Blockade of Myeloid-Derived Suppressor Cells after Induction of Lymphopenia Improves Adoptive in a Murine Model of This information is current as of September 27, 2021. Krithika N. Kodumudi, Amy Weber, Amod A. Sarnaik and Shari Pilon-Thomas J Immunol published online 24 October 2012 http://www.jimmunol.org/content/early/2012/10/24/jimmun ol.1200274 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 24, 2012, doi:10.4049/jimmunol.1200274 The Journal of Immunology

Blockade of Myeloid-Derived Suppressor Cells after Induction of Lymphopenia Improves Adoptive T Cell Therapy in a Murine Model of Melanoma

Krithika N. Kodumudi, Amy Weber, Amod A. Sarnaik, and Shari Pilon-Thomas

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor- specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to un- derstand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate and suppressive activity of CD4+CD25+Foxp3+ Tregs and CD11b+Gr1+ MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with Downloaded from docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and . Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8+ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL http://www.jimmunol.org/ activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy. The Journal of Immunology, 2012, 189: 000–000.

elanoma is a leading cause of mortality in the myeloid cells. In mice, these cells are broadly defined as CD11b+ United States, resulting in one American dying every Gr-1+ cells. Recent reports have shown that MDSC by themselves M 60 min. Currently, there are few nonsurgical options for can induce tumor-specific Tregs (10). Increased levels of circu- the treatment of metastatic melanoma (1, 2). Cancer immuno- lating MDSC have been correlated with advanced disease stage therapy, which focuses on the induction of immunity against tu- and extensive metastatic tumor burden in cancer patients (11). by guest on September 27, 2021 mor cells, is a promising approach for the treatment of melanoma. There have been several reports showing that lymphopenia in- A major hurdle in the development of effective immunotherapy is duced by nonmyeloablative chemotherapeutic treatments or total tumor-induced suppression, including factors such as regulatory body irradiation (TBI) can modulate immune responses and in- T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that crease antitumor immunity (12–14). These treatments induce a can limit the effectiveness of tumor-specific T cells (3, 4). Tregs severe lymphopenic condition in the host and leads to the subse- have been shown to suppress antitumor activity and ablation of quent expansion of T cells in the periphery in a process known as this population has led to tumor eradication (5). In preclinical homeostatic proliferation. Homeostatic proliferation can lead to melanoma models as well as in cancer patients, depletion of Tregs the proliferation and activation of T cells specific for self-Ags (15, followed by dendritic cell (DC)‑based vaccination has led to en- 16). Studies have shown increased antitumor responses following hanced vaccine-mediated tumor-specific T cell responses (3, 6, 7). irradiation and adoptive T cell transfer in preclinical murine MDSC are implicated in the maintenance of immune tolerance to models (17, 18). Adoptive transfer of autologous tumor-infiltrating tumors (4, 8, 9). MDSC are a phenotypically heterogeneous cell after lymphodepleting has resulted in population consisting of myeloid progenitor cells and immature objective responses in melanoma patients (12, 19). However, only one-fifth of the treated patients demonstrated complete tumor regressions. Although it has been shown that administration of cy- Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, clophosphamide and fludarabine or TBI in mice before adoptive FL 33612 transfer reduces or eliminates immunosuppressive populations (13), Received for publication January 19, 2012. Accepted for publication September 24, little is known about the effects of TBI on reconstitution of Tregs 2012. and MDSC. Hence, it is important to understand the role of these This work was supported by National Institutes of Health Grant R00 CA128825-01 suppressor populations after the induction of lymphopenia to de- (to S.P.-T.). sign effective immunotherapeutic treatments to achieve complete Address correspondence and reprint requests to Dr. Shari Pilon-Thomas, Department tumor regression. of Immunology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Mag- nolia Drive, Stabile Research Building-2, Tampa, FL 33612. E-mail address: Shari. Conventional anticancer agentshavebeenexploredfortheir Pilon-Thomas@moffitt.org ability to target suppressor populations (20–24). Gemcitabine, a Abbreviations used in this article: CM, complete medium; DC, dendritic cell; DCFDA, nucleoside analog, has been shown to reduce splenic MDSC in 29,79-dichlorofluorescein diacetate; DTX, docetaxel; MDSC, myeloid-derived sup- mice bearing large tumors without affecting T cells, NK cells, mac- pressor cell; TBI, total body irradiation; Treg, T regulatory cell. rophages, or B cells (25). Serafini et al. (26) showed that sildenafil Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 downregulates arginase activity and NO synthase expression, thereby

