t cells expressing checkpoint receptor tigit are enriched ... · in acute myeloid leukemia (aml;...

Biology of Human Tumors

T Cells Expressing Checkpoint Receptor TIGITAre Enriched in Follicular Lymphoma Tumorsand Characterized by Reversible Suppression ofT-cell Receptor SignalingSarah E. Josefsson1,2, Kanutte Huse1,2, Arne Kolstad3, Klaus Beiske4,Daniela Pende5, Chlo�e B. Steen1,2,6, Else Marit Inderberg7, Ole Christian Lingjærde1,6,Bjørn �stenstad3, Erlend B. Smeland1,2, Ronald Levy8, Jonathan M. Irish9,10,11, andJune H. Myklebust1,2

Abstract

Purpose: T cells infiltrating follicular lymphoma (FL) tumorsare considered dysfunctional, yet the optimal target for immunecheckpoint blockade is unknown. Characterizing coinhibitoryreceptor expression patterns and signaling responses in FL T-cellsubsets might reveal new therapeutic targets.

Experimental Design: Surface expression of 9 coinhibitoryreceptors governing T-cell function was characterized in T-cellsubsets from FL lymph node tumors and from healthy donortonsils and peripheral blood samples, using high-dimensionalflow cytometry. The results were integrated with T-cell receptor(TCR)-induced signaling and cytokine production. Expression ofT-cell immunoglobulin and ITIM domain (TIGIT) ligands wasdetected by immunohistochemistry.

Results: TIGITwas a frequently expressed coinhibitory receptorin FL, expressed by the majority of CD8 T effector memory cells,which commonly coexpressed exhaustion markers such as PD-1

and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production ofIFNg , while TCR proximal signaling (p-CD3z, p-SLP76) was notaffected. The TIGIT ligands CD112 and CD155 were expressed byfollicular dendritic cells in the tumor microenvironment. Dys-functional TCR signaling correlated with TIGIT expression in FLCD8 T cells and could be fully restored upon in vitro culture. Thecostimulatory receptor CD226 was downregulated in TIGITþ

comparedwith TIGIT�CD8FL T cells, further skewing the balancetoward immunosuppression.

Conclusions: TIGIT blockade is a relevant strategy forimproved immunotherapy in FL. A deeper understanding of theinterplay between coinhibitory receptors and key T-cell signalingevents can further assist in engineering immunotherapeutic regi-mens to improve clinical outcomes of cancer patients. Clin CancerRes; 24(4); 870–81. �2017 AACR.

IntroductionFollicular lymphoma (FL) is the most common subtype of

indolent non-Hodgkin lymphoma. Although outcomes have

improved (1), current chemoimmunotherapy regimens are usu-ally not curative. Additionally, FL patients can transform to moreaggressive histology, leading to rapid progression and need forintensive therapy (2).Ongoing clinical trials to improve treatmentof FL focus on novel targeted agents and various immunomod-ulatory regimens, including immunotherapy with checkpointblockade (3, 4).

Targeting coinhibitory receptors such as PD-1 and CTLA-4by immune checkpoint blockade can restore the function ofexhausted T cells with antitumor reactivity (5, 6). T cells in theFL tumor microenvironment (TME) are considered dysfunc-tional and associated with disease progression (7–9). However,whereas blockade of PD-1 represents a breakthrough for severalsolid cancers (10–12) and for Hodgkin's lymphoma (13), theresponse rate as monotherapy in FL has been lower thananticipated (14), given the high expression of PD-1 in intra-tumor T cells and presence of PD-L1þ histiocytes in the TME(9, 15). However, the influence of different T-cell subsets forlymphomagenesis is complex. While T follicular helper cells(TFH) display PD-1

hi phenotype and are highly functional bysupporting lymphoma B cells through CD40 ligand and secre-tion of cytokines IL4 and IL21 (16–18), exhausted T cellsexpress intermediate levels of PD-1 (15, 19). A hallmark ofT-cell exhaustion is expression of multiple coinhibitory

1Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway. 2Departmentof Cancer Immunology, Institute for Cancer Research, Oslo University Hospital,Oslo, Norway. 3Department of Oncology, Division of Cancer Medicine, OsloUniversity Hospital, Oslo, Norway. 4Department of Pathology, Oslo UniversityHospital, Oslo, Norway. 5Immunology Laboratory, Ospedale Policlinico SanMartino, Genova, Italy. 6Department of Computer Science, University of Oslo,Oslo, Norway. 7Department of Cellular Therapy, Oslo University Hospital, Oslo,Norway. 8Division of Oncology, Stanford School of Medicine, Stanford, Califor-nia. 9Department of Cell and Developmental Biology, Vanderbilt University,Nashville, Tennessee. 10Vanderbilt-Ingram Cancer Center, Vanderbilt UniversityMedical Center, Nashville, Tennessee. 11Department of Pathology, Microbiologyand Immunology, Vanderbilt University Medical Center, Nashville, Tennessee.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: June H. Myklebust, Department of Cancer Immunology,Institute for Cancer Research, Oslo University Hospital, Postboks 4953 Nydalen,0424 Oslo, Norway. Phone: 47-9910-9677; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-2337

�2017 American Association for Cancer Research.

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receptors alongside progressive loss of effector functions(20). Therefore, coblockade of several coinhibitory receptorsmight be necessary to achieve optimal antitumor T-cellresponses. T-cell immunoglobulin and ITIM domain (TIGIT)is a recently identified coinhibitory receptor, expressed bynatural killer (NK) cells, effector T cells (TE), T regulatory cells(Treg) and TFH (21–25). Prior findings suggest TIGIT as acandidate for checkpoint blockade, as TIGIT is frequentlyfound on tumor-infiltrating T cells (TIL) in solid tumors andin acute myeloid leukemia (AML; refs. 26–28), and the TIGITligands, CD155 and CD112, are expressed by different celltypes, including antigen-presenting cells and tumor cells(21, 22, 24, 29).

Numerous genes are recurrently mutated in FL (30–33), cre-ating tumor antigens, including the lymphoma immunoglobu-lins, that may trigger T-cell antitumor responses (34). Antigenrecognition by the T-cell receptor (TCR) initiates a cascade oftyrosine phosphorylations, and the amplitude and duration ofTCR signaling is critical for T-cell effector function (35). Hence,exhausted T cells can be distinguished from functional T cells bylow TCR signaling strength. Upon TCR interaction with peptide–MHC, the immunoreceptor tyrosine-based activation motifs(ITAM) of the TCR-associated CD3 subunits become phosphor-ylated by Src family kinases such as LCK (35, 36). Subsequentrecruitment and phosphorylation of the adaptor protein SH2-domain containing leukocyte protein of 76 kDa (SLP76), andlinker for activation of T cells (LAT), results in formation of theLAT signalosome, which enables activation of multiple down-stream effectors, including activation of the RAS–MEK–ERK,PI3K/AKT and NF-kB pathways. TCR signaling is enhanced bycostimulatory receptors such as CD28, but dampened by coin-hibitory receptors such as CTLA-4 and PD-1 due to recruitment ofphosphatases (37, 38).

The hypothesis underlying this study was that characterizingsignaling responses and coinhibitory receptor expression in intra-tumor T-cell subsets could reveal new targets for immune check-point blockade. Based on previous studies, demonstrating theimportance of PD-1 for T-cell immunosuppression (9), our

approach was to measure functional responses in T cells withdifferential expression of PD-1, while in parallel screening forcoinhibitory receptors that could be of interest for immunecheckpoint blockade in combination with PD-1. This approachidentified TIGIT as the most frequently expressed coinhibitoryreceptor in FL T cells, and the expressionwas associatedwith T-celldysfunction. Taken together, our data suggest TIGIT as a prom-ising new target for immune checkpoint blockade in FL.

Materials and MethodsHuman samples

Specimenswere obtainedwith informed consent in accordancewith the Declaration of Helsinki and with approval from theRegional Committees for Medical and Health Research Ethics(REK S-0749b and 2010/1147a). Malignant LN specimens wereobtained at time of diagnosis from FL patients (n ¼ 12) or aftertreatment (n ¼ 2) at the Norwegian Radium Hospital, Oslo,Norway, and tonsils were obtained from patients undergoingtonsillectomy at Agroklinikken (Asker, Norway). LN and tonsilswere processed to single-cell suspensions by mincing and storedas aliquots in liquid nitrogen. Peripheral blood was collectedfromanonymous, healthy donors at The BloodBank inOslo (REKS-03280), processed to mononuclear cells (PBMC) by Ficollgradient centrifugation (Ficoll-Paque PLUS, GE Healthcare) andcryopreserved in liquid nitrogen.

