cells, whereas in absence ofgdT cells PD-L1 or Galectin-9
blockade offers no additional tumor-protective benefit. Even
more, PD-L1 or Galectin-9 blockade expand and potently acti-
vate PDA-infiltrating CD4+and CD8+T cells ingdT cell-compe-
tent hosts but do not enhanceabT cell immunogenicity in the
absence ofgdT cells. It is perhaps surprising that checkpoint re-
ceptor blockade would not have potency in Tcrd–/–mice consid-
ering the substantial myeloid cell infiltrate in PDA (Liou et al.,
2015; Pylayeva-Gupta et al., 2012). Indeed, we found that
myeloid cells from Tcrd–/–mice have equivalent T cell inhibitory
capacity to their cellular counterparts in WT mice. However, we
foundthat,whereasabTcellsareinintimateproximitytogdTcells
in the PDA TME, myeloid cells are separated by great distances
fromabT cells in situ in human PDA, invasive murine PDA, and
pre-invasive disease suggesting enhanced opportunity forgdT
cell-abT cell interaction and limited opportunity for direct cross-
talk between macrophages andabT cells. Further, tumor cell
expression of exhaustion ligands is also possibly of lesser signif-
icanceasTcellsareexcludedfromdirectcontactwithtumorcells
via CXCL12 produced from FAP-expressing carcinoma-associ-
ated fibroblasts in PDA (Feig et al., 2013; Joyce and Fearon,
2015 ). Collectively, these data may suggest thatabT cells are
prevented from having immunogenic relevance in PDA via a
‘‘double-hit’’: fibroblast-mediated chemokine signaling excludes
abT cells from the direct tumor environs wheregdT cells serve to
check their activation via ligation of inhibitory receptors.
In summary, we show that PDA-infiltratinggdT cells are a
highly influential lymphocyte subset in human and murine PDA,
which promote pancreatic oncogenesis and reduce survival via
novel cross-talk with the adaptive immune compartment. These
data implicategdT cells as high-yield targets for the develop-
ment of experimental therapeutics in PDA and has potential
implications for the mechanistic progression of oncogenesis in
other cancer subtypes. Finally,gdT cells may have prognostic
significance in PDA and may be predictive of response to immu-
notherapeutic regimens.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dCONTACT FOR REAGENT AND RESOURCE SHARING
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
BAnimals and In Vivo Procedures
BHuman and Murine Cellular IsolationdMETHOD DETAILS
BFlow Cytometry and FACS Sorting
BWestern Blotting
BHistology, Immunohistochemistry, and Microscopy
BIntravital Imaging
BIn Vitro T Cell Activation Assays
dQUANTIFICATION AND STATISTICAL ANALYSIS
BStatistical AnalysisSUPPLEMENTAL INFORMATIONSupplemental Information includes seven figures and can be found with this
article online athttp://dx.doi.org/10.1016/j.cell.2016.07.046.AUTHOR CONTRIBUTIONSProject Leadership, Data Collection and Analysis, Manuscript Preparation,
D.D.; Project Conception and Data Collection, C.P.Z.; Data Analysis and
Manuscript Preparation, L.S.; IHC, Flow Cytometry, and In Vivo Experiments,
N.A. and N.M.; Manuscript Preparation, G.W.; In Vivo Experiments and Tissue
Culture, A.A.; Technical Assistance, R.B.; Mouse Breeding and Genotyping,
D.T.; Data Analysis, R.N.; Technical Assistance, A.T.-H., M.H., and V.R.K.M.;
Intravital Imaging, J.-E.J. and E.N.; Human Tissue Procurement (IHC),
V.G.P.; Intravital Imaging, M.L.D.; Intravital Imaging, D.B.-S.; Human Tissue
Procurement and Pathologic Analysis, C.H.; Project Design, Data Analysis,
and Manuscript Preparation, G.M.ACKNOWLEDGMENTSThis work was supported by grants for the German Research Foundation
(L.S.), the National Pancreas Foundation (C.P.Z.), the Pancreatic Cancer Ac-
tion Network (G.M.), the Lustgarten Foundation (G.M.), and National Institute
of Health Awards CA155649 (G.M.), CA168611 (G.M.), and CA193111 (G.M.
and A.T.-H.). We thank the New York University Langone Medical Center
(NYU LMC) Histopathology Core Facility, the NYU LMC Flow Cytometry
Core Facility, the NYU LMC Microscopy Core Facility, and the NYU LMC
BioRepository Center, each supported in part by the Cancer Center Support
Grant P30CA016087 and by grant UL1 TR000038 from the National Center
for the Advancement of Translational Science (NCATS).Received: July 16, 2015
Revised: February 16, 2016
Accepted: July 27, 2016
Published: August 25, 2016REFERENCESAndre ́n-Sandberg, A., Dervenis, C., and Lowenfels, B. (1997). Etiologic links
between chronic pancreatitis and pancreatic cancer. Scand. J. Gastroenterol.
32 , 97–103.
Bayne, L.J., Beatty, G.L., Jhala, N., Clark, C.E., Rhim, A.D., Stanger, B.Z.,
and Vonderheide, R.H. (2012). Tumor-derived granulocyte-macrophageFigure 7. Exhaustion Ligand Blockade Reverses the Direct Suppressive Effects ofgdT Cells onabT Cells and on Pancreatic Tumorigenesis
(A and B) Splenic (A) CD4+or (B) CD8+T cells from untreated WT mice were either unstimulated or stimulated withaCD3/aCD28 alone or in co-culture with PDA-
infiltratinggdT cells (5:1 ratio).aPD-L1 (10mg/ml) was selectively added to each group. The fraction of CD62L–CD44+cells was determined at 72 hr by flow
cytometry. Representative contour plots and quantitative data are shown.
(C) Similarly, CD4+and CD8+T cell expression of TNF-awas measured. Experiments were performed in quadruplicate and repeated three times.
(D) WT and Tcrd–/–mice were orthotopically implanted with KPC-derived tumor cells and serially treated withaPD-L1 oraGalectin-9-neutralizing mAbs, or
respective isotype controls. Pancreatic tumors were harvested at 3 weeks. Representative gross images are shown (experiment #1) as are quantitative data on
tumor weights from two separate experiments using different stocks of KPC-derived tumor cells (n = 5/group for each experiment).
(E–G) WT and Tcrd–/–pancreata were again orthotopically implanted with KPC-derived tumor cells and serially treated withaPD-L1 oraGalectin-9-neutralizing
mAbs or the respective isotype controls. Pancreatic tumors were harvested at 3 weeks. (E) The fraction of PDA-infiltratingabT cells among CD45+leukocytes and
(F) CD8+and (G) CD4+T cell adoption of an activated CD62L–CD44+phenotype were determined by flow cytometry (n = 5/group; p < 0.05, p < 0.01, p < 0.01).
Cell 166 , 1485–1499, September 8, 2016 1497