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200274 2 TBI-INDUCED MDSC AND Tregs reducing the suppressive function of MDSC. Antimicrotubule agents, specific peptides. Cell proliferation was measured by [3H]thymidine uptake. such as docetaxel (DTX) and paclitaxel, have immune-enhancing All experiments were performed in triplicate. properties when used alone or in combination with immunotherapy Functional assays (22, 27, 28). A recent study has shown that paclitaxel promotes differentiation of MDSC into DC in vitro in a TLR4-independent To test the function of Tregs from TBI-treated or untreated tumor-bearing mice, a CD4+CD25+ isolation kit was used to isolate manner (29). Paclitaxel has also been shown to reduce Tregs and Tregs on day 21 after B16 injection. For stimulator cells, T cell-depleted their inhibitory function (28). DTX has been shown to decrease splenic cells from C57BL/6 mice were used and cultured at 5 3 104 cells/ 2 splenic MDSC in mice bearing mammary tumors, leading to an well in a 96 well plate coated with 0.25 mg anti-CD3. Naive CD4+CD25 3 4 + + increased antitumor response (22). Although recent reports have T cells were cocultured at 5 10 cells/well. CD4 CD25 T cells from B16- bearing mice treated with or without TBI were added at 5 3 104cells/well. shown the effect of DTX on MDSC, it has not been used for specific Naive T cells alone, stimulators alone, and inhibitory cells alone served as inhibition of MDSC in the setting of lymphopenia for the treatment controls. After 72 h of culture, proliferation was analyzed by the incorpo- of melanoma. Hence, in this study, we investigated the reconsti- ration of [3H]thymidine during the final 6 h of culture. All experiments were tution of suppressor populations (Tregs and MDSC) after lympho- performed in triplicate. depletion. Furthermore, we investigated whether blockade of re- Reactive oxygen species production constituting MDSC leads to persistence of adoptively transferred T cells and enhances the efficacy of adoptive T cell transfer and The oxidation-sensitive dye 29,79-dichlorofluorescein diacetate (DCFDA) (Molecular Probes/Invitrogen, Eugene, OR) was used for the measurement vaccination in a murine model of melanoma. of reactive oxygen species production by MDSC (30). Cells were incubated at room temperature in serum-free RPMI 1640 medium in the presence Materials and Methods of 3 mmol/l DCFDA with or without 300 nmol/l PMA for 30 min, washed Downloaded from with PBS, and then labeled with anti-CD11b and Gr-1 Abs. After incubation Mice on ice for 20 min, cells were washed with PBS and analyzed using flow Six- to 8-wk-old C57BL/6 mice were purchased from Harlan Laboratories cytometry. (Indianapolis, IN). OT-I mice were purchased from The Jackson Laboratory Arginase activity (Bar Harbor, ME). Mice were housed at the Animal Research Facility of the H. Lee Moffitt Cancer Center and Research Institute. All animal protocols Arginase activity was measured in cell lysates as described previously (31). were reviewed and approved by the Institutional Animal Care and Use Briefly, cells were lysed for 30 min with 100 ml 0.1% Triton X-100. http://www.jimmunol.org/ Committee at the University of South Florida. Subsequently, 100 ml 25 mM Tris-HCl and 10 ml10mMMnCl2 were added, and the enzyme was activated by heating for 10 min at 56˚C. Arginine hy- Cell lines drolysis was conducted by incubating the lysate with 100 ml0.5ML-argi- B16 melanoma was maintained by serial in vitro passages in complete nine (pH 9.7) at 37˚C for 2 h. The reaction was stopped with 900 mlH2SO4 medium (CM) consisting of RPMI 1640 medium supplemented with 10% (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). Urea concentration was measured at heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 mM sodium py- 540 nm after addition of 40 ml of a-isonitrosopropiophenone (dissolved in ruvate, 2 mM fresh L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, 100% ethanol), followed by heating at 95˚C for 30 min. 50 mg/ml gentamicin, 0.5 mg/ml fungizone (all from Life Technologies, Rockville, MD), and 0.05 mM 2-ME (Sigma-Aldrich, St. Louis, MO). B16 NO production expressing OVA (M05) cells were maintained by serial in vitro passages in Equal volumes of culture