Lack of P53 Augments Antitumor Functions in Cytolytic T Cells

Published OnlineFirst July 27, 2016; DOI: 10.1158/0008-5472.CAN-15-1798 Cancer Microenvironment and Immunology Research

Lack of p53 Augments Antitumor Functions in Cytolytic T Cells Anirban Banerjee1, Krishnamurthy Thyagarajan1, Shilpak Chatterjee1, Paramita Chakraborty1, Pravin Kesarwani1, Myroslawa Soloshchenko1, Mazen Al-Hommrani1, Kristina Andrijauskaite1, Kelly Moxley2, Harinarayanan Janakiraman3, Matthew J. Scheffel4, Kristi Helke5, Kent Armenson6, Viswanathan Palanisamy3, Mark P. Rubinstein1, Elizabeth-Garrett Mayer6, David J. Cole1, Chrystal M. Paulos4, Christina Voelkel-Johnson4, Michael I. Nishimura2, and Shikhar Mehrotra1

Abstract

Repetitive stimulation of T-cell receptor (TCR) with cognate mor T-cell function. Our data show that as compared with h3T antigen results in robust proliferation and expansion of the T cells, cells, h3T-p53 KO T cells exhibited enhanced glycolytic com- and also imprints them with replicative senescence signatures. mitment that correlated with increased proliferation, IFNg Our previous studies have shown that life-span and antitumor secretion, cytolytic capacity, expression of stemness gene sig- function of T cells can be enhanced by inhibiting reactive oxygen nature, and decreased TGF-b signaling. This increased effector species (ROS) or intervening with ROS-dependent JNK activa- function correlated to the improved control of subcutaneously tion that leads to its activation-induced cell death. Because established murine melanoma after adoptive transfer of p53- tumor suppressor protein p53 is also a redox active transcrip- KO T cells. Pharmacological inhibition of human TCR-trans- tion factor that regulates cellular ROS generation that triggers duced T cells using a combination of p53 inhibitors also downstream factor–mediating apoptosis, we determined if potentiated the T-cell effector function and improved persis- p53 levels could inﬂuence persistence and function of tumor- tence. Thus, our data highlight the key role of p53 in regulating reactive T cells. Using h3T TCR transgenic mice, with human the tumor-reactive T-cell response and that targeting this path- tyrosinase epitope–reactive T cells developed on p53 knockout way could have potential translational signiﬁcance in adoptive (KO) background, we determined its role in regulating antitu- T-cell therapy. Cancer Res; 76(18); 1–12. 2016 AACR.

Introduction sion and reduced survival of effector T cells in an oxidative tumor microenvironment are the key confounding factors in Adoptive transfer of tumor epitope reactive T cell in cancer immunotherapy (2, 3). We have previously shown that reactive patients has generated much interest due to promising control of oxygen species (ROS) scavengers can inhibit repetitive T-cell tumor growth (1). However, susceptibility to immunosuppres- receptor (TCR) stimulation-mediated activation induced cell death (AICD) of tumor-reactive T cells without interfering with 1Department of Surgery, Medical University of South Carolina, Charles- cytokine production (4), a measure of CTL function, placing ton, South Carolina. 2Department of Surgery, Oncology Institute, redox regulation at a central point for therapeutic intervention. Loyola University, Maywood, Illinois. 3Department of Oral Health The altered expression of a redox active transcription factor p53 Research, Medical University of South Carolina, Charleston, South leads to uncontrolled cell proliferation, senescence, and cell death Carolina. 4Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina. 5Department (5). However, only a handful of studies have reported the role of of Comparative Medicine, Medical University of South Carolina, p53 in shaping T-cell immune response. Grayson and colleagues 6 Charleston, South Carolina. Department of Public Health Sciences, (6) reported slightly higher memory response in p53-KO mice Medical University of South Carolina, Charleston, South Carolina. compared with p53-sufficient mice, and only minor differences Note: Supplementary data for this article are available at Cancer Research in proliferation, apoptosis, or maintenance of "non-self" viral Online (http://cancerres.aacrjournals.org/). antigen-specific T cells. A recent study has shown that in order A. Banerjee, K. Thyagarajan, and S. Chatterjee authors contributed equally to this to mount an effective antigen-specific proliferative response, þ article. CD4 T cell kinetically downregulate the expression of tumor Current address for A. Banerjee: The George Washington University, Washing- suppressor p53 until 72–96 hours (7). Another study showed that ton DC, NW-20037. p53 inhibits systemic autoimmune diseases by inducing regula- Corresponding Author: Shikhar Mehrotra, Department of Surgery, Hollings tory T cells (Treg; ref. 8). Because p53 is also required for TGFb gene Cancer Center (HO 512H), Medical University of South Carolina, 86 Jonathan responses by cooperating with Smads (9), we hypothesized that Lucas Street, Charleston, SC 29425. Phone: 843-792-9195; Fax: 843-792-2556; T cells from p53-KO mice will be less prone to TGFb-mediated E-mail: [email protected] immunosuppression in a tumor microenvironment, and with less doi: 10.1158/0008-5472.CAN-15-1798 incidence of inducible regulatory T cell (iTreg) generation a 2016 American Association for Cancer Research. durable antitumor T-cell response could be mounted by targeting

