Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Clinical Immunology 111 (2004) 69 – 81 www.elsevier.com/locate/yclim

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer Joyce B. Reome, James C. Hylind, Richard W. Dutton, and Mark J. Dobrzanski * Trudeau Institute, Saranac Lake, NY 12983, USA Received 5 August 2003; accepted with revision 19 November 2003

Abstract Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-g, whereas type 2 cells secrete IL-4, IL10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7 – 14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44High and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44High TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-g-producing Th1 cells. T cells co-expressing DX5 (CD3+/DX5+) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-h, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer. D 2004 Elsevier Inc. All rights reserved. Keywords: Tumor immunity; Cytokines; Chemokines; NK cells; Th1/Tc1; Th2/Tc2 TIL cells; Tumor evasion

Introduction Breast cancer is the second leading cause of cancerrelated deaths among women in North America. Initiation of breast cancer is precipitated, in part, by a combination of oncogenic mutational events that promote genetic instability and unimpeded cellular proliferation [1]. Subsequently, many invasive mammary carcinomas are highly dependent on the production of select growth and regulatory factors derived not only from the malignant cells themselves but also by their surrounding stromal cells [2 – 4]. ConcomiAbbreviations: Tc2/Th2, CD8+ or CD4+T cells producing type 2 cytokines; Tc1/Th1, CD8+ or CD4+T cells producing type 1 cytokines; CD3/DX5, NK-like T cell subpopulations; IP-10, interferon-inducible protein-10; MIF, macrophage inhibitory factor; TGF-h, transforming growth factor-h; TIL, tumor infiltrating lymphocytes. * Corresponding author. Trudeau Institute, Algonquin Avenue, Saranac Lake, NY 12983. Fax: +1-518-891-5126. E-mail address: [email protected] (M.J. Dobrzanski). 1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.11.013

tantly, such progressive malignancies may also be influenced by inflammatory responses induced by tumor infiltrating lymphocytes (TIL) and other inflammatory immune cell populations at the site of tumor growth [5]. Phenotypic and functional analysis of such antitumor immune responses indicate that although tumor-reactive T cells can localize and persist in patients with various cancers, they may be ineffective and/or unresponsive to tumor cells in vivo. This may be due in part to alterations in T cell signal transduction [6,7], inhibition through NKlike cell receptors on CD8 T cells [8,9], or the presence of immunosuppressive cytokines and regulatory T cells [10,11]. Alternatively, many breast cancers demonstrate considerable heterogeneity during the clinical course of disease, which may in part, contribute to accelerated tumor progression and metastases. Nonetheless, the factors involved in breast cancer growth and progression, specifically with respect to tumor and endogenous immune effector cell interactions, remain relatively undefined.

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Both CD4 and CD8 T cell populations predominantly fall into two subpopulations based on cytokine secretion [12 – 14]. Type 1 T cells characteristically secrete IFN-g, whereas type 2 T cells secrete IL-4, IL-5, IL-10, and GM-CSF. Upon stimulation, these cells and their cytokines have been shown to have a potent influence on the antitumor response [15 – 17]. Alternatively, NK T cells represent a third effector T cell subpopulation that secretes similar cytokines. However, such cells have been associated with immunosuppressive functions in some, but not all, tumor types [18]. Collectively, this suggests that potential antitumor TIL cell response repertoires are functionally and phenotypically dynamic and are highly dependent, in part, on tumor etiology, location, and stage of tumor maturation/progression. These observations provide a further impetus to characterize endogenous TIL effector cell subpopulations that selectively arise and/or persist in aggressive malignancies, such as breast cancer. In the current study, we evaluated the antitumor effects and biological activities of endogenous type 1 and type 2 effector T cell subpopulations at primary sites of mammary tumor growth among mice with progressive malignancy. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of TIL cell subpopulations in mice orthotopically challenged subcutaneously in the mammary region. We discuss the potential implications of select effector T cell subpopulation recruitment and presence of immunoregulatory factors (i.e., chemokines/cytokines) within the tumor environment at different stages of tumor maturation that contribute, in part, in regulating effective endogenous antitumor responses that result in aggressive breast malignancies.