supernatants (100 ml) were mixed with Greiss by guest on September 27, 2021 CM supplemented with 0.8 mg/ml G418. reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naph- thylethylenediamine dihydrochloride in double-distilled water). After 10 min Reagents of incubation at room temperature, the absorbance at 550 nm was measured For analysis of immune cell populations, the following anti-mouse Abs were using a microplate plate reader (Bio-Rad, Hercules, CA). Nitrite con- purchased from BD Biosciences (San Diego, CA): anti-Ly6G and -Ly6C centrations were determined by comparing the absorbance values for the biotinylated Abs, anti-CD11b PerCP-Cy5.5, anti-CD11b allophcocyanin, test samples to a standard curve generated by a serial dilution of 0.25 mM anti–Gr-1 PE, anti–Gr-1 PE Cy7, anti-CD4 Pacific blue, anti-CD8 Alexa sodium nitrite. Fluor 780, and anti-CD3 FITC. The CD4+CD25+ Treg isolation kit and streptavidin microbeads were purchased from Miltenyi Biotec (Auburn, Flow cytometry CA). The anti-mouse Foxp3 T regulatory staining kit was purchased from Spleens were harvested under sterile conditions. Single-cell suspensions eBioscience (San Diego, CA). Clinical grade DTX (Taxotere; Sanofi-Aventis) were prepared, and red cells were removed using ammonium-chloride- was used in this study. potassium chloride lysing buffer. One million cells (splenocytes or MDSC) were incubated for 30 min on ice in staining medium with relevant Abs for the Lymphopenia model surface expression analysis according to the standard protocols. After A total of 1 3 105 B16 or 3 3 105 M05 tumor cells were injected s.c. in the washing, the samples were analyzed using an LSRII (BD Biosciences), and left flank of C57BL/6 or Ly5.2 mice. Three days later, mice received a the data were analyzed using FlowJo software (Tree Star). sublethal dose (600 cGy) of TBI administered by a [137Cs] gamma radi- ation source. Generation of bone marrow-derived DC and Ag pulsing Erythrocyte-depleted mouse bone marrow cells were cultured in CM sup- Isolation of MDSC and MDSC suppressor assay plemented with 20 ng/ml GM-CSF and 10 ng/ml IL-4 (R&D Systems, MDSC were isolated from the spleens of naive, B16 tumor bearing mice, or Minneapolis, MN) as described previously (18). On day 5, cells were har- B16 tumor-bearing mice treated with TBI. Splenocytes were depleted of vested by gentle pipetting and layered onto an Optiprep gradient (Axis- RBCs using ammonium-chloride-potassium chloride lysis buffer and washed Shield, Oslo, Norway). The low-density cell interface was collected and twice with cold MACS buffer (1% BSA in PBS with 2 mmol/l EDTA). washed twice. Resulting DC were washed one time and resuspended at 1 3 6 Washed cells were resuspended at 2 3 108 cells in 1 ml MACS buffer and 10 cells/ml CM containing 20 ng/ml GM-CSF and 20 ng/ml IL-4. DC incubated with 100 ml biotinylated anti-Ly6C and Ly6G (Gr-1) Abs were pulsed overnight with 10 mcg/ml of OVA 257–264 peptide (Invitrogen, (Miltenyi Biotec) for 20 min at 4˚C. Labeled splenocytes were then in- Carlsbad, CA). cubated at 4˚C with 100 ml streptavidin microbeads (Miltenyi Biotec) for 15 min. Cells were washed, resuspended in 5 ml MACS buffer, and applied Immunization schedule to a MACS column for positive selection, according to the manufacturer’s MDSC blockade in TBI-treated mice in combination immunotherapy. A instructions (Miltenyi Biotec). The purity of cell populations as analyzed total of 3 3 105 M05 tumor cells were injected s.c. in the left flank of by flow cytometery was .95%. For functional assays, splenocytes from C57BL/6 mice. Three days later, mice received a sublethal dose (600 cGy) OT-I mice were used as responder cells. CD8+ T cells from these mice have of TBI administered by a [137Cs] gamma radiation source. Mice received 6 a transgenic TCR that recognize the OVA 257–264 peptide. MDSC from naive, 5 3 10 OT-I T cells/mouse on day 4 and two injections of DC pulsed with 6 tumor-bearing mice with or with out TBI treatment were cultured at different OVA 257–264 peptide (1 3 10 / mouse/ vaccination) on days 4 and 10. Four ratios with 2 3 105 splenocytes from OT-I mice in the presence of control or dosesofDTXat16mg/kgweregiveni.p.,(i.p.),every3dstartingonday4. The Journal of Immunology 3