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p53. Further, p53 negatively regulates glycolysis through activa- consumption or glycolytic flux was measured using the XF 24 tion of TP53-induced glycolysis regulator (TIGAR; ref. 10) and analyzer (Seahorse Bioscience) as described earlier (14). positively regulates oxidative phosphorylation (OXPHOS) through upregulation of SCO2, a member of the COX-2 assembly Flow cytometry involved in the electron-transport chain (11). Because long-term Detailed protocols for staining the cells for surface markers and T-cell effector and memory response is also metabolically regu- intracellular cytokines have been described earlier (15). Data were lated (12), we determined if differences in metabolic signature analyzed with FlowJo software (Tree Star). due to lack of p53 expression correlate to antitumor T-cell function. Real-time quantitative PCR fi Our study demonstrates that p53 de cient T cells exhibited Total RNA was isolated using TRIzol reagent (Invitrogen). enhanced effector function and proliferation while maintaining hi hi cDNA was generated from 1 mg total RNA using iScript cDNA the CD62L CD44 central memory (Tcm) phenotype. Further, Synthesis Kit (BioRad). Quantitative real-time PCR was per- p53-KO T cells are not transformed to iTregs and exhibit elevated formed using a SYBR Green mix (Biorad) in the CFX96 Detec- cytolytic properties with remarkable tumor control in a mouse tion System (BioRad). The fold change in expression of mole- melanoma model. Thus, p53 could serve as target for improving cules in h3T-p53 KO T cells was calculated over h3T cells and ACT. expressed as relative fold change. The TGFb Pathway PCR array (Qiagen) was used to monitor the expression of 84 genes, along Materials and Methods with five housekeeping genes and control for genomic DNA Mice contamination, RNA quality, and general PCR performance. C57BL/6 (cat. # 000664) and p53-KO (cat. # 002101) mice Data analysis was performed using Qiagen's proprietary web- were obtained from The Jackson Laboratory. Development of based analysis tool. h3T TCR transgenic mouse has been described recently (13). Briefly, the class I–restricted human tyrosinase epitope Statistical analysis (YMDTMSQV)368-376 reactive TCR isolated from tumor-infiltrat- All data reported are the arithmetic mean from three or five þ ing lymphocytes of an HLA-A2 metastatic melanoma patient was independent experiments performed in triplicate SD unless used to generate this transgenic mice. Animals were maintained in stated otherwise. The unpaired Student t test was used to evaluate pathogen-free facilities and procedures approved by the Institu- the significance of differences observed between groups, accepting tional Animal Care and Use Committee. P < 0.05 as a threshold of significance. Data analyses were performed using the Prism software (GraphPad). In vivo data Culture conditions were analyzed using Kaplan–Meier methods, and pairwise com- Recombinant cytokines were purchased from BioLegend. Com- parisons of survival distributions were done via the log-rank test. plete IMDM (cIMDM) media containing 10% FBS, penicillin, and Mice that did not reach a tumor size of 400 mm3 by the end of the streptomycin were used for T-cell differentiation. On day 3 of experiment were sacrificed and had survival time censored in the culture, T cells were harvested and either processed for intracel- analysis. lular cytokine analysis, RNA preparation using TRIzol (Invitro- gen) or used for adoptive cell therapy. Results p53 knockout TCR transgenic T cells show increased Adoptive T-cell protocol proliferation, Tcm phenotype, and reduced senescence Mouse melanoma tumor (B16-F10) and human melanoma To determine the role of p53 in tumor epitope-specific T cells, (624-MEL) were maintained in vitro in cIMDM. B16-F10 (0.25 we crossbred p53 knockout (p53-KO) mice with h3T TCR 6 6 10 ) and 624-MEL (2.5 10 ) were injected subcutaneously (s.c.) transgenic mice (13). Supplementary Figure S1A shows the fl / into left ank of C57BL/6 or Rag1 C57BL/6 mice or NSG-A2 PCR-based genotype screening for the h3T-p53 KO. Using cell mice, respectively. Twenty-four hours before adoptive transfer trace violet dye we noticed that upon stimulation with cognate of T cells on day10, the recipient mice were injected cyclophos- antigen, the TCR transgenic T cells from h3T-p53 KO prolifer- phamide (4 mg/mice, i.p.). ated faster until 48 hours (left) as compared with the wild-type (wt) h3T T cells (Fig. 1A). The difference persisted even after 72 Activation-induced T-cell death hours (right) of stimulation showing greater cell division in Three days after TCR activation, transgenic T cells were resti- h3T-p53 KO–derived T cells. This increased proliferation could mulated for 4 hours with either cognate antigen or nonspecific be attributed solely to the absence of p53, because the expres- antigen-loaded T2-A2 cells at a 5:1 ratio. Apoptosis was measured sion of activation-induced cell surface molecules such as CD69 by staining for Annexin V according to the manufacturer's pro- or CD25 (IL2Ra) was similar in h3T-p53 KO– and h3T-derived tocol, followed by flow cytometry. Data were analyzed with T cells (Supplementary Fig. S1B). In keeping with the increase FlowJo software (Tree Star). in proliferation, a higher number of total splenocytes and thymocytes were retrieved from h3T-p53 KO mice (Fig. 1B; Glucose consumption, oxygen consumption, and glycolytic flux Supplementary Fig. S1C). Our data show that TCR activated Cells were stained with fluorescent-labeled deoxy-glucose ana- h3T-p53 KO–derived T cells have higher expression of Cyclin D, log, 2NBDG (Cayman Chemicals) according to the manufac- a key cyclin protein involved in regulating cell-cycle progres- turer's protocol. Cells were washed and stained with other fluo- sion, and is repressed by p53 (16). The expression of cyclin- rochrome-conjugated antibodies and acquired by flow cytometry. dependent kinase inhibitors CDKn1a, CDKn2a,andCDKn2b, All analyses were done on viable cells. Mitochondrial oxygen which are regulated by p53, was also significantly reduced in