Evaluation of tumor growth in vivo Syngeneic female BALB/c mice were injected subcutaneously with 0.2 ml of a single-cell suspension containing 1  105 TS/A adenocarcinoma cells in the right anterior mammary region. Tumors were measured every 3 or 4 days following tumor challenge using vernier calipers. Tumors volumes were obtained by multiplying the measured length by the measured width by the calculated mean of these measured values and were presented as the mean F SEM. For detection of disease progression and metastases, cytospin preparations of single cell suspensions from spleen, lung, and draining lymph nodes were obtained, fixed with methanol, and stained with eosin and methylene blue (Fisher, Pittsburgh, PA). Although tumor cells appeared heterogeneous in size, they were easily differentiated as predominately larger cells with an elevated nuclear to cytoplasm ratio. Counts were performed on a total of 200– 300 cells on coded slides. Cell preparation At specified time intervals, mice were sacrificed and single tumor cell suspensions were obtained by mechanical dispersion through nylon mesh screens in RPMI 1640– 5% FCS. After three washes in RPMI 1640 –5% FCS, lymphoid cells were resuspended in RPMI 1640 (Gibco, Grand Island, NY), supplemented with 2 mM pyruvate, 100 units/ml penicillin, 100 Ag/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated fetal calf serum (FCS; Gibco). Flow cytometric analysis

Materials and methods Animals Female BALB/c mice (H-2d), 6 to 10 weeks of age, were obtained from the Animal Breeding Facility at the Trudeau Institute. Animals were maintained and treated according to animal care committee guidelines of the National Institutes of Health and Trudeau Institute. Tumor cells The weakly immunogenic TSA tumor cell line that is syngeneic to the BALB/c background was kindly provided by Dr. Laura Carter (Wyeth, Cambridge, MA). This highly aggressive tumor cell line was established from a moderately differentiated mammary adenocarcinoma that arose spontaneously in the thoracic region of a multiparous BALB/c female mouse [19]. Subconfluent monolayers, in log growth phase, were harvested by addition of 0.25% solution of trypsin in HBSS and washed three times in serum-free HBSS before use in all in vitro and in vivo experiments.

Single-cell suspensions of tumor were obtained and washed three times in a 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. Lymphocytes (106), pretreated with FcR block, were incubated for 20 min on ice with 100 Al of FAB containing 1 Ag of various mAbs conjugated to either Biotin, PE, FITC, or Cychrome. For biotinylated mAbs, streptavidin APC or streptavidin was used as a second step reagent. The mAbs used include anti-CD8 (Caltag Laboratories, Bulingame, CA) or anti-CD4 (Pharmingen), anti-CD44 (Pharmingen, clone IM7), anti-CD3 (Pharmingen), anti-CD122 (Pharmingen), anti-CD25 (Pharmingen), anti-CD69 (Pharmingen), anti-CD95 (Pharmingen), CD19 (Pharmingen), or DX5 (Pharmingen) mAbs. 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). For TSA tumor cells, direct immunoflourescent staining with either FITC- or PE-conjugated-Class I H2-Kd, -Class I H2-Dd, -Class II Iad, CD95, -FasL, or CD44. Ten thousand cells were analyzed

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per sample with dead cells excluded by FSC/SCC profiles. Surface marker analysis was performed using Cell Quest Software and the percent positive and absolute cell numbers were determined.

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.

Intracellular cytokine staining

Statistical analysis

Single-cell suspensions of primary tumor were obtained from mice as described above. Cells were stimulated with either PMA (10 7 M) and ionomycin (1 Ag/ml) or platebound anti-CD3 for 5 h. Two hours before harvesting, brefeldin A (10 Ag/ml) was added to cultures to retain cytoplasmic cytokines. Cells were pretreated with FcR block followed by either Cy-chrome-conjugated anti-CD8 or anti-CD4 (Pharmingen) and FITC-conjugated-anti-CD44 (Pharmingen). Subsequently, cells were fixed with 2% paraformaldehyde followed by intracellular staining in permeabilization buffer containing 0.5% saponin and 1% BSA in PBS, and either PE-conjugated IFN-g, IL-4, IL-10, GMCSF, or TNF-a (BD Pharmingen). Cells were washed and resuspended in 1% BSA/PBS solution and analyzed by flow cytometry. Data were analyzed using CELLQUEST software (Becton Dickinson).

For statistical analysis, the two-tailed Student’s t test or nonparametric Mann – Whitney Rank Sum test was used.