Control groups received T cells alone, DC alone, DTX alone, or PBS. Tumor size was measured and recorded every 2 d. In another set of experiments, splenocytes were harvested from these mice 1 wk after the final immuni- zation for in vitro assays.

[51Cr] release cytotoxicity assay A[51Cr] release assay was done as described previously (18). M05 cells were used as targets. Spleens of treated mice were harvested on day 21, and purified T cells were used as effector cells. T cell purity was measured by flow cytometry and cells were 95% positive for CD90 (data not shown). Briefly, target M05 cells were labeled with 100 mCi [51Cr] (Amersham Biosciences) at 37˚C in a 5% CO2 atmosphere for 1.5 h. The labeled tumor cells were washed three times and added to the effector cells in triplicate wells of 96-well round-bottom microplates at 100:1, 50:1, and 25:1 E:T ratios. After 5 h, the percentage of specific [51Cr] release was determined by the following equation: ([experimental cpm 2 spontaneous cpm]/[total cpm incorporated 2 spontaneous cpm]) 3 100. All determinations were done in triplicate, and the SE of all assays was calculated and was typically 5% of the mean or less.

Cytometric bead array

Briefly, splenocytes were plated at 2 3 106, cocultured with 2 3 105 ir- Downloaded from radiated M05 melanoma tumor cells, and incubated for 48 h. Culture supernatants were analyzed for IFN-g production using commercially avail- able cytometry bead array kit (BD Biosciences). Briefly, 25 ml mixed capture beads were incubated with 25 ml culture supernatant and 25 mlPEdetection FIGURE 1. Reconstitution of splenic immune subsets after TBI. Mice reagent for 2 h at room temperature. The immunocomplexes were then received 1 3 105 B16 tumor cells on day 0, followed by 600 rad TBI on day washed and analyzed using a FACSCalibur affixed with a 488-nm laser (BD 3. Spleen cells from wild-type (A) and B16 tumor-bearing (B) mice were

Biosciences), according to the manufacturer’s protocol. analyzed for CD4+,CD8+, CD11b+Gr-1+ MDSC, and CD4+CD25+Foxp3+ http://www.jimmunol.org/ Statistical analysis Tregs on days 3, 7, 10, 14, and 21. Data represent the percentage of normal compared with splenocytes from mice not treated with TBI. Data are rep- A Mann–Whitney U test (unpaired) or a Student t test was used to compare resentative of three independent experiments (n =4). differences between two treatment groups. All statistical evaluations of data were performed using GraphPad Prism software. Statistical significance was achieved at p , 0.05. CD4+CD252 cells in the absence of CD4+CD25+ Tregs had sig- nificant proliferation (p , 0.01). This result shows that reconstituting Results Tregs post-TBI are suppressive and behave similarly to endogenous Reconstitution of immune subsets after lymphopenia Tregs from tumor-bearing mice. by guest on September 27, 2021 To evaluate the reconstitution of immune subsets after induction of Repopulating MDSC are highly immunosuppressive after TBI lymphopenia, naive and B16 tumor-bearing mice received 600 rad TBI. We observed lymphopenia and leukopenia in both naive and Next, we evaluated the function of TBI-induced MDSC. We pu- + B16 tumor-bearing mice, evidenced by total splenocyte counts rified Gr-1 cells from the spleens of naive and tumor-bearing post-TBI (data not shown). As shown in Fig. 1A, CD4+ and CD8+ mice that received TBI on day 21 posttumor injection. B16 tumor- T cells were detected on day 7 in the wild-type mice, with 60% of bearing mice with no TBI treatment were used as a control. MDSC CD4+ T cells reconstituted (percentage of normal) and 40% of were cocultured with OT-I splenocytes at various ratios in the + CD8 T cells reconstituted by day 21. In B16 tumor-bearing mice, presence of OVA257–264 peptide, and T cell proliferation was eval- 30% reconstitution of CD4+ T cells and 20% CD8+ T cells was uated. OT-I cells alone and MDSC alone served as controls. We observed (Fig. 1B). CD4+CD25+Foxp3+ Tregs were reconstituted to observed that TBI-induced MDSC from tumor-bearing mice were 70% in wild-type and 100% in B16 tumor-bearing mice by day 10. significantly more suppressive compared to the endogenous MDSC Although, CD11b+Gr-1+ MDSC were reconstituted to 100% by day from tumor-bearing mice that did not receive TBI treatment (Fig. 10 in both wild-type and B16 tumor-bearing mice. Alternatively, the 2B). Even at the lowest ratio of 1 MDSC:12 T cells, there was cell numbers of splenic immune subsets per million of splenocytes significant (p , 0.01) suppressive activity, whereas the endogenous were calculated post-TBI are shown as mean 6 SD (n =4)inTableI. MDSC from tumor-bearing mice were not able to inhibit T cell These findings demonstrate that in a lymphopenic environment, proliferation. MDSC derived from TBI-treated naive mice (not MDSC and Tregs recover from lymphodepletion faster than CD4+ bearing tumor) did not show any suppressive activity compared with and CD8+ T cells, and this difference is further accentuated in tumor- control OT-I cells pulsed with peptide alone. To further analyze bearing mice, with proliferation resulting in higher inhibitory cell suppressive activity, MDSC were added at various ratios to OT-I frequency compared with basline by 21 d. splenocytes in the presence of OVA257–264 peptide, and IFN-g production was evaluated. We observed significantly suppressed Repopulating Tregs are suppressive after TBI IFN-g production by OT-I cells when cocultured with MDSC from Because Tregs and MDSC undergo rapid reconstitution after TBI, tumor-bearing mice with or without TBI at 1:3 ratios (p , 0.01; we sought to determine the suppressive function on T cell prolif- Fig. 2C). However, at both 1:6 and 1:12 ratios, no suppressive ac- eration. Tregs were purified from the spleens of B16 tumor-bearing tivity was observed in MDSC from non–TBI-treated tumor-bearing mice with or without TBI on day 21 posttumor injection. Inhibitor mice, whereas a significant reduction of IFN-g production was alone, stimulator alone or responder alone served as controls. As observed in OT-I cells when cocultured with MDSC from TBI- showninFig.2A,CD4+CD25+ Tregs from the spleens of TBI-treated treated tumor-bearing mice. Collectively, these data show that repop- B16 tumor-bearing mice were as suppressive as the CD4+CD25+ ulating MDSC post-TBI has enhanced immunosuppressive activity Tregs from tumor-bearing mice with no TBI treatment, whereas of MDSC on a per cell basis. 4 TBI-INDUCED MDSC AND Tregs