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h3T-p53 KO cells as compared with h3T T cells (Fig. 1C). In T cells as compared with the h3T T cells (Fig. 3B). Specifically, addition, higher proliferation rate could lead the T cells close to glycolysis genes, hexokinse (HKII), phosphofructokinase (Pfk), replicative senescence with increased CD62Llo phenotype and lactate dehydrogenase A (LDHA; Fig. 3B, i), and key glycolysis susceptibility to cell death (3). A recent study has also shown regulator hypoxia-inducing factor (HIF1a; Fig. 3B, ii; , P 0.05; that p53 isoform switching regulates tumor-associated replica- ref. 19) were found to be significantly upregulated. Further, the tive senescence in T cells (17). However, we observed that h3T- expression of TIGAR (Tp53-induced glycolysis and apoptosis þ p53 KO T cells not only exhibit higher percentage of CD62L regulator), a known negative regulator of glycolysis that is acti- þ CD44 T central memory (Tcm) phenotype as compared vated by p53 (10), was also reduced in h3T-p53 KO T cells as with h3T T cells (Fig. 1D), but also showed lower expression compared with h3T T cells (Fig. 3B, ii). Increased expression of of senescence-associated b-galactosidase and increased CD28 glycolytic genes was also observed when comparing magnetic þ þ expression (Fig. 1E). Thus, reduced expression of p53 modu- bead-sorted CD8 T cells from p53-KO T cells to the wt CD8 T lates cell-cycle progression of T cells without inducing replica- cells (Supplementary Fig. S1E). However, expression level of tive senescent phenotype. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), a key regulator of mitochondrial biogenesis, Decreased cell death in h3T-p53 KO T cells correlates with was significantly decreased in h3T-p53 KO T cells as compared higher antioxidant capacity with h3T T cells (Fig. 3B, iii). Further, we also observed increased Because Tcm phenotype is associated with higher antioxidant expression levels of key enzymes involved in the regulation of capacity and reduced cell death (3), we determined ROS/RNS pentose phosphate pathway (PPP) that are required for nucleo- levels and AICD levels between h3T-p53 KO versus h3T T cells. tide synthesis. In concordance with a recent study that showed p53 Upon TCR restimulation with cognate tyrosinase antigen, h3T- inhibits PPP (20), we observed that the mRNA expression of p53 KO T cells secreted less ROS (measured using DCFDA dye), glucose-6 phosphate dehydrogenase (G6PD) was 4-fold higher, and RNS (measured using DAF dye), as compared with h3T and that of ribose-5-phosphate isomerase (RPIA) was about 3- T cells (Fig. 2A). This also correlated to increased antioxidant fold higher in activated h3T-p53 KO T as compared with h3T T levels as determined by cell surface thiol (c-SH; measured using cells (Fig. 3B, iv). Because cells with glycolytic phenotype exhibit a melamide dye) and intracellular glutathione (iGSH; measured significantly higher extracellular acidification rate (ECAR) than using monocholorobimane dye; Fig. 2B) in p53-KO T cells. those dependent upon oxidative phosphorylation, which display Further, a quantitative real-time analysis revealed that antioxidant higher oxygen consumption rate (OCR; ref. 14), we determined enzymes catalase and superoxide dismutase (SOD) levels were the ECAR and OCR levels in real time using bioanalyzer (Sea- also elevated in activated h3T-p53 KO T cells as compared with horse). Our data show that 3-day activated h3T-p53 KO T cells h3T T cells. While TCR restimulation-induced ROS/RNS levels exhibit higher ECAR as compared with h3T T cells (Fig. 3C). Thus, could affect downstream signaling that involves JNK and leads to enhanced glycolysis accompanied by increased commitment to T cell death (4), we observed that upon TCR restimulation, h3T- PPP could be contributing to T-cell anabolism (21). The higher p53 KO T cells exhibit reduced JNK phosphorylation (Fig. 2D, degree of glycolysis also correlated to the higher usage of the top), and cell death, as indicated by reduced loss of mitochondrial mTOR pathway as observed by elevated phosphorylation levels of membrane potential (measured using DiOC6; Fig. 2D, bottom). ribosomal protein S6 (Fig. 3D), which is reported to mediate Phosphatidyl serine upregulation (using Annexin V) among the glycolysis (22). Transgenic T cells at the basal level or stimulated þ þ Vb12 CD8 TCR transgenic T cells was also reduced (Fig. 2E). with control peptide showed lower pS6 staining, indicating that þ Admittedly, while the difference between the Annexin V cells in the increase pS6 in h3T-p53 KO was antigen specific. Thus, these p53-KO versus WT was about 10% to 15%, the number of cells data suggest that increased antioxidant capacity and glycolytic lo 2 that were Annexin V (between 0 and 10 on the x-axis) was commitment of p53-KO T cells could be due to increased expres- appreciable (Supplementary Fig. S1D). To further confirm if ROS/ sion of PPP molecules—as G6PD reduces nicotinamide adenine RNS levels are important mediators of p53 phosphorylation, the dinucleotide phosphate (NADP) to NADPH, and NADPH in TCR-activated wt T cells were either pretreated for 45 minutes with turn maintains the level of glutathione to help protect against antioxidant compound L-NAC (10 mmol/L), or left untreated oxidative damage—a scenario that could be useful in maintaining before TCR restimulation. We observed that reduced RNS accu- persistence of tumor-reactive T cells in the oxidative tumor mulation (determined by DAF staining) after antioxidant L-NAC microenvironment. pretreatment also correlated with reduced p53 phosphorylation (Fig. 2F). Thus, the loss of p53 in T cells results in their increased p53 expression inversely correlates to cytokine response and þ antioxidant capacity, which renders them less susceptible to effector function in CD8 T cells oxidative stress–mediated cell death. Because loss of p53 results in increased glycolysis, a key metabolic pathway that regulates cytokine IFNg (23), we compared Loss of p53 enhances glycolysis and pentose phosphate expression of effector molecules between h3T-p53 KO and h3T T þ pathway activity in stimulated CD8 T cells cells. Our data demonstrate that upon TCR stimulation with Recent studies have shown that p53 is also involved in regu- tyrosinase antigen, the fraction of T cells secreting cytokines IL2, lating various metabolic pathways (18), by balancing glycolysis IFNg, and TNFa were about 2-fold more as compared with the and oxidative phosphorylation, regulating the production of h3T T cells (Fig. 4A). Overnight antigen stimulation also con- ROS. While determining how p53 loss regulates T-cell metabo- firmed that h3T-p53 KO T cells secreted twice the amount of IFNg lism, we observed that uptake of fluorescent glucose 2-NBDG was as compared with h3T T cells (Fig. 4A, right). Importantly, h3T- higher in TCR-activated h3T-p53 KO T cells as compared with h3T p53 KO T cells also exhibit increased externalization of lysosomal T cells (Fig. 3A, i). Next, we observed significantly higher mRNA protein CD107a (Fig. 4B), an indicator of enhanced cytotoxic levels of glycolytic pathway enzymes in TCR-activated p53-KO granule exocytosis (24), which indicates increased cytolytic ability

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A 60 100 48 Hours 72 Hours * B h3T 50 80 * h3T-p53 KO 40

h3T-p53 KO 6 30 60 20 Figure 1. 40 h3T 10 p53 KO T cells preserve Tcm phenotype cells aer 72 hrs % of CTV % of diluted 0 Count×10 20 despite increased proliferation. A, splenocytes from the h3T and h3T-p53 h3T CTV 0 KO mice were harvested and stained Splenocytes with cell trace violet (CTV) dye before h3T-p53KO stimulating with human tyrosinase peptide-pulsed irradiated splenic feeder C cells from HLA-A2 mouse. The dilution of 3 1 D Basal cell trace violet dye with time was * determined using FACS to evaluate 100 2 * * antigen-speciﬁc proliferation. Adjacent

