Comparison of immune infiltrates in melanoma andpancreatic cancer highlights VISTA as a potentialtarget in pancreatic cancerJorge Blandoa,1, Anu Sharmab,1, Maria Gisela Higaa, Hao Zhaoa, Luis Vencea, Shalini S. Yadava, Jiseong Kima,Alejandro M. Sepulvedac, Michael Sharpc, Anirban Maitrad,e, Jennifer Wargof, Michael Tetzlaffd, Russell Broaddusd,Matthew H. G. Katzf, Gauri R. Varadhacharyg, Michael Overmang, Huamin Wangd, Cassian Yeeh, Chantale Bernatchezh,Christine Iacobuzio-Donahuei, Sreyashi Basua, James P. Allisona,j,2, and Padmanee Sharmaa,b,j,2

aThe Immunotherapy Platform, The University of Texas MD Anderson Cancer Center, Houston, TX 77054; bDepartment of Genitourinary Medical Oncology,The University of Texas MD Anderson Cancer Center, Houston, TX 77030; cJanssen Oncology Therapeutic Area, Janssen Research and Development, LLC,Pharmaceutical Companies of Johnson & Johnson, Spring House, PA 19477; dDepartment of Pathology, The University of Texas MD Anderson Cancer Center,Houston, TX 77030; eDepartment of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030;fDepartment of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; gDepartment of Gastrointestinal MedicalOncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; hDepartment of Melanoma Medical Oncology, The University of TexasMD Anderson Cancer Center, Houston, TX 77030; iDavid Rubenstein Pancreatic Cancer Research Center, Memorial Sloan Kettering Cancer Center, New York,NY 10065; and jDepartment of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030

Contributed by James P. Allison, November 30, 2018 (sent for review July 19, 2018; reviewed by Nina Bhardwaj and Aatur D. Singhi)

Immune checkpoint therapy (ICT) has transformed cancer treatmentin recent years; however, treatment response is not uniform acrosstumor types. The tumor immune microenvironment plays a criticalrole in determining response to ICT; therefore, understanding thedifferential immune infiltration between ICT-sensitive and ICT-resistant tumor types will help to develop effective treatmentstrategies. We performed a comprehensive analysis of the immunetumor microenvironment of an ICT-sensitive tumor (melanoma, n =44) and an ICT-resistant tumor (pancreatic cancer, n = 67). We foundthat a pancreatic tumor has minimal to moderate infiltration of CD3,CD4, and CD8 T cells; however, the immune infiltrates are predom-inantly present in the stromal area of the tumor and are excludedfrom tumoral area compared with melanoma, where the immuneinfiltrates are primarily present in the tumoral area. Metastatic pan-creatic ductal adenocarcinomas (PDACs) had a lower infiltration oftotal T cells compared with resectable primary PDACs, suggestingthat metastatic PDACs have poor immunogenicity. Further, a signif-icantly higher number of CD68+ macrophages and VISTA+ cells (alsoknown as V-domain immunoglobulin suppressor of T cell activation)were found in the pancreatic stromal area compared with mela-noma. We identified VISTA as a potent inhibitory checkpoint thatis predominantly expressed on CD68+ macrophages on PDACs.These data suggest that VISTA may be a relevant immunotherapytarget for effective treatment of patients with pancreatic cancer.

pancreatic cancer | immune monitoring | immune checkpoints |immunopathology | immune infiltrate

Pancreatic cancer patients have poor prognoses with limitedtreatment options, having a 5-y survival rate of less than 7%

(1–4). Whereas, other immunogenic tumor types, such as mela-noma, have demonstrated dramatic clinical responses to immunecheckpoint inhibitors, including anti–CTLA-4 and anti–PD-1antibodies (5–7). In contrast, clinical studies have reported aminimal clinical benefit in response to these antibodies in pa-tients with pancreatic cancer (3, 4, 8). A greater understanding ofthe tumor immune microenvironment in pancreatic cancer mayenable development of effective immunotherapeutic strategies. Im-mune infiltration into pancreatic tissues has not been well charac-terized. Some studies have reported that the pancreatic tumormicroenvironment (TME) is poorly infiltrated with effector lympho-cytes and has multiple immunosuppressive cells, including myeloid-derived suppressor cells and regulatory T cells (9–11). Previouspublications largely indicate that pancreatic tumors have minimalinfiltration of immune cells (9, 10, 12–14). In contrast, reports fromother groups suggest that, apart from a regulatory immune signature,