Table I. Cell number (3104) per million of splenocytes

CD4 CD8 CD11bGr-1 CD4CD25Foxp3

Naive B16 Naive B16 Naive B16 Naive B16 Pre-TBI 25 6 2.3 16.3 6 1.52 2.5 6 0.31 1.2 6 0.03 Post-TBI Day3 0.5 6 0.01 0.2 6 0.04 0.8 6 0.01 0.6 6 0.02 0.1 6 0.02 0.2 6 0.01 0.1 6 0.01 0.3 6 0.01 Day 7 2.2 6 0.03 2.3 6 0.12 1.9 6 0.02 3.5 6 0.01 0.9 6 0.01 0.9 6 0.05 0.4 6 0.02 0.4 6 0.02 Day 10 9.4 6 0.80 7.8 6 0.61 5.8 6 0.05 6.5 6 0.21 2.5 6 0.31 2.6 6 0.26 0.9 6 0.1 1.3 6 0.11 Day 14 10.1 6 1.21 11.3 6 1.10 6.7 6 0.11 4.5 6 0.80 2.9 6 0.21 3.5 6 0.97 1.1 6 0.32 1.5 6 0.21 Day 21 16.5 6 0.34 12.5 6 0.20 7.8 6 0.21 4.9 6 0.12 3.8 6 1.14 6.3 6 0.03 1.4 6 0.66 1.8 6 0.05 n = 4/group. Data compiled from three independent experiments.

Next, we sought to understand the mechanism for the increased could abrogate their reconstitution with DTX treatment. To eval- suppressive activity. Several factors have been implicated in uate the effect of DTX on reconstituting MDSC post-TBI, C57BL/6 MDSC-mediated immune suppression, including the production of mice were injected s.c. with B16 cells on day 0 and received 600 rad arginase, NO, and ROS (30–32). We compared the production of TBI on day 3. Mice received four doses of DTX injected i.p. at these factors in MDSC from B16 tumor-bearing mice treated with 3 d or weekly intervals. Spleens were harvested on days 3, 7, 10, 14, Downloaded from or without TBI. Gr-1+ cells were purified from the spleens of TBI- and 21 posttumor injection, and immune subsets were analyzed by treated or untreated tumor-bearing mice on day 21 posttumor in- flow cytometry. As shown in Fig. 4A, B16 tumor-bearing mice that jection. OT-I splenocytes were cultured at different ratios of MDSC received DTX had a significant reduction in TBI-induced MDSC, inthepresenceofOVA257–264 peptide for 48h. OT-I splenocytes regardless of whether they received the treatment on a weekly basis alone and MDSC alone served as controls. OT-I splenocytes cul- orevery3d(p , 0.05). By day 10, we observed a reduction in TBI- tured with different ratios of MDSC in the presence of OVA257–264 induced MDSC reconstitution in DTX-treated mice compared with peptide had a significant increase in the NO production compared the TBI-treated tumor-bearing mice that did not receive DTX. By http://www.jimmunol.org/ with the endogenous MDSC from the tumor bearing mice (p , day 21, mice that received DTX every 3 d had a significant reduction 0.05; Fig. 3A). The oxidation-sensitive dye DCFDA was used for in their MDSC reconstitution, up to 50% (p , 0.002) compared with the measurement of ROS production by MDSC and analyzed by the untreated mice (250%), while we observed 100% reconstitution flow cytometry. As shown in Fig. 3B, there was an increased ROS in mice receiving the treatment on a weekly interval (p , 0.01). production in TBI-induced MDSC compared with endogenous Previous studies have shown that DTX treatment can increase MDSC from tumor-bearing mice. Next, arginase activity in cell the number of CD4+ and CD8+ T cells (22, 27). To determine whether lysates was also measured. We observed a similar trend as that seen DTX, given every 3 d, had an effect on reconstituting T cells, we in the NO and ROS levels, with increased arginase activity in the analyzed the CD4+ and CD8+ T cell reconstitution in TBI-treated by guest on September 27, 2021 TBI-induced MDSC compared with the endogenous MDSC (p , tumor-bearing mice after DTX treatment. As shown in Fig. 4B and 0.01; Fig. 3C). 4C, we did not see any significant difference in reconstitution of CD4+ and CD8+ on days 7, 10, 14, and 21 in mice that received DTX. DTX reduces reconstitution rate of MDSC post-TBI These data show that DTX given at 3-d intervals significantly re- Because MDSC from TBI-treated tumor-bearing mice demonstrated duced the MDSC reconstitution post-TBI without modulating T cell enhanced suppressive activity, we wanted to evaluate whether we populations.