0.5 Cells 80 bar diagram shows percentage increase + in proliferating cells from different 1 60 CD44 experiments. B, bar diagram CD44 + Fold increase 40 representing the total viable 0 0 splenocytes obtained from three Cyclin D CDKn1a h3T- p53 KO 20 individual h3T and h3T-p53 KO mice as

% CD62L 0 counted using trypan blue dye. C, real- 1 1 time quantitative PCR analysis for cyclin h3T D and cyclin inhibitors (CDKn1a, CDKn2a, and CDKn2b) was done using RNA

0.5 0.5 h3T- p53 KO obtained from h3T and h3T-p53 KO h3T

** mice–derived splenic T cells. Data from

** two repeat experiments are shown. D, Fold increase 0 0 basal cell surface expression of CD44 CDKn2a CDKn2b and CD62L was determined using FACS þ on Vb12 gated splenic T cells from h3T h3T CD62L and h3T-p53 KO mice. Adjacent bar h3T-p53 KO diagram shows percentage difference in CD62LþCD44þ T cells from repeat experiments. E, TCR-activated splenic E T cells from h3T and h3T-p53 KO mice were used to determine expression of 1,600 12,000 senescence-associated b-galactosidase

10,000 ** as per the manufacturer's protocol and 1,200 * 8,000 CD28 expression using FACS. Numerical ﬂ -gal cells 800 6,000 value represents mean uorescence intensity. Adjacent bar diagram shows 4,000 h3T 1104 400 3908 CD28 MFI cumulative data from different 2,000 experiments (N ¼ 3; , P < 0.05; 0 MFI of SA b 0 , P < 0.01). MFI, mean ﬂuorescence h3T-p53 KO 9284 intensity. 862 h3T h3T SA β-Galactosidase CD28 h3T- p53 KO h3T-p53KO h3T h3T-p53 KO

of h3T-p53 KO T cells over p53-sufﬁcient h3T T cells. We also in HLA-A2 mice for 14 days before transferring HLA-A2–restricted þ þ observed that the expression of signature transcription factors for tyrosinase reactive Vb12 TCR transgenic CD8 splenic T cells type-1 cytotoxic (Tc1) cells as T-bet and IRF-4 was higher in p53- from h3T-p53 KO or h3T mouse (schema in Fig. 5A). We observed þ KO CD8 T cells (Fig. 4C). In addition, an increased expression of that h3T-p53 KO T cells showed long-term tumor control than genes related to key effector molecules such as GM-CSF, Gran- those that received h3T T cells (Fig. 5B; Supplementary Fig. S2A). zyme B, IL1Rn, IL23R, and IL22 was noticed in h3T-p53 KO T cells At the experimental endpoint, 10-fold higher transferred T cells than in h3T T cells (Fig. 4D). These data indicate that h3T-p53 KO were tracked in the peripheral blood of the recipient group T cells are highly poly-functional cells and exhibit increased that received h3T-p53 KO splenic T cells (Fig. 5C), which exhibited þ þ effector function as compared with h3T T cells. CD62L CD44 central memory phenotype (16% in h3T vs. 44% in h3T-p53 KO; Fig. 5D). The retrieved h3T-p53 KO transgenic þ Adoptive transfer of p53-KO TCR transgenic CD8 T cells T cells continued to produce more effector cytokines IFNg improves tumor control and TNFa than retrieved h3T T cells upon restimulation To determine the antitumor potential of p53-deﬁcient T cells, (Fig. 5E). We also noted that a fraction (4%–11%) of h3T-p53 þ B16-A2 tumor melanoma cells were established subcutaneously KO–transferred T cells exhibited CD44 CD62L phenotype that

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3,000 A 10,000 8,000 2,000 6,000 * * 4,000

DAF MFI DAF 1,000 2,000 h3T 2655 DCF-DA MFI h3T 7027 0 0 h3T-p53 KO h3T h3T-p53 KO 4455 1819 h3T RNS (DAF) ROS (DCF-DA) h3T- p53 KO h3T- p53 KO B 5,000 80,000 * 4,000 * 60,000 3,000 40,000 2,000 2347 51404 iGSH MFI 20,000 h3T 1,000 h3T

c-SH (ALM 633) MFI 0 0 3208 h3T-p53 KO p53 h3T

h3T h3T- KO 61754 c-SH iGSH h3T- p53 KO h3T- p53 KO 1,000 C * D E 2 800 * 331 60 600 209 50 * 400 *

pJNK MFI 40 1 h3T 200 30 p53 h3T- KO 0

Fold change 20

pJNK h3T Annexin V (%) 0 10 Catalase SOD1 h3T- p53 KO 0 CD8+Vb12+ h3T h3T-p53 KO 663 1,500 * h3T 1,000 h3T-p53 KO MFI

1089 6 h3T 500 DiOC p53 h3T- KO 0

DiOC6 F 150 2,000 WT WT WT + L-NAC + L-NAC 1,500 100 159529 1033 WT 1,000

DAF MFI 50 + L-NAC 500 Phospho-p53 MFI 129230 877 + L-NAC 0 0 100 102 104 106 100 102 104 106 DAF (NO) Phospho-p53

Figure 2. Increased antioxidant capacity and lower cell death in p53-KO T cells. TCR-activated splenic T cells from h3T and h3T-p53 KO mouse at day 3 were used. A, after restimulation with cognate human tyrosinase antigen-pulsed T2-A2 cells for 4 hours and staining using DAF (for NO) or DCF-DA (for H2O2) dyes before analyzing by FACS (A). B, staining with Alexa Fluor–labeled maleamide dye to determine the expression of cell surface thiols (c-SH) and with monochlorobimane dye for determining intracellular glutathione (iGSH) levels using FACS. C, real-time quantitative PCR analysis of antioxidant genes catalase and superoxide dismutase using