ReagentsStimulation reagents: TCR activation (a-TCR): anti-CD3 bio-

tin and anti-CD28 biotin labeled antibodies were used at 5 mg/mL each and avidin (Thermo Fischer Scientific) was used at 50mg/mL. Phorbol 12-myristate 13-acetate (PMA) was used at 125ng/mL and ionomycin was used at 500 ng/mL (Sigma-Aldrich).GolgiPlug was from BD Biosciences. Cells were stained usingfluorochrome-coupled antibodies (Supplementary Table S1).Antibody used to detect FoxP3 was added after fixation andpermeabilization according to the eBioscience protocol. Bril-liant Stain Buffer (BD biosciences) was used as staining buffer.Pacific Blue used for fluorescent barcoding of cells was from LifeTechnologies, Molecular probes.

Activation of T-cell signaling and phospho-specific flowcytometry

Activation of signaling and detection by phospho-specific flowcytometry were performed as described (9, 39, 40). Specimenswere thawed, and cells were allowed to rest at 37�C for 4 hours,before redistribution into v-bottomed 96-well plates and givenanother 20 minutes rest. For functional studies over time, cellswere cultured for 48hours at 37�C, at 2.5�106/mL inCellGroDC(CellGenix) supplemented with 5% human serum (DiaserveLaboratories). IL2 (20 U/mL; Chiron) was added in some experi-ments as specified. Signaling was activated by a-TCR for 1, 4, or10 minutes (details in Supplementary methods). Signaling wasstopped by adding paraformaldehyde (PFA; 1.6%), followed bycentrifugation and permeabilization in >90% freezer-cold meth-anol. After rehydration, the cells were stained with antibodies, or"barcoded" with Pacific Blue prior to staining with antibodiesas previously described (9). The samples were collected on aLSR II flow cytometer (BD Biosciences). Data were analyzed usingCytobank Software, https://community.cytobank.org. Relativephosphorylation changes (fold changes) were calculated using

Translational Relevance

Immunotherapeutic regimens targeting coinhibitory recep-tors, such as PD-1, have emphasized the role of immunecheckpoints in sustaining T-cell immunosuppression. How-ever, the response rate of PD-1 blockade has been lower thananticipated in FL, providing a rationale to investigate therole of other coinhibitory receptors. Here, in-depth char-acterization of coinhibitory receptor expressionwas combinedwith functional assessment of intratumor T cells from FLpatients. This approach provided new insights into mechan-isms that may contribute to immunosuppression in FL byidentifying T-cell immunoglobulin and ITIM domain (TIGIT)as a commonly expressed coinhibitory receptor in FL T cells,and the expression correlated with reduced effector function.Our results suggest that the potential relevance of TIGITinhibition as a novel form of checkpoint therapy is high andsupport clinical investigation of TIGIT blockade in FL,possibly in combination with blockade of PD-1.

TIGIT Is an Abundant Coinhibitory Receptor in FL

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arcsinh transformation of median fluorescence intensity (MFI) ofthe cell population of interest.

viSNE analysisThe computational tool viSNE (41) was used for visualization

of immunophenotype data, see Supplementary Methods.

Stimulation of cytokine productionSamples were incubated for 6 hours in the presence of PMA

and ionomycin, with GolgiPlug present for the last 4 hours. PFA(1.6%) was added to stop activity, followed by centrifugationand permeabilization in >90% freezer-cold methanol. At thispoint, the samples could be stored at �80�C, before stainingwith antibodies and flow cytometry acquisition.

Gene expression analysisGene expression data were obtained from two different

datasets; Dave and colleagues (7) and Brodtkorb and collea-gues (42), and included pretreatment FL biopsies only, seesupplementary methods.