pancreatic tumors also contain an effector immune infiltrate (11,15–18). A previous genomic study in human pancreatic tumorsidentified an immunogenic subtype of pancreatic cancer withup-regulated immune networks (19). The expression of specificimmune checkpoints in the pancreatic TME, which may representnovel immunotherapy targets, has not been investigated systematically.Compared with melanomas, pancreatic and other solid tumors

have some important architectural histologic differences. In addition,

Significance

The study demonstrates that V-domain immunoglobulin sup-pressor of T cell activation (VISTA) is an immune checkpointthat is preferentially expressed at higher levels in pancreaticcancer. The study gives a detailed analysis of immune in-filtration in primary and metastatic pancreatic tumors com-pared with melanoma, which differs in its tumor/stromaldistribution. Our data indicate that human pancreatic tumorsexpress CD68+ macrophages and highlight the inhibitorycheckpoint molecule VISTA as a potential immunotherapeutictarget in pancreatic cancer.

Author contributions: P.S. and J.P.A. designed research and supervised the study; A.M.,J.W., G.R.V., M.O., C.Y., C.B., C.I.-D., J.P.A., and P.S. managed patients and provided analyt-ical and material support; A.M.S. and M.S. contributed new reagents/analytic tools; J.B.,A.S., M.G.H., J.K., and S.B. performed research; J.B., A.S., H.Z., L.V., S.S.Y., S.B., J.P.A., andP.S. analyzed data; J.B., M.T., R.B., M.H.G.K., and H.W. reviewed pathology and immuno-histochemistry data; and J.B., A.S., S.S.Y., and P.S. wrote the paper.

Reviewers: N.B., Mt. Sinai School of Medicine; and A.D.S., University of Pittsburgh Schoolof Medicine.

Conflict of interest statement: J.P.A. is an inventor and recipient of royalties from in-tellectual property licensed to Bristol-Meyer Squibb, Merck, and Jounce. He is a memberof the scientific advisory board for Jounce Therapeutics, Neon Therapeutics, Amgen,Apricity, BioAlta, Forty-Seven, Tvardi Therapeutics, TapImmune, ImaginAB, Codiak Biosci-ences, and Marker Therapeutics. J.P.A. and P.S. own a patent licensed to Jounce Thera-peutics. P.S. serves as a consultant for Constellation, Jounce Therapuetics, Kite Pharma,Neon Therapeutics, BioAtla, Pieris Pharmaceuticals, Oncolytics Biotech, Merck, BioMx,Forty-Seven, Polaris, Apricity, Marker Therapeutics, Codiak, ImaginAB, and TapImmune.She also has stock ownership in Jounce, Neon Therapeutics, Constellation, Oncolytics,BioAtlanta, Forty-Seven, Apricity, Polaris, Marker Therapeutics, Codiak, ImaginAB, andTapImmune. A.S. is an employee of Janssen.

Published under the PNAS license.1J.B. and A.S. contributed equally to this work.2To whom correspondence may be addressed. Email: jallison@mdanderson.org orpadsharma@mdanderson.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811067116/-/DCSupplemental.

Published online January 11, 2019.

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morphological differences exist among different types of melanomas(20–25) and pancreatic cancers (26–28). Cancers do not just comprisemalignant cells, but are rather complex matrix-like “organoid for-mations” defined in the TME (29–32). The two main regions thatcomprise this complex matrix are parenchymal tumor cells (tumorarea) and stromal cells (stromal area). Melanomas are generallydescribed as having melanocytes in an aberrant distribution andgrouped nests of melanocytes, which are normally surrounded by orembedded in a thinner matrix of stromal component largely invadedby immune cells. In contrast, pancreatic ductal adenocarcinomas(PDACs) comprise a thicker desmoplastic structure composed offibrous connective tissue populated with myeloid cells and erraticallydistributed lymphoid components (see representative micro-photographs, SI Appendix, Fig. S1A–C and refs. 20–25).Melanomas originate in one of the most immunogenically ac-