RNA. D, staining with fluorochrome-conjugated phospho-JNK antibody (left) and membrane potential dye DiOC6 (right) after 4 hours of restimulation, E, determination of AICD 4 hours after TCR restimulation by staining with Annexin V and using FACS. Numerical value represents mean fluorescence intensity (MFI) in FACS overlay panels, and adjacent bar diagram represents cumulative data from different experiments (, P < 0.05). F, wild-type splenic T cells were TCR stimulated and cultured for 72 hours in the presence of IL2 (100 U/mL), after which these were harvested, washed, and then either incubated with NAC (10 mmol/L) for 45 minutes or left untreated. Cells were further TCR restimulated and stained with DAF and phospho-p53 antibody following the manufacturer's protocol (BD Phosflow), and CD8þ T cells were gated for FACS analysis. N ¼ 2. www.aacrjournals.org Cancer Res; 76(18) September 15, 2016 OF5

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A 15,000 * 10,000

5,000 2NBDG MFI

h3T 0 p53

h3T- KO h3T 2NBDG h3T- p53 KO

B i ii 2.5 2.5 h3T-p53 KO h3T 1.0 h3T * * 2.0 * 2 * h3T-p53 KO 1.5 * 1.5 0.5 1.0 1

Fold increase 0.5

Fold increase 0.5 0.0 0.0 0 HIF1a TIGAR pfk HKII LDHA PDK-1

h3T-p53 KO iii h3T 1 iv 6 h3T ** h3T-p53 KO * 4

0.5 * 2 Fold increase Fold increase 0 0 PGC-1a

C D 120 ** 20 ** 16 17.8% 80 12

6.05% 8

40 4

p53 for phospho-S6

ECAR (mpH/min) h3T- KO % of Cells posive 0 h3T 0 h3T Phospho-S6 h3T-p53 KO

Figure 3. Increased glycolytic commitment in p53-KO T cells. Splenic T cells from h3T and h3T-p53 KO mouse were TCR activated for 3 days and used. A, determination of the ﬂuorescent glucose (2NBDG) uptake using FACS as detailed in Material and Methods. MFI, mean ﬂuorescence intensity. B, to obtain RNA for analyzing the expression of key glycolytic genes (i), HIF1a and TIGAR (ii), mitochondrial biogenesis regulator PGC1-a (iii), and pentose phosphate pathway genes (iv). C, determination of basal ECAR using Seahorse assay bioanalyzer as per the manufacturer's protocol. D, determination of phosphorylation level of S6 protein after intracellular staining and analysis using FACS (, P < 0.05; , P < 0.01). Bar diagram on the right of each overlay represents cumulative data from different experiments.

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A T2/Tyrosinase T2/Tyrosinase h3T 60 * h3T-p53 KO

IL2 50 25,000 * TNF- 40 ** 20,000 30 h3T-p53 KO h3T-p53 KO 20 * 15,000 10 % of Posivecells 0 10,000 h3T h3T IFN g Conc. (pg/mL)

Figure 4. TNF a 5,000 p53- 2/IFN g KO T cells exhibit enhanced IL effector functions.A, splenic T cells from IFNg IFNg 0 h3T and h3T-p53 KO mouse activated IFNg for 3 days were either restimulated with cognate human tyrosinase antigen- B pulsed T2-A2 cells for 6 hours before C 2 * 3 performing intracellular staining using 16,000 ﬂuorochrome-conjugated anti- * * 12,000 h3T cytokine (IL2, IFN , and TNF ) antibody g a 2 h3T-p53 KO (left) or were restimulated overnight to 8,000 determine the IFNg secretion in the 4,000 1 supernatant by ELISA (right). CD107a MFI B, the degree of degranulation was 0 1 determined in na€ve or 3-day activated KO Fold increase h3T and h3T-p53 KO splenic T cells by h3T staining for CD107a expression. p53 0 0 C and D, RNA isolated from 3-day h3T- Tbet IRF4 activated h3T and h3T-p53 KO mice were used to determine expression of transcription factors T-bet and IRF4 (C) and effector molecules and cytokine, D cytokine receptors (D). The fold change 10.0 in expression of these molecules in 5.00 h3T ** h3T-p53 KO T cells was calculated over h3T ** h3T-p53 KO p53 h3T cells ( , P < 0.05; , P < 0.01). h3T- KO ** * 5.0 2.50 * increase * * Fold

0.00 0.0 Tbx21 csf-2 Lrmp GzmB IL-1rn IL10 IL3IL9 IL23r IL22 is known to harbor stem-cell memory phenotype cells (25), which antigen in the presence or absence of inhibitors. We observed that correlated with 2-to-3-fold higher expression of stemness genes as compared with the activated T cells alone, the p53 inhibitor Tcf7, Lef1, and PRDM1 as compared with h3T T cells (Fig. 5F). pretreated T cells resulted in a significantly improved control of These data indicate that the quantitative and qualitative differ- tumor growth and thus survival of the tumor-bearing mouse (Fig. ences between the h3T and h3T-p53 KO T cells may account for 5H). These data show that pharmacological inhibition of p53 differences in ability to control tumor growth in vivo. Further, to could be a clinically translatable ACT approach. confirm the feasibility of this approach using TCR-engineered T cells, we used the splenic T cells from the C57BL/6 wild-type mice Reduced TGFb signaling in p53-KO T cells and p53-KO mice that were rendered tumor antigen specificby Next, we addressed if the improved tumor control exhibited by þ using retroviral transduction of tyrosinase reactive HLA-A2 h3T-p53 KO T cells is due to reduced susceptibility to immuno- TIL1383I TCR (26). Adoptive transfer of the tyrosinase reactive suppression or reduced plasticity toward iTregs conversion. TIL1383I TCR-transduced T cells also showed long-term control Importantly, h3T-p53 KO T cells exhibited reduced expression of of subcutaneously established B16-A2 tumors in the HLA-A2 TGFbRI and TGFbRII as compared with the h3T-derived splenic recipient mice (Fig. 5G). Next, we tested the efficacy of p53 T cells (Fig. 6A). Further, ex vivo programming conditions that use inhibitors pifithrin-mu (PFT-m) and pifithrin-alpha (PFT-a)in TGFb and IL2 (Fig. 6B, i) showed that p53-KO–derived splenic þ controlling tumor growth. For this purpose, subcutaneously CD4 T cells exhibit less susceptibility to iTreg conversion established B16-F10 murine melanoma in C57BL/6 mice was (Fig. 6B, ii). The quantitatively reduced iTregs also corresponded treated by adoptively transferring 1 106 melanoma epitope to the reduced FoxP3 expression in the p53-KO–derived splenic þ gp100 reactive T cells that were activated for 3 days with cognate CD4 T cells as compared with wild-type T cells (Fig. 6B, iii). A