ImmunohistochemistrySerial sections of cryopreserved FL tissue were stained with

antibodies for CD155 (L95) and CD112 (L14) as previouslydescribed (43), in addition to CD21 (2G9).

ResultsFL CD8 T-cell composition is skewed toward PD-1int

phenotypeTo explore if PD-1 was more frequently expressed in intra-

tumor T cells from FL than in corresponding subsets fromhealthy tissues, LN specimens from 14 FL patients were immu-nophenotyped and compared with 11 tonsillar and 7 PBMCsamples from healthy donors. In order to distinguish TFH fromother subsets, distribution of T cells was characterizedbased on differential expression of PD-1 and ICOS in CD4(PD-1�ICOS�, PD-1intICOS�, PD-1intICOSþ, and PD-1hiICOSþ (TFH)) and CD8 (PD-1

�ICOS� and PD-1intICOS�)T-cell subsets (Fig. 1A). We found that neither the TFH com-partment nor the CD4þ PD-1int T-cell subsets were significantly

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Skewing toward PD-1int phenotype and reduced IFNg production in CD8 FL T cells. Single cell suspensions from FL LN and healthy donors (tonsils andPBMC) were analyzed by fluorescence flow cytometry. A, CD8 and CD4 T cells were divided into subsets based on expression of PD-1 and ICOS. B,Distribution of T-cell subsets. FL (n ¼ 14), tonsils (n ¼ 11) and PBMC (n ¼ 7). C, Cells were cultured with or without PMA and ionomycin, and intracellularIFNg was measured by flow cytometry. Each data point represents a single donor. FL (n ¼ 9), tonsils (n ¼ 13), and PBMC (n ¼ 7). Statistical differencescalculated using Mann–Whitney nonparametric test; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ��, P < 0.0001.

Josefsson et al.

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different between FL tumors and tonsil controls. In contrast, theCD8þ PD-1int subset was markedly increased in FL tumorscompared to healthy PBMC or tonsils (P < 0.003 and P <0.0001, Fig. 1B), suggesting a larger fraction of exhausted CD8T cells in FL.

CD8 T cells from FL display reduced IFNg productionWe next measured cytokine production in relation to PD-1

expression in CD4 and CD8 T cells. We observed that reducedpercentage of CD8 T cells from FL patients produced IFNg ,compared with healthy individuals. Interestingly, IFNg produc-tionwas reduced in PD-1� as well as PD-1int CD8 T cells (Fig. 1C),indicating that PD-1–negative CD8 FL T cells were also sup-pressed. This finding suggests presence of other inhibitorymechanisms in PD-1� CD8 FL T cells, leading to reduced func-tionality. Production of IL4 and IL21 was also measured, but wasnot significantly different in CD8 T cells from FL LN and healthydonors (Supplementary Fig. S1). In CD4 T cells, IL4 productionwas low but at comparable levels in FL and tonsillar subsets,whereas IL21 production was reduced in all FL subsets except forPD-1�ICOS� cells (Supplementary Fig. S1).

TCR-induced p-ERK is highly reduced in FL T cellsAs functional TCR signaling is critical for generation of

effective antitumor T-cell responses, including production ofIFNg , we next investigated TCR-induced signaling in T cellsfrom FL tumors (Fig. 2A). TCR signaling was activated usinga-CD3 and a-CD28 biotinylated antibodies, followed by avi-din crosslinking. To identify optimal time points to detectmaximal phosphorylation levels, TCR was activated for 1, 4,and 10 minutes. Whereas p-CD3z and p-SLP76 peaked at 1minute post stimulation, p-ERK signaling was undetectable at 1minute and reached the maximal level 4 minutes after stimu-lation (Supplementary Fig. S2). A comparison of TCR-inducedsignaling responses in FL and healthy individuals revealed thatT cells from FL patients were distinguished by highly reducedTCR-induced p-ERK, while p-SLP76 and p-CD3z levels werecomparable (Fig. 2B). The low levels of TCR-induced p-ERK wasevident in CD8þPD-1int FL T cells, with a relative median foldchange (FC) of 0.18 as compared with 0.56 and 0.34 in PBMCand tonsils, respectively (Fig. 2C). Strikingly, TCR-induced p-ERK was low in all CD4 FL T-cell subsets (range, 0.2–0.4; Fig.2C). In contrast, TCR proximal signaling, as determined by p-SLP76, was comparable in FL and tonsillar T cells, with medianFC ranges of 1.7–2.0 and 1.9–2.2, respectively (Fig. 2C). Phos-phorylation of CD3z was also potent in FL, similar to the levelsobserved in tonsillar T-cell subsets (Fig. 2C). Interestingly, thelow TCR-induced p-ERK observed across all T-cell subsets fromFL LN indicated a block in the distal part of the pathway. Thiscorresponded with the observed reduction in IFNg production.