tive organs of the body, the skin, which is traversed by a networkof lymphatic and blood vessels and contains a large number ofimmune-competent cells (33), whereas the pancreas has a lym-phatic network in which peripheral extensions can be found withinthe lobules, but intralobular lymphatic extensions are relativelysparse (34). We have no clear understanding of the distribution ofimmune infiltrates in these architecturally different tumor types,and dissecting these differences may enable development of noveland effective treatment strategies for pancreatic cancer. A de-tailed analysis of the immune infiltration of pancreatic tumors hasnot been done, and the extent of infiltration in pancreatic tumorsis unclear. The differential distribution of immune cells within thetumor versus the stromal area of the TME of PDACs may have animpact on response to immune checkpoint therapy (ICT). In ad-dition, we do not have a clear understanding of the differences intumor immune microenvironment of primary (resectable) andmetastatic PDACs. In the clinical scenario, treatment with neo-adjuvant chemotherapy as standard-of-care may alter or impactthe immune TME, which may ultimately impact response to ICT.To gain more insight into the immune contexture of pancreatic

tumors, we compared (i) immune infiltration in pancreatic cancer,which responds poorly to ICT, with that of melanoma, which re-sponds favorably to ICT, and (ii) immune infiltration in primaryuntreated to primary neoadjuvant-treated PDACs as well as met-astatic treated PDACs. We analyzed the microlocalization of theimmune infiltration in the TME to determine whether immunecells are localized in close contact with tumor cells or within the

tumor (tumor area) or are in the stromal component of the tumor(stromal area). We found that both tumor types have a hetero-geneous distribution of immune infiltrates. PDAC was found tohave a wide range of CD3, CD4, and CD8 T-cell infiltration, butthese subsets were mainly found in the stromal area of the TME.In addition, our data clearly highlight CD68+ macrophages thatexpress the inhibitory immune checkpoint V-domain Ig suppressorof T cell activation (VISTA) as a subset of immune cells that arehighly expressed in pancreatic tumors and are frequently present inthe stromal area of the tumor.

ResultsExpression of T-Cell Coinhibitory Genes Correlates with Overall Survivalin Pancreatic Cancer. In a cohort of 23 untreated primary PDACsamples, we evaluated the gene expression of nine T-cell coinhibi-tory genes (LAG-3, PD-L1, VISTA, BTLA, CTLA-4, TIGIT, TIM-3, CD244, and CD160) that were previously published (19) to beassociated with the immunogenic subtype of pancreatic cancer.Principal component analysis (PCA) separated patients into twocohorts: those with high T-cell coinhibitory gene expression (n =11) and those with low T-cell coinhibitory gene expression (n =12) (Fig. 1A). The expression of the inhibitory immune checkpointgenes was inversely correlated with survival. The median survivalof patients whose tumors had low expression of these T-cell coinhibi-tory genes was significantly higher than patients whose tumors had highexpression of these genes (37 mo versus 20 mo; P = 0.016) (Fig. 1B).

Immune Infiltration in Human Pancreatic Cancer Is Mainly Located inthe Stromal Component of the TME. We next compared density ofmultiple immune subsets in untreated primary PDAC (n = 29)and untreated metastatic melanoma (n = 44). Compared withmelanoma, which has a higher overall infiltration, untreatedprimary PDAC had a broad range of immune cell subset in-filtration (Fig. 2). As expected, we observed that melanoma tu-mors had significantly higher density of T cells represented byCD3, CD4, and CD8 markers (Fig. 2). Melanoma tumors alsoshowed significantly higher density of memory T cells (CD45RO),B cells (CD20), cells expressing the activation markers ICOS andOX40, cytotoxic cells (Gr-B), and regulatory T cells (FoxP3) (SIAppendix, Fig. S3). The density of immune cells expressing PD-1and PD-L1 was also significantly higher in melanomas comparedwith PDAC (Fig. 2). The protein expression of PD-L1 on tumor

Fig. 1. Expression of T-cell coinhibitory genes correlates with overall survival in pancreatic cancer. (A) PCA showing coinhibitory genes that are associatedwith the immunogenic subtype of pancreatic cancer (LAG-3, PD-L1, VISTA, BTLA, CTLA-4, TIGIT, TIM-3, CD244, and CD160). Gene expression data from acohort of 23 untreated pancreatic tumors were analyzed. PCA separated patients into two cohorts: those with high T-cell coinhibitory gene expression (n =11) and those with low T-cell coinhibitory gene expression (n = 12). (B) Kaplan−Meier survival curves for patients with high (red) or low (blue) T-cell coin-hibitory gene expression.