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A B16-A2 D d0 (3.5 × 105) h3T-p53 KO CD44 Cells Figure 5. transferred Improved tumor control by adoptively p53 106 V 12+ transferred h3T- KO T cells. d14 A, schematic representation of the Splenocytes from experimental protocol. B, tumor growth either h3T-p53 KO h3T Cells Tumor curve (in mm2) obtained after treating or h3T mouse transferred tracked subcutaneously established melanoma in C57BL/6 recipient mice by adoptively transferring either 1 106 h3T or h3T-p53 B 500 Control p53KO h3T No Cells KO T cells. Nine mice per group were )

2 transferred treated using ACT. The peripheral blood 400 CD62L was obtained from the recipient mice, þ 300 and the adoptively transferred Vb12 E T cells were evaluated for total 200 percentage population (C); cell surface 20.2 expression of CD44 and CD62L (D); 100 h3T-p53 KO cytokine IFNg and TNFa secretion upon *** Tumor size (mm Tumor restimulation (E). F, activated h3T or h3T- 7.94 h3T 0 p53 KO splenic T cells were used to 1 7 14 21 28 35 IFNg prepare RNA and determine the C expression of genes related to stem cell Days þ 18.5 phenotype using qPCR. G, splenic CD8 T h3T-p53 KO cells obtained from C57BL/6 WT or h3T-p53 KO CD8 C57BL/6-p53 KO mouse strains were Cells 8.44 h3T transduced with HLA-A2þ human transferred TNFa tyrosinase epitope reactive TIL1383I TCR splenocytes and 107 cells were adoptively F transferred to the Rag-A2 recipients with 4 h3T Cells h3T * subcutaneously established murine p53 transferred h3T- KO melanoma B16-A2. The tumor growth in 3 * various groups of recipient mice that * were either treated or left untreated is 2 shown. Seven mice in each group No Cells between two experiments were used and transferred 1 showed identical response. H, melanoma epitope gp100 reactive T cells were V 12 obtained from Pmel TCR transgenic mice 0 and activated for 3 days with cognate TCF 7Lef1PRDM1 antigen either alone or in the presence of G p53 inhibitors (5 mmol/L Pif-a þ Pif-m). H The activated T cells were transferred to 500 Untreated control ) 2 T cells alone the B16-F10 murine melanoma-bearing 400 Control p53 Inhibitor pre-treated T C57BL/6 host and tumor growth was B6 cells 300 ** measured twice weekly. A total of 12 to 16 * p53 0.6 0.8 1.0 -KO mice in each group were treated in two **** 200 experiments with similar results 100 ( , P < 0.05; , P < 0.005; , P < 0.00; Tumor size (mm Tumor , P ¼ 0.0006). Probability of survival 0 0.0 0.2 0.4 0 50 100 150 0 10 20 30 40 50 60 Days Time (days)

detailed analysis of differences in the TGFb signaling pathway was and mitosis was also upregulated in p53-KO T cells. (29). Sim- performed using the TGFb Signaling Targets RT2 Proﬁler PCR ilarly, Ctnnb1, a gene that encodes b-catenin protein, is upregu- Array (Qiagen). Our data in Fig. 6C show that a number of lated in p53-KO T cells. Ptk2, protein tyrosine kinase 2 (also signaling molecules were differentially expressed in T cells known as focal adhesion kinase), which plays an important role obtained from p53-KO mice, which may have contributed to the in T cell–antigen-presenting cell conjugation, and HMOx1 enhanced antitumor phenotype. For example, Furin, is a direct encoded heme-oxygenase-1 levels, a target of p53 (30), were also target gene of the IL12/STAT4 pathway, regulates Th1/2 cell increased in p53-KO T cells. The expression of inhibitor of balance by limiting conversion to Th2 phenotype, and its expres- dna binding 2 (Id2), which promotes generation of distinct þ sion directly correlates to the stability and long-term secretion of CD8 T-cell memory, was also increased in p53-KO T cells þ IL2 by CD4 T cells (27). Similarly, increased expression of (31). Tumor suppressor p53 is an essential partner of Smads, activating transcription factor (ATF) 3 that is a positive regulator affecting TGFb signaling at various points in the pathway (32). of IFNg gene expression (28) supports our observation of Importantly, the expression of mitogen-activated protein kinase increased Th1 cytokine response in p53-KO T cells. The cell kinase kinase 7 (Map3K7) was reduced in p53-KO T cells. This division cycle 6 (Cdc6), a target of p53 that coordinates S phase kinase mediates the signal transduction induced by TGFb and

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A C 1 1 h3T h3T-p53 KO Ifrd1 * Furin Atf3 * Cdc6 0.5 0.5 Ctnnb1 Emp1 Figure 6. Ptk2

Altered TGFb signaling in p53-KO T cells. Fold increase Hmox1 A, activated h3T or h3T-p53 KO splenic Id2 CD8þ T cells obtained after FACS sorting 0 0 Smad3 were used to prepare RNA and to TGF-β RI TGF-β RII E2f4 determine the expression of TGFb B i Gadd45b receptors (TGFbRI and TGFbRII). B, h3T Tnfsf10 or h3T-p53 KO splenic T cells were +Anti CD3 and Myc Total anti CD28 cultured for 3 days under iTreg polarizing Day 3 evaluation Bhlhe40 splenocytes conditions, and FoxP3 expression Rara Spleen in culture Treg polarizing condition II10 analysis was done using intracellular h3T-p53 KO/h3T IL2 (100 U/mL)+TGF-b1 (5 ng/mL) staining (left) or real-time PCR (right). Wfs1 a-IL4 (10 µg/mL)+ a-IFNg Cebpb C, RNA from A was also used to run the (10 µg/mL) Runx1 TGFb signaling real-time PCR-based 84- iTreg Ep300 gene array. The data obtained are ii iii h3T Brd2 presented in fold change with genes Hes1 KO CD4 h3T-p53 KO grouped for pathways indicated on left. Creb1 The data are representative from one of 1 Hey1 two experiments, and genes with similar p53 Serpine1 results in both array experiments are Vegfa presented. h3T- Ptk2b 0.5 Mbd1 Notch1 Id3