TIGIT is frequently expressed in T cells from FLWe hypothesized that multiple coinhibitory receptors might

play a role in dampening T-cell antitumor responses in FL. Wetherefore used 11-parameter flow cytometry panels to achieve anin-depth characterization of coinhibitory receptor expressionpatterns in FL T-cell subsets, and compared patterns with healthydonor samples as before. A viSNE analysis, based on the expres-sion of 6 lineage markers (CD4, CD8, CXCR5, ICOS, CD45RA,and CCR7) was used to visualize the data and to identify con-ventional T-cell subsets, as well as T-cell subsets identified based

on PD-1 and ICOS expression (Supplementary Fig. S3A–S3C).The expression pattern of 9 coinhibitory receptors—PD-1, TIGIT,TIM-3, CTLA-4, LAG-3, BTLA, CD244, LAIR-1, and CD160—wasthen identified in the conventional T-cell subsets (Fig. 3). Strik-ingly, TIGIT was an abundant coinhibitory receptor in FL T cellsand was expressed by the majority of CD4 and CD8 T effectormemory (TEM) cells (Fig. 3). Furthermore, TIGIT

þ CD8 TEM cellsfromFL coexpressed several exhaustionmarkers, such as PD-1 andCD244 (Fig. 3; Supplementary Fig. S3D and S3E), suggesting thatTIGIT marks exhausted CD8 T cells in FL.

Detailed analysis revealed that TIGIT was expressed at signif-icantly higher levels across all T-cell subsets in FL tumorscompared with healthy donor tonsils or PBMC, but with con-trasting expression pattern across distinct subsets: low expres-sion in na€�ve T cells and highest in TEM and TFH cells. On average,80% and 79% of CD8 and CD4 TEM cells from FL expressedTIGIT (Fig. 4A–C). This is an important finding as TEM was themajor subset of CD4 and CD8 T cells in FL tumors (Supple-mentary Fig. S4). The majority of FL CD8 and CD4 TEM cellsalso expressed PD-1 (80% and 65%, respectively), and someexpressed BTLA (10% and 42%; Fig. 4B and C). TIM-3, CTLA-4,LAG-3, LAIR-1, and CD160 were all less frequently expressed inFL CD4 and CD8 TEM cells (in average

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Figure 2.

Intratumor FL T cells are distinguished by lowlevels of TCR-induced distal signaling. Singlecell suspensions from FL LN (n ¼ 9), andhealthy donor tonsils (n ¼ 11) and PBMC(n ¼ 9) were cultured with or without a-CD3and a-CD28 antibodies for 2 minutes,followed by avidin crosslinking for 1, 4, or10 minutes and then assayed for TCR-inducedphosphorylation of CD3z, SLP76, and ERKusing phospho-flow cytometry. A, Schematicoverview of TCR signaling. B, Representativehistograms of TCR-induced phosphorylationin CD3þ T cells from one FL patient samplecompared with one healthy donor tonsil.Shown is median fold change (FC) inductionrelative to unstimulated cells, using arcsinhtransformed data. C, TCR-induced p-ERK(40), p-SLP76 (10), and p-CD3z (10) in CD8 andCD4 T-cell subsets shown as median FCinduction relative to unstimulated cells.Each data point represents a single donor.Statistical differences calculated usingMann–Whitney nonparametric test;� , P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001.

Josefsson et al.