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cells was not significantly different between the tumor types (SIAppendix, Fig. S3).Conventional methods for IHC analyses, such as density (positive

cells per square millimeter area of analysis) and percentage

(number of positive cells per total number of cells) are ap-propriate for comparing samples from the same tumor type,since they have similar features, but these measures are in-adequate for comparing tumors of different cytomorphologies

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Fig. 2. Comparison of immune infiltrates in the total TME in pancreatic cancer and melanoma. Bar and dot plots comparing immune subsets (CD3, CD4, CD8,and CD68) and immune cells expressing VISTA, PD-1, and PD-L1 evaluated by IHC at 10× (large square) and 20× (small square) magnification. Each dotrepresents a patient, and bars with lines indicate median with confidence interval (CI). Only significant P values between groups are indicated. Statisticalsignificance is defined as P < 0.05. **P ≤ 0.01; ***P ≤ 0.001.

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Fig. 3. Distribution of immune infiltrates in the tumor and stromal compartments in pancreatic cancer compared with melanoma. (A) Tumor/stroma ratio foreach tumor type. (B) Bar plots comparing the median expression of the selected immune subsets (CD3, CD4, CD8, CD68, and VISTA) in each compartment ofthe total TME. Statistical significance is defined as P < 0.05. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, nonsignificant.

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or that have different tumor/stroma distributions. A de-scription of the analysis of the tumor and stromal areas in theTME is given in SI Appendix, Supplementary Materials andMethods. We observed that melanomas have a higher tumor/stroma ratio, with tumor cells occupying ∼70% of the area,whereas, in PDAC, tumor cells occupy ∼30% of the area (Fig.3A). To understand whether differences in immune in-filtration of melanoma and PDAC are reflected in the dif-ferential distribution of various immune cells within the tumorversus stroma, we computed the absolute numbers of cellsexpressing immunologic markers in the two compartments.CD3, CD4, and CD8 T cells were observed in both tumoraland stromal components for melanoma, but these T cells werepredominantly found in the stromal component for PDAC,with minimal infiltration into the tumoral component ofPDAC (Fig. 3B). Detailed examination of the other immunesubsets (CD45RO, Gr-B, OX40, ICOS, FOXP3, PD-1, PD-L1,and CD20) also demonstrated significantly higher density ofthese cells in the tumoral component, and, for some subsets,the stromal component, of melanoma compared with PDAC(SI Appendix, Fig. S4).Next, we evaluated macrophage infiltration as defined by

CD68 expression and the inhibitory checkpoint VISTA that ispredominantly expressed on macrophages (35–37). Total CD68+

macrophages did not significantly differ between melanoma andPDAC (Fig. 2), but we did find higher density of CD68+ mac-rophages in the tumoral component of melanoma comparedwith PDAC (Fig. 3B). Despite lower density of CD68+ mac-rophages, PDAC had significantly higher density of VISTAexpression (Figs. 2 and 3B), which highlights VISTA as a po-tential immunotherapeutic target.

Immune Infiltrates Differ in Primary Untreated, Primary Neoadjuvant-Treated, and Metastatic Treated Pancreatic Tumors. In a comple-mentary analysis, we also compared three cohorts of PDACs: (i)untreated resectable primary PDACs (n = 29); (ii) neoadjuvant-treated primary PDACs (n = 28); and (iii) treated metastaticPDACs (n = 10), which were pancreatic tumor samples obtained

from patients with metastatic disease (SI Appendix, Tables S1 andS2 A–C). The density of CD3 T cells in both the neoadjuvant-treated primary tumors (∼250 cells per square millimeter) anduntreated primary tumors (∼350 cells per square millimeter) weresignificantly higher than that in the metastatic tumors (∼100 cellsper square millimeter) (Fig. 4). Similarly, the density of CD4 T cellswas significantly higher in both the neoadjuvant-treated primarytumors and untreated primary tumors than in the metastatic tumors(Fig. 4). A similar trend for CD8 T cells was also observed (Fig. 4).Although metastatic tumors had lower density of T cells comparedwith primary tumors, we did observe higher density of ICOS andGr-B subsets in metastatic tumors (SI Appendix, Fig. S5), which mayindicate activated T cells. Overall, immune infiltration was found tobe lower in metastatic PDACs compared with primary PDAC (SIAppendix, Fig. S5). Metastatic PDAC also had lower density ofCD68+ macrophages compared with primary PDAC, but metastaticPDAC had similar density of VISTA+ cells compared with primaryuntreated PDAC (Fig. 4).