Analysis of tumor cytokine/chemokine mRNA expression Total RNA from either tumor cell cultures or whole tumor tissue at different time points were extracted by tissue homogenation in TRIzol reagent (Gibco). Cytokine/chemokine mRNA expression levels were quantitated using the RiboQuant Multiprobe Ribonuclease Protection Assay (RPA) system (PharMingen) with the mCK-1, mCK-3, and mCK-5 cytokine/chemokine mRNA detection probe

Results Phenotypic characterization of TS/A mammary tumor cells in vitro Since tumor-derived cytokines have been shown to play a potential role in both early tumor cell growth and immune cell regulation [5], we assessed whether these tumor cells produced select pro-inflammatory cytokines associated with tumor progression. Total RNA was prepared from tumor cells in log growth phase and RNase protection assays were performed. As shown in Fig. 1A, tumor cells produced substantial levels of TGF-h1, TGF- h3, and MIF. In contrast, mRNA from other cytokines, such as TGF-h2, IFN-h, TNF-a, LTb, and TNF-h were not detectable, suggesting that TS/A tumor cells selectively produced specific pro-inflammatory cytokines in vitro. In parallel cultures, TS/A tumor cells were harvested to assess tumor cell surface antigen expression by flow cytometry. As shown in Fig. 1B, all tumor cells expressed MHC class I (Kd and Dd) and CD44, but not MHC Class II (I – Ad) or CD95 cell surface antigens. Moreover, smaller proportions

Fig. 1. TSA mammary tumor cells express select pro-inflammatory cytokine genes in vitro. TSA tumor cells (2  106) were cultured and harvested at various time intervals. In A, tumor cells were harvested and mRNA was prepared as described under Materials and methods. Pro-inflammatory cytokine mRNA was detected by RNase protection assays using the mCK-1 multiprobe template set. Values were normalized against the L32 housekeeping gene for direct quantitation. B, cells from corresponding wells were labeled with either FITC-conjugated mAbs specific for MHC Class I and Class II or PE-conjugated mAbs for CD95, FasL or CD44. Live cells were distinguished by forward/side light scatter profiles and analyzed by flow cytometry. Isotype controls are shown as unshaded curves. Results are representative of two similar experiments.

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of tumor cells expressed low, yet detectable, cell surface levels of Fas L. Results are representative of two independent experiments. Assessment of tumor infiltrating lymphocyte (TIL) kinetics in mice at various stages of tumor cell maturation in progressive breast cancer To assess potential endogenous T cell responses to progressive breast cancer, we characterized, in part, a murine orthotopic mammary tumor model using the TS/A adenocarcinoma. Local disease was established in normal syngeneic BALB/c mice by a unilateral subcutaneous injection of 1  105 TS/A tumor cells into the right anterior mammary region. At 3-day intervals following tumor challenge, tumor growth was measured using vernier calipers and tumor volumes were obtained as described in Materials and methods. As shown in Fig. 2A, TS/A tumor cells progressively grew in vivo without evidence of spontaneous regression. Concomitantly, regional lymph node and systemic tumor involvement were determined by either gross or microscopic assessment. Cytospin preparations of single cell suspensions from draining lymph nodes, spleen, and lung tissues at various time intervals showed a markedly detectable and progressive tumor cell infiltration that was initially present in draining lymph nodes by days 18– 21 following tumor challenge (data not shown). This suggested that orthotopically injected TS/A mammary tumor cells undergo spontaneous metastases to regional lymph nodes and systemic organs that is both grossly and microscopically evident at later times (> 18 days) of disease progression. To assess and characterize lymphocyte infiltration kinetics at the site of primary tumor growth at various stages

Fig. 2. Tumor infiltrating lymphocyte (TIL) kinetics during progressive TSA mammary tumor growth. Syngeneic Balb/c mice (n = 8 – 10/gp) were injected subcutaneously with 1  105 TS/A tumor cells in the mammary fat pad region. At 3-day intervals, local tumor volumes were determined as described in Materials and methods. In parallel studies, regional draining lymph node (DLN) involvement from tumor-bearing mice was determined by either gross or microscopic assessment. Cytospin preparations of single cell suspensions from tissues at various time intervals showed that tumor cell infiltration was initially detectable in DLNs by days 18 – 21 post tumor challenge (A). In B, mice (n = 3/time point/experiment) were injected subcutaneously in the mammary region with 1  105 TS/A cells. At various time intervals, tumors were extirpated and single cell suspensions were labeled with FITC-anti-CD3 and PE-CD19 (B) or FITC-anti-CD3 and either Cy-anti-CD4 or-anti-CD8 (C) mAbs. Viable lymphocytes, distinguished by their forward/side light scatter profiles, were analyzed by multicolor flow cytometry. The absolute cell numbers were calculated as the percentage of positive staining cells times the total number of mononuclear cells per tissue. Data are representative of three independent experiments.