TIGITþCD8 T cells display TCR distal signaling defects that canbe restored

To further investigate the relationship between TIGIT expres-sion and dysfunctional TCR-induced signaling, we includeddetection of TIGIT in our signaling assay. Distinguishing betweenTIGIT� and TIGITþ cells among the CD8 FL T cells revealed thatTIGITþ cells had reduced TCR-induced p-ERK compared withTIGIT� cells (Fig. 6A and B). This contrasted TCR proximalsignaling, demonstrated by high levels of TCR-induced p-SLP76regardless of TIGIT expression (Fig. 6B). These results indicate thatTIGIT plays a role in dampening signaling distal to the TCR.

To test whether the dysfunctional TCR signaling could berestored, we studied signaling responses after 48 hours in vitroculture (Fig. 6C). Detection of TIGIT revealed that the percentage

of TIGITþ CD8 FL T cells was stable over time (SupplementaryFig. S8). Interestingly, while TCR-induced p-SLP76 was compa-rable inCD8T cells at day0 andafter 2days,weobserved a strikingincrease in TCR-induced p-ERK, from 1.03 to 2.01 FC (Fig. 6D).Importantly, the recovery of TCR-induced p-ERK was highlyreproducible and remarkably high in TIGITþ CD8 FL T cells(median fold change from 0.8 at day 0 to 2.1 at day 2) ascompared with TIGIT� CD8 FL T cells (from 1.6 at day 0 to2.7 at day 2; Fig. 6E and F; Supplementary Fig. S9). As TIGITligands were expressed by FDC (Fig. 5A), which are tightlyadhered to the stroma, these cells were not preserved and aretherefore not present in the cryopreserved single cell suspensionsused in the functional assays. In conclusion, our results showedthat the highly reduced TCR-induced p-ERK in FL could be

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Expression patterns of coinhibitory receptors in CD8 and CD4 T-cell subsets. Eleven-parameter fluorescence flow cytometry was used to identify coinhibitoryreceptor expression in conventional T-cell subsets from FL LN (n ¼ 4) and healthy donor tonsils (n ¼ 2), using single cell suspensions. Results arevisualized by viSNE (gating shown in Supplementary Fig. S4). Scale maximum is set to highest measured signal for each marker, or a minimum of 3,000.The manually added line in the viSNE plots marks the distinction between CD8 and CD4 T cells.






recovered upon in vitro culturewhenTIGIT ligand–expressing cellsare not present, suggesting that FL T cells receive suppressivesignals through TIGIT via ligandþ cells in the TME in vivo.

DiscussionImmune checkpoint blockers have shown impressive clinical

benefits in several tumor types. Despite frequent expression of

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Figure 4.

TIGIT is frequently expressed in FL TE, TEM, TFH, and Tregs. Surface expression of coinhibitory receptors was analyzed in single cell suspensions from FL LN, andhealthy donor controls (tonsils and PBMC) by fluorescence flow cytometry. A, Plots show CD3þ T cells. B and C, Coinhibitory receptor expression wasmeasured in conventional CD8 and CD4 T-cell subsets. Each data point represents a single donor. FL (n ¼ 14), tonsils (n ¼ 11), and PBMC (n ¼ 7). Statisticaldifferences calculated using Mann–Whitney nonparametric test; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001. D, FL LN samples (n ¼ 3) wereassayed for the contribution of TIGITþ Tregs. Tonsils and PBMC from healthy donors were included for comparison. Bar graph shows mean � SEM.

Josefsson et al.





PD-1 in intratumor T cells in FL (9, 15), a significant proportion ofpatients do not respond to anti–PD-1 blockade (14, 45). Tumorgenomic landscape, mutational load and tumor specific neoanti-gens are potential determinants of the response to immune check-point blockade, as well as characteristics of the TME (34, 46–49).As T-cell exhaustion might relate to coexpression of several coin-hibitory receptors, identification of the most relevant types astargets for immune checkpoint blockade in FL patients will beimportant in order to fully unleash the antitumor response. In thisstudy, we performed a multidimensional functional and pheno-typical characterization of intratumor T cells from FL patients andcomparedwith tonsils and PBMC fromhealthy donors to identifyrelevant targets for immune checkpoint blockade in FL. Thisapproach identified TIGIT and PD-1 as the most frequentlyexpressed coinhibitory receptors. In FL CD8 T cells, we observedreduced production of IFNg as well as highly reduced TCR-induced p-ERK,which correlatedwith TIGIT expression and couldbe fully restored by in vitro culture in absence of TIGIT ligandsCD155 andCD112. The TIGIT ligandswere expressed by FDCandendothelial cells in FL tumors. Together, these results indicate thatTIGIT is a relevant target for immune checkpoint blockade in FL.