FIGURE 2. The suppressive activity of recon- stituted Tregs/MDSC (A)CD4+CD25+ cells were purified on day 20 from B16 tumor-bearing mice treated with or without TBI. T cell-depleted splenic cells from C57BL/6 mice were used as stimulators and were cultured at 5 3 104 cells/ well in a 96-well plate coated with 0.25 mganti- CD3. Naive CD4+CD252 T cells were cocultured at 5 3 104 cells/well. CD4+CD25+ T cells were added at 5 3 104 cells/well. After 72 h of culture, proliferation was analyzed by the addition of [3H]thymidine during the final 6 h of culture. All experiments were performed in triplicate. (B) CD11b+Gr-1+ MDSC were purified using a MACs column and were cultured at various ratios with 2 3 105 splenocytes from OT-I mice in the pres- ence of OVA257–264 peptide. After 72 h of culture, proliferation was analyzed by the addition of [3H]thymidine during the final 6 h of culture. (C) IFN-g production was measured in the super- natants after 48 h of coculture with a cytometric bead array assay as described in Materials and Methods.*p , 0.01. The Journal of Immunology 5

FIGURE 3. TBI-induced MDSC express high levels of NO, ROS, and arginase. (A)NO production (data represent the mean 6 SD of triplicates; *p , 0.05). OT-I cells (1 3 105 cells/well) were stimulated in triplicates for

48 h with OVA257–264 peptide in the presence of different ratios of MDSC, and NO levels were measured. (B) The levels of ROS pro- duction in MDSC from untreated or TBI- treated mice bearing B16 tumor was measured using DCFDA staining and flow cytometry. *p , 0.05. Data represent one of the three in- dependent experiments. (C) Arginase activity was measured in untreated and TBI-treated 6

mice bearing B16 tumors. Mean SD of two Downloaded from independent experiments is shown. *p , 0.01. http://www.jimmunol.org/

Blockade of TBI-induced MDSC with DTX improves the ment with DCOVA or T cells alone had a significant reduction in antitumor efficacy of adoptive T cell transfer and DC tumor growth compared with the untreated groups (p , 0.001; immunotherapy Fig. 5B). These data suggest that blockade of TBI-induced MDSC Next, we evaluated whether blocking TBI-induced MDSC with can improve the efficacy of adoptive T cell transfer and DC vac- DTX would improve the efficacy of adoptive T cell transfer and DC cination. immunotherapy. C57BL/6 mice were injected s.c. with M05 cells on day 0 and received 600 rad TBI on day 3. Mice received 5 3 106 OT-I Blockade of TBI-induced MDSC enhances the persistence of adoptively transferred T cells and improves CTL function by guest on September 27, 2021 T cells on day 4 and were vaccinated with DC pulsed with OVA257–264 peptide (DCOVA) on days 4 and 10. DTX was given every 3 d starting We next examined whether adoptively transferred T cells persisted on day 4 for a total of four doses. As shown in Fig. 5A, after four for a longer duration in mice treated with DTX. Ly5.2 mice were doses of DTX treatment, a significant reduction in tumor size was injected with 3 3 105 M05 cells followed by TBI on day 3. Mice 6 observed in mice that received OT-I T cells and DCOVA immu- received 5 3 10 OT-I (Ly5.1 mice) T cells on day 4. DCOVA vac- notherapy compared with mice that received only T cells and DCOVA cinations were given on days 4 and 10. Four doses of DTX were vaccination (p , 0.001). However, mice that received DTX treat- given every 2–3 d beginning on day 4. Splenocytes were collected at

FIGURE 4. DTX treatment in combi- nation with TBI reduces MDSC in tumor- bearing mice. Mice were injected with B16 tumor cells on day 0, followed by TBI on day 3. Mice received DTX treat- ment on a weekly basis or every 3 d starting on day 4. Spleen cells from mice that received 600 rad alone or 600 rad + DTX were analyzed for CD11b+Gr-1+ MDSC (A), CD4+ T cells (B), and CD8+ T cells (C) on days 3, 7, 10, 14, and 21 posttumor injection. Graphs represents the percent of normal of CD11b+Gr-1+ MDSC, CD4+, and CD8+ T cells (n =4/ group). *p , 0.01. 6 TBI-INDUCED MDSC AND Tregs