Fig. 3. Activated TIL cell subpopulation kinetics during progressive TSA mammary tumor growth. Mice (n = 3/time point/experiment) were injected subcutaneously in the mammary region with 1  105 TS/A tumor cells. At various time intervals, primary tumors were extirpated and single cell suspensions were labeled with Cy-anti-CD8 or-anti-CD4, PE-anti-CD44, and FITC-anti-CD3 mAbs. Gates were set on CD8 and CD4 TIL cell populations and CD44 staining profiles within these populations were assessed by multicolor flow cytometry. Absolute cell numbers of ‘‘activated’’ CD8 and CD4 T cells co-expressing high levels of CD44High are shown. Data are representative of three independent experiments.

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of progressive disease, TS/A tumors were extirpated at various time intervals following tumor challenge and lymphocyte populations were enumerated by multicolor flow cytometric analysis. As shown in Fig. 2B, CD3+ T

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cells infiltrated the site of tumor growth by days 7 – 14 following TS/A tumor challenge and showed a progressive elevation in cell number and frequency with time. In contrast, CD19+ cell numbers were markedly and consis-

Fig. 4. Activated CD8 and CD4 TIL cell surface marker profiles during progressive mammary tumor growth. Mice (n = 3/time point/experiment) were injected subcutaneously in the mammary region with 1  105 TS/A mammary tumor cells. At various time intervals, tumors were extirpated and single-cell suspensions were labeled with Cy-anti-CD8 or-anti-CD4, PE-anti-CD44 and either FITC-conjugated anti-CD69, anti-CD95, anti-CD122, or anti-CD25 mAbs. Gates were set on CD8 (A) or CD4 (B) TIL cell populations co-expressing CD44High, and surface marker staining profiles within these populations were assessed by multicolor flow cytometry. Histogram numbers indicate the percentage of CD8/CD44High or CD4/CD44High TILs expressing specified cell surface activation markers. At corresponding time intervals, tumor volumes were measured as described in Fig. 1A. Data are representative of three independent experiments.

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tently lower at all corresponding time points suggesting that B cells played little, if any, role during progressive TS/A mammary tumor growth. Moreover, the number and the frequency of CD3+ TIL were predominantly CD4+ whereas, CD3+/CD8+ TIL cells were at markedly lower cell numbers and with later kinetics than that of corresponding CD3+/CD4+ TIL subpopulations (Fig. 2C).

Activated CD4 and CD8 TIL cell subpopulation kinetics during progressive TS/A mammary tumor growth Because up-regulated CD44 expression is indicative of both activated effector and long-lived T cells thought to be responsible for effective T cell-mediated tumor immunity [20], we assessed the numbers of CD8+ TIL cells co-

Fig. 5. CD8 and CD4 TIL cell cytokine-releasing profiles during progressive mammary tumor growth. Tumor-bearing mice (n = 3/time point/experiment) were injected subcutaneously in the mammary region with 1  105 TS/A tumor cells. At various time intervals, single-cell suspensions from primary tumors were obtained and cultured with PMA and ionomycin for 4 h in the presence of brefeldin A. Cells were harvested and labeled with Cy-anti-CD8 or-anti-CD4, FITCanti-CD44 and either PE-conjugated anti-IFN-g, anti-IL-4, anti-IL-10, anti-TNF-a, or anti-GM-CSF. Gates were set on CD8 (A) or CD4 (B) T cell populations, and intracellular cytokine staining profiles within these populations were assessed by multicolor flow cytometry. Numbers indicate the percentages of specified TIL cells expressing intracellular IFN-g, IL-4, IL-10, TNF-a and GM-CSF. At corresponding time intervals, tumor volumes were measured as described in Fig. 1A. Data are representative of three independent experiments.