h3T ** Aipl1

Fold increase Dnaja1 0 Map3k7 FoxP3 Foxp3 Min Avg Max controls a variety of cell functions, including transcription regu- with the cognate tyrosinase epitope (Fig. 7C). Additionally, p53 lation and apoptosis (33). Expression of E2F4—a transcription inhibition not only downregulated ROS and RNS, but also factor that plays a crucial role in the control of cell cycle and reduced the expression of CD95, CD95L, exhaustion molecules þ regulating antigen recall response in CD8 T cells—was also Lag3 and PD1 on TCR-activated T cells (Supplementary Fig. S2B) elevated in p53-KO T cells (34). The p53-KO T cells expressed Thus, pharmacologically inhibiting p53 in human TCR-trans- higher Gadd45b, which augments antitumor immune response by duced cells mimicked results of increased metabolic activity in enhancing the expression of IFNg, granzyme B, CCR5 in T cells form of glycolysis, with increased effector functions and lower cell (35), and protecting from apoptosis by p38 activation and JNK death. Further, we observed that pretreatment with either PFT-m or inhibition (36). WFS1, a gene encoding an endoplasmic reticu- PFT-a results in fewer human T cells exhibiting FoxP3 expression lum (ER) membrane protein and involved in survival of pancre- under iTreg ex vivo programming condition (Supplementary Fig. atic b-cells, was also found to be upregulated in p53-KO T cells. As S2C). Lastly, tracking studies using human T cells transduced with expected, we also found that Myc levels were enhanced in p53-KO melanoma reactive TIL1383I TCR that were pretreated with a T cells, which also showed higher HIF1a and glycolytic commit- combination of p53 inhibitors and transferred into NSG-A2 mice ment. Thus, targeting p53 in T cells result in modulating TGFb- showed increased persistence at 72 hours as compared with the mediated signaling molecules. untreated counterparts (Fig. 7D). Thus, we believe that the strat- egy to inhibit p53 expression is potentially translatable and could Pharmacological inhibition of the p53 inhibitor PFT-m alters improve the efﬁcacy of ACT. þ functionality of human TCR-transduced CD8 T cells To determine the translational potential of inhibiting p53, pharmacological inhibitors PFT-m and PFT-a pretreated human Discussion tyrosinase TCR TIL1383I transduced T cells were characterized. p53 is regarded as the "guardian of genome integrity" due to its Upon p53 inhibition, we noticed a signiﬁcant increase in glucose complex role in regulating cellular differentiation (37). More than uptake as measured by 2-NBDG (Fig. 7A), which also correlated to 50% of tumors have a direct mutation of p53, which promotes an increased fraction of IFNg secreting cells upon antigen restim- invasion, metastasis, proliferation, and cell survival (38). p53 is ulation (Fig. 7B). Importantly, AICD was also reduced when PFT- also a central regulator of the glycolysis and TGFb signaling m pretreated TIL1383I TCR-transduced T cells were restimulated pathways (9, 19). Therefore, we hypothesized that rendering

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A B No inhibitor 4,000 1,000 3,500 * * * * PFT- 800 PFT- 3,000 2,500 600 No inhibitor MFI 2,000 1,500 400 IFN g 2NBDG MFI 1,000 PFT- 200 500 0 PFT- 0 μ a α m IFNg PFT PFT PFT 2NBDG PFT inhibitor No inhibitor C 60 D No 50 μ μ μ PFT- (10 mol/L) + (30 mol/L) Injected pretreated T cells 40 * T Cells (-) ve Control cells 0.9 0.2 0.5 0.3 30 20 CD8

Annexin V % 10 0 CD8+Vβ 12+ CD4

PFT-µ No inhibitor

Figure 7. p53 inhibitor-treated TCR-transduced T cells exhibit increased effector function and persistence. Human peripheral blood T cells from normal healthy individuals were retrovirally transduced with melanoma epitope tyrosinase reactive TIL1383I TCR and were either left untreated or were pretreated for an hour with p53 inhibitors PFT-a (30 mmol/L) or PFT-m (10 mmol/L) before TCR restimulation with tyrosinase peptide-pulsed T2-A2 cells for 4 to 6 hours to analyze glucose uptake using 2NBDG assay (A), cytokine secretion by intracellular IFNg staining (B), and susceptibility to AICD by Annexin V staining (, P < 0.01; C). Bar diagram on the right of each overlay represents cumulative data from different experiments. D, ten million human T cells engineered with tyrsoinase reactive TIL1383I TCR were either untreated or pretreated with a combination of p53 inhibitors PFT-a and PFT-m and adoptively transferred to NSG-A2 mice. Peripheral blood and spleens of recipient mice were stained for human Vb12, human CD8, and CD4 for tracking the persistence of the transferred cells. Data were acquired using FACS. Numerical value is the average from three mice in similar groups. MFI, mean ﬂuorescence intensity.