FIGURE 5. DTX treatment in combination with TBI enhances the immunotherapeutic efficacy of adoptive T cell transfer and DC vaccination. Mice received 3 3 105 M05 tumor cells on day 0, followed by TBI on day 3. On day 4, 5 3 106 OT-I T cells were adoptively transferred followed by two doses of DC pulsed with

OVA 257–264 peptide on days 4 and 10. Four doses of DTX treatment were given every 3 d starting from day 4. (A) Tumor growth. (B) Survival curve. *p , 0.001. Downloaded from http://www.jimmunol.org/ various time points, and the percentage of OT-I T cells was mea- IFN-g production as a measure of T cell function. As shown in sured by flow cytometry. Mice that received therapy with OT-I Fig. 6B, restimulation with OVA peptide for 48 h resulted in higher T cells and DCOVA in combination with DTX had a higher per- IFN-g production by splenocytes from mice that received DTX centage of OVA tetramer specific T cells on days 8, 15, and 20. As treatment in combination with OT-I T cells and DCOVA vaccination shown in Fig. 6A, a higher percentage of CD8+ OVA-specific T cells (980 6 35.4 pg/ml) compared with splenocytes from mice that were observed in mice that received DTX treatment compared with received OT-I T cells and DCOVA vaccination alone (597 6 30.4 pg/ml; the mice that received immunotherapy alone. We next evaluated p , 0.01). These data suggest that blockade of TBI-induced MDSC by guest on September 27, 2021

FIGURE 6. Addition of DTX treatment in combination with adoptive T cell transfer and DC vaccination increases the persistence OT-I T cells in mice. 5 6 Ly5.2 mice were injected with 3 3 10 M05 cells, followed by TBI on day 3. Mice received 5 3 10 OT-I (Ly5.1 mice) T cells on day 4. DCOVA vac- cinations were given on days 4 and 10. Four doses of DTX were given every 2–3 d beginning on day 4. (A) The percentage of CD8+ and OVA tetramer- positive cells was measured by flow cytometry. (B) IFN-g production was measured in the splenocytes of treated mice. Splenocytes were plated at 2 3 106, cocultured with 2 3 105 irradiated M05 cells, and incubated for 48 h. Culture supernatants were analyzed for IFN-g production using commercially available cytometry bead array kit. Data shows the mean 6 SD of triplicates. *p , 0.01. (C) A 5-h 51Cr release assay was performed using M05 tumor cells as targets. Spleens of treated mice were harvested on day 21, and purified T cells were used as effector cells. Data show the mean 6 SE of triplicates from a chromium release assay. *p , 0.05. The Journal of Immunology 7 with DTX enhanced the persistence and activity of adoptively little ROS but high levels of NO (31). These suppressive factors have transferred T cells. been shown to have a direct effect on the inhibition of T cell Next, we examined whether reduction of TBI-induced MDSC by function (41–44). Although it has been shown in several studies that DTX enhances the function of CD8+ T cells. Splenic T cells from MDSC use multiple factors such as NO, ROS, and arginase for their mice receiving T cells and DCOVA, treated with or without DTX, suppressive function on T cells, this study shows that after the in- were purified on day 21 and cocultured with 51Cr-labeled M05 duction of lymphopenia, MDSC have increased production of these melanoma tumor cells at E:T ratios of 100:1, 50:1, or 25:1. T cells factors compared with the endogenous MDSC from non–TBI- from mice that received DCOVA and OT-I T cells in combination treated tumor-bearing mice. It has been shown that various tumor- with DTX treatment exhibited higher cytotoxicity against M05 derived factors define the expansion of these MDSC subsets. melanoma tumor cells at a 100:1 ratio compared with mice that did However, in our study, we observed elevated levels of all of these not receive DTX treatment (p , 0.05; Fig. 6C). However, no sig- suppressive factors. It is possible that both monocytic and granu- nificant differences were measured at lower ratios. These data locytic MDSC are being reconstituted following lymphodepletion suggest that blockade of TBI-induced MDSC with DTX improves and that each population contributes to increased immunosuppres- the CTL function in vaccinated mice against M05 melanoma tumor sive function. Studies to define the factors responsible for the rapid cells. expansion of these MDSC subsets in a lymphopenic environment are ongoing. Discussion MDSC frequency is increased in melanoma patients and is as- Impaired immune function is often associated with negative regu- sociated with disease progression (45, 46). Strategies to target latory factors such as Tregs and MDSC (3, 9, 33). These populations MDSC include inhibition of expansion or promotion of differen- Downloaded from can inhibit T cell activation and may limit the efficacy of immu- tiation into mature cells that no longer possess suppressive activity notherapies (34). Induction of lymphopenia by TBI or chemo- (21, 47). All-trans-retinoic acid has been shown to induce MDSC therapy can eliminate these immunosuppressive populations. Our differentiation that leads to neutralization of ROS production in laboratory and others have shown that adoptive T cell therapy and both mice and cancer patients (23, 48). Several studies have also DC vaccination after the induction of lymphopenia enhances shown that MDSC can be directly eliminated using chemother-