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expressing elevated levels of CD44 surface antigen at various stages of tumor progression. As shown in Fig. 3, the absolute cell numbers and percentages of activated CD4/CD44High TIL cell populations were detectable within 7 –14 days with peak levels at day 21 following tumor challenge. However, at later stages of tumor progression (>day 21), cell numbers and frequencies substantially decreased with time. In contrast, activated CD8/CD44High TIL cell numbers were detectable by day 14 following tumor challenge and further increased with tumor maturation and progression. However, at substantially lower levels and with later kinetics than that of corresponding activated CD4 TIL cell populations. In parallel studies, we further evaluated the accumulation and kinetics of such CD4/CD44High and CD8/CD44High effector TIL cell populations and their state of acute activation at sites of primary tumor growth during progressive mammary disease. As shown in Fig. 4, either CD8/ CD44High or CD4/CD44High TIL effector cell subpopulations co-expressed CD69, CD95, and CD25 surface activation antigens. Interestingly, in spite of the presence and ongoing infiltration by both highly activated CD4 and CD8 effector cells, primary tumor cell growth and progression remained unimpeded.

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influence both antitumor responses and tumor progression in vivo [15 – 17,21,22], we next assessed the cytokinereleasing profiles of CD4+ and CD8+ TIL cell subpopulations from sites of primary mammary tumor growth at various stages of disease progression. Type 1 (IFN-g) and type 2 (IL-4, IL-10, and GM-CSF) T cell cytokine profiles were assessed at the single cell level by intracellular staining and flow cytometry. As shown in Figs. 5A and 6, the frequency and cell numbers of CD8+ TIL cells producing either type 1 (Tc1) or type 2 (Tc2)-related cytokines, at specified time points following tumor challenge, were similar with noticeably elevated levels at day 21. In contrast, the proportions and cell numbers of CD4+ TIL cells predominantly producing IL-4 (Th2) and TNF-a were markedly higher than that of corresponding populations producing IFN-g, IL-10, and GM-CSF (Figs. 5B and 6). Moreover, peak intracellular cytokine levels in the former occurred at earlier time points (day 14) when compared to that of the latter (day 21). This suggests that IL-4-producing Th2 immune responses were elicited and with earlier kinetics than that of IFN-g-producing Type 1 responses during progressive TS/A mammary tumor growth.

Assessment of CD4 and CD8 TIL cell cytokine-releasing profiles during progressive mammary tumor growth

Kinetics and cytokine-releasing profiles of distinct CD3+ TIL cell subpopulations expressing DX5 during progressive mammary tumor growth

Since type 1 and type 2 CD8 and CD4 T cell subpopulations and their cytokines have been shown to

Since NK T cells represent a third effector T cell population that has been associated with both agonistic

Fig. 6. CD8 and CD4 cytokine-releasing TIL cell subpopulation kinetics during progressive TSA mammary tumor growth. Tumor-bearing mice (n = 3/time point/experiment) were injected subcutaneously in the mammary region with 1  105 TS/A tumor cells. At various time intervals, tumor cell suspensions were obtained and cultured for intracellular cytokine staining, as described in Fig. 5. Gates were set on either CD4 or CD8 TIL cell populations, and intracellular cytokine staining profiles within these populations were assessed by multicolor flow cytometry. Data are presented as the absolute cell numbers for either CD4 or CD8 TIL cells expressing intracellular IFN-g, IL-4, IL-10, TNF-a and GM-CSF. Data are representative of three independent experiments.

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Fig. 7. CD3/DX5 TIL cell kinetics and cytokine-releasing profiles during progressive mammary tumor growth. Mice were injected subcutaneously in the mammary region with 1  105 TS/A mammary tumor cells. (A) Primary tumors at a representative time point were extirpated and single cell suspensions were labeled with anti-DX5 mAb and either anti-CD3, anti-CD4 or anti-CD8 mAbs. Lymphocytes, distinguished by their forward/side light scatter profiles, were analyzed by multicolor flow cytometry. Numbers indicate the percentage of CD3, CD4, and CD8 TIL cells co-expressing DX5. (B) Primary tumors were extirpated at various time intervals and single cell suspensions were labeled with CY-anti-CD3, APC-Biot-anti-DX5, and either PE-conjugated anti-IFN-g, antiIL-4, anti-IL-10, anti-TNF-a, anti-GM-CSF. Gates were set on CD3 and DX5 TIL cell populations, and intracellular cytokine staining profiles within these populations were assessed by multicolor flow cytometry. Histogram numbers indicate the percentage of CD3/DX5 TIL cells expressing intracellular IFN-g, IL4, IL-10, TNF-a, and GM-CSF. At corresponding time intervals, tumor volumes were measured as described in Fig. 1A. (C) Data are shown as the absolute cell numbers of cytokine-releasing CD3/DX5 T cell subpopulations at specified times and were calculated as previously described. Data are representative of three mice per time point in two independent experiments.

