antitumor responses [16–20], we assessed the

215 infiltration and presence of differentiated T effector cells at different stages of breast tumor progression following combinatorial therapy with both MTX and Tc1 cell transfer. Our results show that co-therapy with MTX, prior to Tc1 cell transfer, diminished “early” infiltration and localization levels of differentiated endogenous CD4/CD44High TIL cell subpopulations co-expressing CD25. While CD25 is generally regarded as an activation marker, it has also been demonstrated that such CD4+ and CD8+ T cell subpopulations are immunosuppressive and can effectively down-regulate immune responses in vivo [16– 20,23]. Our observations of differences in cell numbers of differentiated CD4 and/or CD8 T cells co-expressing CD25 and/ or CD69 at early stages following single-dose therapy with MTX alone may indicate a select modulation in regulatory T cell subpopulations undergoing acute activation via TCR-mediated mechanisms. Subsequently, such changes in the local cellular dynamics following Tc1 effector cell co-therapy suggests that MTX-mediated ablation and/or modulation of such T cell subpopulations, may aid in promoting more effective Tc1mediated antitumor responses at sites of primary tumor growth. Alternatively, in the absence of MTX, Tc1 effector cell transfer may induce and/or maintain the recruitment, accumulation and early localization of phenotypically different regulatory CD4 and CD8 Tcells that may actually promote local immunosuppression and aid in promoting tumor progression by maintaining immune tolerance to tumor cell growth through “active” homeostatic immune mechanisms [21–23,52]. Aside from changes in the local balance and type of endogenous effector TIL cells following combinatorial treatment, enhanced therapeutic efficacy by such infiltrating T cells may be further dependent on different spatial and temporal patterns of cell recruitment and Ag encounter during the effector phase of Tc1-mediated antitumor responses [8,11,52,53]. Moreover, such endogenous effector T cell subpopulations and their immunoregulatory factors may be responsible for recruiting other functionally diverse immune cell repertoires within the tumor environment that may aid in promoting and extending adoptively transferred Tc1-mediated antitumor responses. Thus, our observations on the early cytoablative effects of MTX on differentiated CD4 and CD8 tumor-infiltrating T cell subpopulations may enhance not only donor Tc1 effector cell recruitment and activation, but also enhance participation of innate and/or non-T cell populations, such as tumor-reactive NK, NKT effector cells and macrophages, that may further facilitate more effective endogenous T cell responses. Differences in the local cellular dynamics may further induce changes in the balance of inflammatory mediators, cytokines and chemokines induced by adoptively transferred Ag-specific Tc1 cells [44–46,54–57]. The chemokine, IP-10, has been shown to play distinct roles in the regulation of local immune responses by influencing the balance between pro-inflammatory and antiinflammatory T cells through specific cellular recruitment mechanisms [44–47]. Moreover, IP-10 chemokine expression has not only been shown to be preferentially regulated by local IFN-γ production, but also enhances tumor regression by promoting type 1-like tumor immunity via Th1 or Tc1 cell recruitment [41–45,58,59]. In this study, we show that IP-10 production by endogenous T cells, following administration of both MTX and IFN-γ-producing CD8 effector cells, was markedly up-regulated at both “early” and “late” stages following Tc1 cell transfer. Moreover, such up-regulation of T cell-derived IP-10

216 within the tumor environment appeared to correlate with the appearance and accumulation of more “differentiated” and/or “acutely activated” endogenous T lymphocyte subpopulations. It is interesting to suggest that the emergence of such endogenous Tcell subpopulations and their capacity to produce IP-10 may further orchestrate and/or facilitate tumor infiltration by various Tcell repertoires that may further promote type 1 CD8-mediated antitumor responses. Alternatively, IP-10 has been shown to profoundly affect tumor vascularization in a dose-dependent manner [41,43,59]. The “late” emergence and elevation of such endogenous IP-10 producing Tcell populations may represent regulatory T cells that utilize chemokine production to subsequently diminish tumor vascularization that may directly affect either tumor cell growth or keep effector T cell infiltration, function or survival limited at later stages of tumor progression. Overall, our observations suggest that co-therapy with MTX and Tc1 effector cell transfer is initially dependent, in part, on (i) the localization and persistence of activated donor CD8 T cells and their capacity to produce IFN-γ that may enhance cytokine-inducible chemokines, such as IP-10, (ii) contributions by endogenous chemokine-secreting T cells and their quantitative, spatial and/or temporal patterns of chemokine expression within the tumor environment and (iii) MTX-mediated ablation and/or modulation of endogenous TIL cell subpopulations, at select times prior to adoptive T cell transfer may aid in promoting and/or facilitating IP-10 producing T cells at sites of primary tumor growth. Such interactions may potentially enhance CD8-mediated type 1 antitumor responses by modulating T cell recruitment, persistence, fate and function within the tumor environment in vivo. Although we observed substantial delays in tumor growth following combinatorial treatment, complete disease-free responses remained elusive. The resistance of solid tumors to these and other cellular immunotherapeutic strategies has been attributed, in part, to the phenotypic and/or intrinsic properties of the tumor-infiltrating T cell populations, the immunoregulatory microenvironments of the tumor, the presence or absence of pertinent cytokines and/or regulatory cells, such as Th2/Tc2, Th3, NKT cells and macrophages induced by endogenous homeostatic immune responses to progressively growing tumor [12–23]. Alternatively, many breast cancers demonstrate considerable phenotypic heterogeneity during the clinical course of disease, which may in part, contribute to accelerated tumor progression and diminished antitumor responses [1,3,6,7]. Although our studies showed only partial responses and delays in tumor growth, they prompt for the investigation and use of other conventional and/or experimental therapies in combination with T cell immunotherapy to enhance response rates among cancer patients. Collectively, chemotherapy remains the major approach for treatment of advanced stage solid tumors, such as breast cancer. T cell immunotherapy is a less conventional form of therapy and is also rarely curative. Since chemotherapy is generally associated with immunosuppression that would generally negate the benefits of immunotherapy, few studies have investigated the relationship between these treatments. In this study, we show that co-therapy with MTX preferentially influences the infiltration and localization of select endogenous CD4 and CD8 T cell subpopulations and facilitate the enhancement of chemokines that promote type

M.J. Dobrzanski et al. 1 CD8-mediated antitumor responses. We propose that early removal and/or select ablation of acutely activated (CD25 and/or CD69) and/or differentiated (CD44High) CD4 T cell subpopulations directly and/or indirectly affect adoptively transferred Ag-specific Tc1 effector cell fate, persistence and function that further enhance endogenous CD8mediated type 1 antitumor responses to aggressive malignancy. Such co-therapeutic strategies may aid, in part, in enhancing T cell-mediated therapeutic responses to aggressive malignancies and offer insight into potential mechanisms for enhancing T cell immunotherapy in breast cancer patients.

Acknowledgments We thank C. Eaton (Trudeau Institute) and S. Adams (Texas Tech University Health Science Center of Amarillo) for their excellent assistance with the generation of HA-BALB/C TCR transgene-positive mice. We are particularly grateful to Dr Laura Carter for providing the TS/A mammary adenocarcinoma cell line.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clim.2008.03.518.

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