T cell expansion and activation and leads to improved antitumor apeutic drugs such as gemcitabine, DTX, suntinib, or flurouracil http://www.jimmunol.org/ immune responses against melanoma (13, 17, 18). Adoptive (20, 22, 23, 26, 28). Treatment of mice bearing large tumors with transfer of T cells following the induction of lymphopenia has led chemotherapeutic drugs has been shown to result in dramatic re- to enhanced antitumor immunity in animal models and significant ductions in the number of splenic MDSC and a marked improve- clinical responses in patients with melanoma (14, 17, 19, 35). ment in immunotherapeutic efficacy (24, 25). To block rapidly However, there are few reports on the reconstitution and function reconstituting MDSC in our model, we used DTX, a chemothera- of suppressor populations after the induction of lymphopenia. peutic drug previously shown to block MDSC expansion (22). In In this study, we demonstrate that in the setting of lymphopenia, melanoma-bearing lymphopenic mice, DTX treatment decreased MDSC and Tregs reconstitute faster than CD4+ and CD8+ T cells. MDSC reconstitution and increased the persistence of adoptively TBI-induced Tregs were as suppressive as the endogenous Tregs. transferred T cells, resulting in delayed tumor growth and enhanced by guest on September 27, 2021 Depletion of TBI-induced Tregs with anti-CD25 Abs enhanced the survival. These are the key findings that can potentially improve the antitumor efficacy of adoptive T cell transfer and DC immuno- design of clinical trials in patients with melanoma. therapy in melanoma-bearing mice (data not shown). Our find- Although lymphodepletion strategies are an attractive tool for ings suggest that depletion of TBI-induced Tregs improves the adoptive immunotherapy protocols, our results demonstrate that antitumor efficacy of adoptive T cell transfer and DC immuno- MDSC and Tregs are rapidly reconstituted and are highly immu- therapy. These data are supported by a recent report showing an nosuppressive after the induction of lymphopenia. It is possible that increase in the frequency of Tregs in B16 tumor-bearing mice the rapid reconstitution and suppressive functions of TBI-induced after temozliamide-induced lymphopenia. Systemic Treg deple- MDSC and Tregs may limit the effectiveness of immunotherapy to tion during lymphopenia resulted in enhanced antitumor immunity induce antitumor T cell responses, which in turn contribute to failure (36). It has been shown that lymphopenia induces expansion of at inducing complete tumor regressions. Blocking the reconstitution Tregs during immune reconstitution in individuals with cancer (36, of TBI-induced MDSC or Tregs enhanced antitumor immunity in 37). A study has shown that Denileukin diftitox (Ontak) adminis- a murine model and may improve current adoptive therapy strategies tration to stage IV melanoma patients depletes peripheral blood for the treatment of metastatic melanoma. Tregs and causes the regression of metastatic tumors in a subset of patients (38). CD25 depletion in combination with lymphopenia has Acknowledgments been shown to result in a potent tumor rejection in a B16 melanoma We thank Dr. Dmitry Gabrilovich for valuable comments during the prep- model and may enhance immunity in lymphopenic patients with aration of this manuscript. We also thank the Flow Cytometry Core Facility metastatic melanoma (39, 40). at the H. Lee Moffitt Cancer Center and Research Institute for contribu- Our findings demonstrate that TBI-induced MDSC are more tions to this work. immunosuppressive on a per cell basis compared with endogenous MDSC isolated from B16 tumor-bearing mice. This is a novel finding Disclosures that repopulating MDSC after the induction of lymphopenia are The authors have no financial conflicts of interest. highly immunosuppressive. On the basis of these initial findings, this study focused on evaluating the immunosuppressive function of TBI- induced MDSC. We tested NO, arginase, and ROS production as References possible mechanisms for the observed increase in suppressive 1. Jemal, A., S. S. Devesa, P. Hartge, and M. A. Tucker. 2001. Recent trends in cutaneous melanoma incidence among whites in the United States. J. Natl. function and found increased production of all three suppressive Cancer Inst. 93: 678–683. factors in the reconstituted MDSC following lymphodepletion. It has 2. Jemal, A., R. Siegel, E. Ward, Y. Hao, J. Xu, and M. J. Thun. 2009. Cancer statistics, 2009. CA Cancer J. Clin. 59: 225–249. been reported that granulocytic subsets of MDSC express a high level 3. Viguier, M., F. Lemaıˆtre, O. Verola, M. S. Cho, G. Gorochov, L. Dubertret, of ROS and very little NO, whereas the monocytic subsets have very H. Bachelez, P. Kourilsky, and L. Ferradini. 2004. Foxp3 expressing CD4+ 8 TBI-INDUCED MDSC AND Tregs

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