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and antagonistic roles in tumor regression [18], we next investigated the accumulation and kinetics of CD3+ TIL cell subpopulations co-expressing DX5 at sites of primary tumor growth. Using multicolor flow cytometric analysis, CD3+ TIL cells, co-expressing DX5 cell surface markers, were detectable in tumors of mice with progressive disease (Fig. 7A). Whereas, levels of corresponding DX5-expressing cells among CD4+ or CD8+ TIL cell populations were negligible. As shown in Figs. 7B and C, high proportions of low, yet detectable, CD3+/DX5+ T cell numbers producing IFN-g, IL-4, IL-10, TNF-a, and GM-CSF were detectable by day 14 following tumor challenge. However, with time (>21 days post tumor challenge), numbers of CD3+/DX5+ T cells producing IFN-g, IL-4, IL-10, TNF-a, and GM-CSF were markedly elevated and correlated with progressive tumor cell growth, suggesting a recruitment of select ‘‘regulatory’’ cytokine-producing NK-like T cell subpopulations at later stages of primary tumor progression.

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Selected up-regulation of IP-10, MIF, and TGF-b gene expression at primary tumor sites during progressive disease Since chemokines and select cytokines have been previously shown to regulate and influence both immune function and tumor growth [23,24], we next investigated the expression of various chemokine/cytokine genes in primary tumors at various stages of mammary tumor progression. RNase protection assays were performed to assess intratumoral chemokine gene expression among groups of mice at various time intervals following tumor challenge. As shown in Fig. 8 (upper panel), tumors among TS/A tumor-bearing mice showed an early (days 4– 7 post-tumor challenge) and preferential elevation in the antiangiogenic chemokine IP-10. However, gene expression levels of IP-10 were markedly lower among groups of mice at later time points of tumor progression. In contrast, the immunoregulatory cytokine MIF, and to a lesser extent TGF-b3, showed a consistent and elevated level of gene expression at all time points tested (Fig. 8, lower panel). This suggested that within the tumor environment, select chemokine and/or cytokine genes are differentially up-regulated at different stages of tumor growth that may aid, in part, with immune cell localization/regulation and tumor cell progression in vivo.

Discussion

Fig. 8. Up-regulation and kinetic profiles of select chemokine and cytokine genes during progressive mammary tumor growth. Mice (n =2/time point/ experiment) were injected subcutaneously in the mammary region with 1  105 TS/A mammary tumor cells. At various time intervals, tumors were extirpated and total RNA from whole tumor homogenates was prepared. Both chemokine (upper panel) and pro-inflammatory cytokine (lower panel) mRNA was detected by RNase protection assays and normalized against the L32 housekeeping gene as relative units for comparative analysis. Data are representative of two independent experiments.

In the current study, we assessed the phenotype and kinetics of endogenous tumor infiltrating effector T cell subpopulations at sites of primary tumor growth at different stages of mammary tumor maturation and progression. We show that T cells do not infiltrate the tumor site until days 7 –14 following tumor challenge. Both CD4 and CD8 TILs were predominately CD44High and expressed CD25, CD95, and CD69 cell surface acute activation markers. Activated CD4/CD44High TIL cell subpopulation numbers and frequencies reached peak levels at day 21, then markedly diminished with time whereas corresponding CD8 TIL cell numbers progressively increased, however, at lower levels and with later kinetics. Cytokine-releasing profiles of CD4 TILs showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFNc-producing Th1 cells. Over time (>21 days post tumor challenge), numbers of T cells co-expressing DX5 (CD3+/ DX5+) and producing IFN-c, IL-4, IL-10, and GM-CSF were markedly elevated, suggesting a recruitment of NKlike effector T cells at later stages of tumor progression. Moreover, tumors selectively produced TGF-b, MIF, and IP-10 in vivo at times as early as day 4, with peak levels at day 7 following tumor challenge. Such chemokines/cytokine gene expression at sites of primary tumor growth remained markedly elevated and correlated with a continued progression in tumor growth and metastases.