VISTA Is Expressed on CD68+ Macrophages, and Blocking VISTAbut Not PD-L1 Inhibits Cytokine Production by Tumor-InfiltratingLymphocytes in Vitro. To determine which cells expressed in-hibitory checkpoint molecules and whether there is coex-pression of VISTA on CD68+ macrophages, we subjected sevenpancreatic tumor samples to mass cytometry (also known as cytom-etry by time-of-flight or CyTOF) analysis. CD45+ immune infiltrateswere further analyzed for the expression of immune checkpointsfound to be expressed on different immune cell types. The analysisof a representative case is shown in Fig. 5. We observed a greaterfrequency of VISTA+ cells than PD-L1+ cells (Fig. 5). VISTAexpression was predominantly present on CD68+ macrophages(Fig. 5). A pooled analysis of the seven tumors demonstrated thepresence of a CD68+ macrophage cluster (cluster 18) that waspredominantly VISTA+ (SI Appendix, Fig. S6).The differential expression of PD-L1 and VISTA on distinct

CD68+ subsets suggests that PD-L1 and VISTA represent sepa-rate inhibitory pathways that are capable of suppressing antitumorT-cell responses in pancreatic cancer. However, to date, ICT

Fig. 4. Immune infiltrates in the total TME in primary untreated, neoadjuvant-treated, and metastatic pancreatic tumors. Bar and dot plots compare immunesubsets (CD3, CD4, CD8, CD68, and VISTA) evaluated by IHC. Each dot represents one patient; bars with lines indicate medians with confidence intervals. Onlysignificant P values between groups are indicated. Statistical significance is defined as P < 0.05. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ca, cancer.

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targeting the PD-1–PD-L1 axis has not been successful in pan-creatic cancer (3, 4, 8). To dissect the role of each of these in-hibitory pathways in pancreatic cancer and to test the idea thatboth PD-L1 and VISTA can inhibit T-cell responses, we isolatedand expanded tumor-infiltrating lymphocytes (TILs) from meta-static pancreatic tumors of three different patients and culturedthe CD8+ T cells from each patient’s tumor separately with plate-bound VISTA−Ig and/or or PD-L1–Ig. The proliferation of T cellswas not altered upon treatment with control Ig, as shown by theKi67+ CD8+ T-cell percentage upon stimulation with anti-CD3(∼80%; SI Appendix, Fig. S7). Degranulation, as measured byCD107a, and cytokine production, as measured by IFN-γ andTNF-α, were significantly inhibited in the presence of PD-L1–Ig(∼1.5× to 2×) or VISTA−Ig (∼3× to 10×); however, the engage-ment of the VISTA inhibitory pathway resulted in greater de-crease in CD8+ T-cell responses than the engagement of PD-L1pathway (Fig. 6). Furthermore, the presence of PD-L1-Ig andVISTA-Ig did not result in a significant decrease in T cell responseswhen compared to VISTA-Ig alone (Fig. 6). These data suggest that

monotherapy blockade of the PD-1/PD-L1 inhibitory pathway isunlikely to restore effective T-cell responses, because the VISTAinhibitory pathway is also highly expressed and capable of sup-pressing T-cell responses in human pancreatic cancer.

DiscussionA detailed analysis and understanding of the immune infiltrationof pancreatic tumors is essential for immune targeted therapy. Acomparison of immune infiltration in the TME of an ICT-responsive tumor like melanoma with an ICT-unresponsive tu-mor like pancreatic cancer may provide insights for improvingICT responsiveness. In the current study, we observed thatpancreatic tumors have a broad range of immune infiltrationlevels but that main players such as CD3, CD4, and CD8 T cellsare preferentially distributed in the stromal area of the TME(Fig. 3B); therefore, these findings suggest that not all pancreatictumors have low immunogenicity. The gene and protein ex-pression data presented here are in agreement with previouslypublished gene expression data suggesting an immune gene

Fig. 5. CyTOF analysis of CD68, PD-L1, and VISTA expression in human pancreatic tumors. CyTOF data are from a representative primary pancreatic, showingCD68+ macrophages and the inhibitory immune checkpoints PD-L1 and VISTA. Each point on the viSNE maps represents a single cell. Red indicates a high levelof marker expression; blue indicates no marker expression.