properties of higher proliferation, lower cell death, and increased sion of stem-cell–specific transcription factors such as Tcf7, Lef-1, persistence to CTLs by lowering its p53 expression could improve and PRDM1. Notably, p53 has been shown to bind at the pro- adoptive T-cell immunotherapy. Our data establish that p53- moter region of Oct4 and Nanog, which are required for self- þ deficient tumor-specific CD8 T cells exhibit increased glycolytic renewal and maintenance of embryonic stem cells in an undif- commitment that correlates to higher IFNg secretion, increased ferentiated state, and reduce their gene transcription (40). Given a persistence due to high stem-cell–related gene expression, recent report that a subset of memory T cells with stem-cell–like reduced susceptibility to immunosuppression and iTreg conver- phenotype (referred to as Tscm) exists within the Tcm fraction sion due to reduced TGFb signaling. All these features result in a (41), it is likely that the Tscm phenotype is increased in the p53- robust effector phenotype that leads to improved tumor control. KO T cells. A recent study showed that antigen-specific proliferative p53 also regulates aerobic respiration at the glycolytic and þ responses of CD4 T cells require downregulation of tumor OXPHOS steps via transcriptional regulation of its downstream suppressor p53 (7), and that inhibiting p53-regulating protein genes TIGAR and SCO2 (10, 11). In line with the role of p53 as a Mdm2 resulted in its sustained expression and prevented prolif- negative regulator of glycolysis (11), our data show that p53-KO T eration. Our data show that T cells obtained from the TCR cells exhibit higher glycolytic commitment as observed by glucose transgenic mice h3T developed on p53-KO background (i.e., uptake and increased expression of key glycolytic genes. This h3T-p53 KO) proliferate rapidly, and maintain antigen specificity increase in glycolysis corresponds to the reduced expression of upon TCR stimulation. Given the role of p53 in negatively TIGAR, an inhibitor of the fructose-2, 6-bisphosphate, which is regulating cell-cycle progression by blocking cyclin D1 (16), we normally activated by p53 to regulate glycolysis (11). Another observed that h3T-p53 KO T cells exhibited higher expression of property of p53-deficient T cells was their ability to persist longer cyclin D and lower levels of cyclin inhibitors that correlate to and exhibit lower degree of AICD. It has been shown that p53 increased proliferation leading to enlarged spleen and thymus. leads to upregulation of a number of prooxidant enzymes as However, increased proliferation was not associated with shed- quinone oxidoreductase, proline oxidase, BAX, and PUMA, lead- ding of CD62L molecule (3, 39), because we observed that h3T- ing to oxidative stress and consequently to apoptosis (42–44). p53 KO T cells exhibit CD62Lhi central memory (Tcm) phenotype. Upon its mitochondrial translocation, p53 binds to and inhibits Importantly, h3T-p53 KO T cells also exhibited elevated expres- MnSOD, playing a direct role in promoting ROS formation and

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eventually in apoptosis (45). Thus, an inverse correlation between Disclosure of Potential Conflicts of Interest p53 and antioxidant capacity may have contributed to the No potential conflicts of interest were disclosed. increased persistence and antitumor T-cell response. Interestingly, a detailed necropsy of the tumor-bearing recipient animals Authors' Contributions showed increased inflammatory reactions, without any evidence Conception and design: A. Banerjee, K. Thyagarajan, C.M. Paulos, C. Voelkel- Johnson, S. Mehrotra to suggest that this was due to the transfer of p53 KO T cells (data Development of methodology: A. Banerjee, K. Thyagarajan, P. Chakraborty, not shown). P. Kesarwani, K. Moxley, M.P. Rubinstein, C.M. Paulos, M.I. Nishimura, Another confounding factor that limits long-term tumor con- S. Mehrotra trol by ACT is suppressive tumor microenvironment, which is Acquisition of data (provided animals, acquired and managed patients, abundant with suppressive cytokines such as TGFb. Importantly, provided facilities, etc.): A. Banerjee, K. Thyagarajan, S. Chatterjee, P. Kesar- key cellular responses to TGFb signals have been shown to rely on wani, M. Soloshchenko, M. Al-Hommrani, K. Andrijauskaite, K. Moxley, H. Janakiraman, M.J. Scheffel, K. Helke, M.I. Nishimura, S. Mehrotra p53 family members (9). This study shows that p53-KO T cells Analysis and interpretation of data (e.g., statistical analysis, biostatistics, display an impaired response to TGFb signals. Additionally, Smad computational analysis): A. Banerjee, K. Thyagarajan, S. Chatterjee, P. Chak- and p53 protein complexes converge on separate cis binding raborty, P. Kesarwani, K. Helke, K. Armenson, M.P. Rubinstein, E.-G. Mayer, elements on a target promoter and synergistically activate S. Mehrotra TGFb-induced transcription (6). Thus, it is likely that p53-KO T Writing, review, and/or revision of the manuscript: A. Banerjee, K. Thyagar- cells displayed diminished transcriptional activation of key TGFb ajan, S. Chatterjee, K. Helke, M.P. Rubinstein, D.J. Cole, C. Voelkel-Johnson, M.I. Nishimura, S. Mehrotra target genes (32). It has also been shown that p53 enhances Administrative, technical, or material support (i.e., reporting or organizing the transcription of Treg signature transcription factor Foxp3 data, constructing databases): K. Thyagarajan, M. Soloshchenko, by binding to the promoter and the conserved noncoding M. Al-Hommrani, K. Moxley, K. Helke, V. Palanisamy, M.P. Rubinstein, DNA sequence-2 of the Foxp3 gene (8), and that fewer nTregs C.M. Paulos, S. Mehrotra and iTregs are obtained from p53-KO mice. Our data also Study supervision: S. Mehrotra confirm this observation, which implies that impaired TGFb signaling molecules may be responsible for reduced plasticity Acknowledgments in p53-deficient T cells. It is also likely that increased glycolytic The authors acknowledge Drs. Zihai Li, Radhika Gudi, and Ephraim Ansa- fi Addo in the Department of Microbiology and Immunology at MUSC for their commitment identi ed in h3T-p53 KO T cells by elevated levels help with this article. of ECAR values, glycolytic genes, and HIF1a metabolically downregulates Treg differentiation, because HIF1a has been Grant Support shown to attenuate Treg development by binding Foxp3 and The work was supported in part by NIH grants R21CA137725 and targeting it for proteasomal degradation (46). Importantly, p53 R01CA138930 to S. Mehrotra and P01CA154778 to M.I. Nishimura. The Cell pharmacological inhibitors also improved T cell–mediated Evaluation and Therapy Shared Resource is supported by P30 CA138313. tumor control, and pifithrin-pretreated murine and human T The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in cells exhibited increased persistence in vivo.Overall,thisstudy accordance with 18 U.S.C. Section 1734 solely to indicate this fact. shows that p53 is a central regulator of multiple pathways (such as glycolysis, ROS, TGFb signaling), and its inhibition could be Received July 2, 2015; revised June 29, 2016; accepted July 8, 2016; important for ACT of tumor. published OnlineFirst July 27, 2016.

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OF12 Cancer Res; 76(18) September 15, 2016 Cancer Research

Lack of p53 Augments Antitumor Functions in Cytolytic T Cells

Anirban Banerjee, Krishnamurthy Thyagarajan, Shilpak Chatterjee, et al.

Cancer Res Published OnlineFirst July 27, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1798

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