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Effective tumor regression in both experimental and human breast cancer studies have shown to be highly dependent, in part, on the trafficking and localization of both CD4 and CD8 TIL cell populations at the site of primary tumor growth. Although our studies show that there was a delay in T cell infiltration and select differences in localization kinetics among effector T cell subpopulations at the site of tumor growth, both CD4 and CD8 TIL cells appeared highly activated yet ineffective in preventing tumor progression. One possible explanation is that preexisting tolerance prevents rapid activation and/or delays in expansion of select high affinity tumor-reactive T cells required for successful tumor regression. This would result in a state of immune ignorance and/or anergy among tumor infiltrating lymphocyte subpopulations and subsequent tumor growth and progression [5,25 – 27]. Alternatively, progressive malignancy may also be attributed to the type of endogenous immune response elicited at the site of primary tumor growth. Although multiple hypotheses have been proposed to explain why tumors grow unimpeded in the presence of ‘‘activated’’ TIL cell populations in vivo, it has been suggested that one mechanism may be due, in part, to a lack of effective induction or maintenance of type 1 T cell responses [28 –32]. However, some tumor models have demonstrated favorable contributions by type 2-like immune responses in tumor rejection [16,17,21]. Notably, in studies conducted by Forni et al. [33 –35] using mice challenged subcutaneously in the flank with TS/A, host-derived immune responses were successfully elicited in TS/A tumor-bearing mice through a variety of effective cytokine and whole gene-modified tumor cell vaccine strategies utilizing in part, IL-4 and type 2-like immune responses. In contrast, our experiments assessing local TIL cell response kinetics and immunosurveillance in the mammary region of untreated TS/A tumor-bearing mice show that greater numbers of endogenous IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFNc-producing Th1 and Tc1 TIL cell subpopulations. Furthermore, these effector cell cytokine-releasing profiles and kinetics appeared to correlate with progressive tumor growth and metastases among such untreated tumor-bearing mice. Conceivably, these discrepancies between the deleterious versus favorable effects of type 2-like immune responses in this tumor model could be related to differences in spatial and/or temporal patterns of cytokine expression and/or local responding cell population differences during the priming and/or effector phases of the endogenous antitumor immune response [36 – 38]. For example, Bronte et al. [39 – 41] have shown that IL-4 can selectively upregulate the activity of myeloid suppressor cells that may control, in part, T cell function and antitumor responses. Moreover, differences in tumor location, maturation, site, and dose of vaccination employed may also contribute to enhancing different antitumor responses. Nonetheless, we suggest that this initial rise in IL-4-producing Th2 cells may antagonize and/or delay the emergence of a more favorable

type 1 T cell immune response pathway among untreated TS/A tumor-bearing animals. Aside from ‘‘early’’ IL-4 production by Th2 TILs, CD8 Tc2 effector TIL cell subpopulations also localized and produced elevated levels of both IL-4 and tumor-potentiating IL-10 at later time points of tumor progression. Thus, suggesting that failure to protect and initiate effective tumor regression may be due, in part, to the nature and kinetics of the endogenous immune response and not its absence. It has long been thought that suppressor cells play a role in the progression of cancer [42]. Since many progressive malignancies have been shown to be influenced by inflammatory responses induced by tumor-infiltrating lymphocytes (TIL) and/or other inflammatory immune cell populations at the site of primary tumor growth, we assessed the presence of potential regulatory effector cells that may, in part, promote tumor progression. Recently, it has been demonstrated that a subpopulation of suppressor cells manifest the phenotype CD3/CD4/CD25 [43 – 46] that may prevent endogenous immune responses to autoantigens, such as tumor antigens, and subsequently lead to failure of tumor immunosurveillance and/or enhanced tumor growth. In our studies, using an orthotopic breast cancer model, we show that a large proportion of CD3/CD4 T cells co-expressing CD25 are present and remain at elevated levels as early as day 14 post tumor challenge. Although CD25 is generally regarded as an activation marker, the elevated numbers of CD4/CD25 TIL cell subpopulations observed in our studies may indicate the existence of intrinsic immune responses that promote local immunosuppression and aid in inducing or maintaining tolerance among tumor-bearing mice. We are currently investigating whether removal of these select T cell subpopulations will enhance endogenous antitumor responses that control mammary tumor progression. Others have shown that NK T cells existing among TIL cell populations can suppress the local generation of tumorreactive T cells, and as a result, facilitate the outgrowth of tumor in vivo [47 – 49]. Interestingly, at later stages of tumor maturation (>21 days post tumor challenge), endogenous NK-like effector T cell numbers (CD3/DX5) emerged with a concomitant decrease in CD4 TIL cell numbers and an elevation in tumor cell growth and progression. Moreover, we show that upon restimulation, CD3/DX5 cells have a remarkable capacity to produce the potent immunoregulatory cytokines IL-4, IL-10, GM-CSF, IFN-c, and TNF-a that can influence not only the nature of the local immune response but also survival and differentiation of other effector T cell subpopulations present within the tumor [5,18,20]. However, in other tumor models, it has been suggested that NK T cells also express a wide variety of cell death-inducing effector molecules and have further been shown to aid, in part, in efficient tumor eradication [18]. Although we are not in a position to preferentially support either view, our data indicate that such late-emerging NKlike effector T cell subpopulations are unable to directly or indirectly control progressive tumor growth. Investigations