Fig. 6. Engagement of VISTA inhibits cytokine production by TILs. TILs from metastatic pancreatic tumors (n = 3) were cultured independently in differentexperiments with plate-bound VISTA−Ig and/or PD-L1−Ig to evaluate the function by CD107a degranulation assay and intracellular cytokine staining for IFN-γand TNF-α. Data are expressed as mean with SD for the three independent experiments and represent percentage changes after normalization to control Ig.The differences in degranulation and cytokine release are compared with the control Ig group. Only significant P values between groups are indicated.Statistical significance is defined as P < 0.05. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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signature-rich subtype of pancreatic cancer (19). Approximatelyone-third of the human pancreatic tumors in the present studyhad immune infiltration similar to that of melanoma; however,unlike melanoma, the immune infiltrates were primarily dis-tributed within the stromal area of the TME, with fewer cells inthe tumor area (or tumoral parenchyma). Additionally, pancre-atic tumors have high numbers of CD68+ macrophages, and, inthe present study, we show that these macrophages are prefer-entially distributed in the stromal area. This may have an im-portant impact on clinical outcomes with ICT therapies. Thus,even for moderately infiltrated pancreatic tumors, we may needapproaches which will help mobilize these moderately infiltratedimmune cells toward the tumor area for clinical benefit.A recent study demonstrated that a higher degree of myeloid

cell infiltration is correlated with poor clinical responses in pa-tients with pancreatic cancer who received GVAX vaccination,an allogeneic, GM-CSF-secreting cellular immunotherapy (11).Our study highlights VISTA+ macrophages as one of the po-tential inhibitory cell types, which may affect clinical outcomesin patients with pancreatic cancer. VISTA is predominantlyexpressed on myeloid cells (35). Preclinical data have establishedthat VISTA can suppress T-cell activation and hinders the de-velopment of tumor-specific immunity, but data on the functionalityof VISTA+ cells in human cancer are lacking (35, 38). Our grouppreviously reported that anti−CTLA-4 therapy can increase VISTAexpression on CD68+ macrophages, which have an M2 phenotype,as a resistance mechanism in prostate tumors and represents animmune checkpoint target (36). Recently, another group reportedthat pancreatic tumors with high cytolytic activity have increased

expression of multiple immune checkpoint genes such as CTLA4,TIGIT, TIM3, and VISTA (39).Here, we show that pancreatic tumors express significantly higher

levels of VISTA than melanoma tumors (Fig. 2), and VISTA ex-pression was found predominantly on CD68+ macrophages (Fig. 5).We additionally show that engagement of VISTA diminishes cyto-kine production by T cells isolated from metastatic pancreatic tu-mors (Fig. 6). Therefore, anti-VISTA antibodies may be an effectiveimmunotherapeutic strategy for patients with pancreatic cancer.

Materials and MethodsThe details of complete materials and methods are available in SI Appendix.Details of patient materials used and clinicopathological information onpancreatic cases are provided in SI Appendix, Tables S1 and S2 A–C, re-spectively. The study was approved by MD Anderson Cancer Center’s In-stitutional Review Board, and all relevant data were retrieved from thecomputerized database of the MD Anderson Cancer Center ImmunotherapyPlatform and from the Multidisciplinary Pancreas Database. Nanostringanalysis was performed on untreated primary pancreatic tumor samples (n =23). IHC staining and data analysis on formalin-fixed paraffin-embedded(FFPE) samples was performed on cohorts of pancreatic and melanoma pa-tients as detailed in SI Appendix, Supplementary Materials and Methods.CyTOF analysis was performed on fresh pancreatic tumor samples, and T-cellactivation assays were performed on expanded CD8 TILs.

ACKNOWLEDGMENTS. We thank Ashura Khan, Sheila Duncan, Sumit Subudhi,Jianjun Gao, Nicolle Patterson, and Alexsandra Espejo for technical support. P.S.,J.P.A., J.W., and C.Y. are members of the Parker Institute for Cancer Immuno-therapy. This research work was supported, in part, by the MD Anderson CancerCenter Immunotherapy Platform, the MD Anderson Pancreatic Cancer MoonShot program, and National Institutes of Health/National Cancer Institute GrantR01 CA1633793 (to P.S.).

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