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to elucidate the role of CD3/DX5 effector T cell subpopulations and their regulatory role during progressive breast cancer are currently underway. It has been previously shown that many invasive mammary carcinomas are highly dependent on the production of select growth and regulatory factors derived not only from malignant cells themselves but also by their surrounding stromal cells [2– 4]. Such factors may not only induce local suppression of tumor-reactive T cell trafficking, activation, and function, but may also affect both immune and tumor cell survival and proliferation, tumor neo-angiogenesis and subsequent tumor progression. Our current studies show that TS/A mammary carcinoma cells selectively produce substantial and prolonged amounts of the pro-inflammatory cytokines TGF-b and MIF at the sites of primary tumor growth that correlate with tumor progression and metastases in mice with established tumor. It has been shown in other tumor models that TGF-b can directly inhibit both T cell differentiation and CTL generation among CD8 T cells in vivo [11,50]. Moreover, others have shown that elevated levels of MIF in vivo may provide additional mechanisms for tumor evasion by influencing CD8 T cell survival and migration and suppressing the generation of type 1-like immune responses to promote a less favorable type 2-like antitumor response at the site of tumor growth [51 – 54]. This may explain, in part, our observations that CD8 T cells infiltrate the tumor at later times than that of CD4 T cell populations and that higher numbers of IL-4 producing Th2 cells emerge at the site of tumor growth with earlier kinetics than that of IFN-c producing Th1 subpopulations. It is interesting to speculate that established MHC Class I+/Class II mammary tumor cells can initially induce less effective Th2 CD4 effector T cell subpopulations and concurrently down-regulate CD8 effector T cell infiltration. This would suggest that the tumor may initially orchestrate infiltration of select effector cell subpopulations that are less effective in tumor eradication and thus support further tumor growth and maturation. The failure to reject mammary tumor growth and progression may be due, in part, to the nature and kinetics of the endogenous immune response that may be influenced by tumor-mediated evasion mechanisms. Various chemokines/cytokines have been shown to play distinct roles in the regulation of local immune reactions by influencing the local balance between pro-inflammatory and antiinflammatory regulatory T cell subpopulations by either the recruitment and/or selective repulsion of T cell subpopulations [55 – 58]. Moreover, tumor-derived chemokines have been shown to illicit, in a concentration-dependent manner, pro-carcinogenic biological functions resulting in either tumor enhancement and/or maturation by regulating tumor vascularization [23,59]. In our current study, we show that the antiangiogenic chemokine, IP-10 is selectively upregulated at early time points following tumor challenge and diminish to substantially lower levels with time and tumor maturation and growth. We suggest that heightened levels of IP-10 within the tumor environment may initially block

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angiogenesis and T lymphocyte infiltration into the tumor. Concomitantly, at early stages of tumor growth (4– 7 days following tumor challenge), the tumor establishes itself by producing select cytokines associated with immunosuppression, such as MIF and TGF-b, that enables ineffective antitumor responses and unimpeded tumor progression. Over time, we show that IP-10 chemokine gene expression is substantially diminished which may, in part, aid in tumor vascularization and metastases at later stages of tumor maturation. This may support, in part, a proposed mechanism for our observations that endogenous tumor-reactive T cells are unable to infiltrate the site of early tumor growth and that tumor metastases are not readily evident until later time points following tumor challenge. Collectively, these findings establish a correlation that preferential effector cell recruitment and production of select immunoregulatory factors within the tumor environment at select stages of tumor maturation may aid, in part, in regulating effective endogenous antitumor responses that result in aggressive malignancy. These studies will aid in understanding breast cancer cell growth, with respect to endogenous immune effector cell interaction, and aid in the development of more effective therapeutics to breast malignancies.

Acknowledgments We are particularly grateful to Dr. Laura Carter for providing the TSA mammary adenocarcinoma cell line. This work was supported by United States Army Medical Research and Development Command Grant DAMD17-011-0429.

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