Strikingly, our results showed that TIGIT in average wasexpressed in more than 80% of CD8 and CD4 TEM cells from FLtumors, which accounted for 50% and 60% of CD8 and CD4 Tcells, respectively. Furthermore, more than 95% of Tregs and TFHcells from FL LN expressed TIGIT. Importantly, TIGIT mightpotentially have divergent functions in different T-cell subsets.Agonistic anti-TIGIT antibody had direct inhibitory effects onT-bet expression and IFNg production in CD4þ TE cells (50), andloss of TIGIT in vivo increased T-cell proliferation and proinflam-matory cytokine production (25). In contrast to the unresponsivephenotype of TIGITþ TE cells, TIGIT

þ Tregs are highly functionalcells. Several studies have demonstrated that TIGITþ Tregs haveincreased expression of effector molecules and are more potent

suppressors of TE proliferation than TIGIT� Tregs (28, 51, 52). As

the frequency of Tregs is increased in FL LN, TIGITþ Tregs are likelyto contribute to sustained immune suppression in FL. In addition,TIGIT is frequently expressed by tonsillar TFH (53), and weobserved that the majority (>95%) of TFH from FL LN as well astonsils from healthy donors expressed TIGIT. Previous studiessuggest that TIGIT mediates adhesion of TFH to FDC in germinalcenters (23), and TIGIT is required for efficient B-cell helperfunction of peripheral blood circulating TFH (54). Furthermore,TIGIT can outcompete the costimulatory receptor CD226 dueto its higher affinity for the same ligand and by blocking dimer-ization of CD226, thus preventing its costimulatory function(21, 44, 50, 55). Our results revealed that TIGITþ CD8 FL T cellsrarely expressed the competing costimulatory receptor. This indi-cated an imbalance between costimulation and coinhibition inthese cells, further suggesting that TIGIT plays a role in dampeningCD8 T-cell antitumor responses in FL. Altogether, this suggeststhat immune checkpoint blockade targeting TIGIT should enablehighly potent T-cell antitumor responses in several ways, includ-ing restoring antitumor potential of T effector cells, dampeningthe Treg immunosuppressive effect and by reducing the tumorsupporting effects of TFH cells. In addition to the direct effects ofTIGIT in T cells, TIGIT candirectly restrainNKcell activity (56) andindirectly exert inhibitory effects by activating immunoregulatorydendritic cells upon ligand interaction (21). Hence, blockingTIGIT in these cells may also be pivotal for efficient immuno-therapy responses.

By combined detection of TIGIT, T-cell markers and phos-phorylation of signaling effectors post TCR activation, weidentified a clear correlation between TIGIT expression andTCR signaling dysfunction in CD8 FL T cells. However, TIGITneeds to be ligated to exert its suppressive function. Our resultsshowed that less than 5% of FL tumor cells expressed the TIGITligands CD155 and CD112. Instead, immunohistochemical

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Figure 5.

TIGIT ligands are expressed in FL andTIGITþ CD8 T cells are CD226low. A, FLtissue sections were stained withantibodies against CD155, CD112, andCD21. The tissue sections are closelyneighbored to each other, enablingthe comparison of identical structures.Staining pattern of CD155 and CD112 infollicles (arrows) suggests expressionby FDC, confirmed by staining of thesame follicles with FDC marker CD21.Endothelium (arrowheads) alsoexpressed CD155 and CD112. Imageobjective �10. B and C, TIGIT andCD226 expression was measured inCD8 andCD4T cells fromFL LN (n¼ 7)using flow cytometry. Healthy donorPBMC was included for comparison.Bar graphs show mean � SEM.� , P < 0.05; �� , P < 0.01; �� , P < 0.001by Mann–Whitney test.






staining revealed the presence of CD155 and CD112 on FDCand on endothelial cells in FL tumors. These cells are tightlyadhered to the stroma and were not detectable in the cryopre-served samples used for immunophenotyping by flow cytome-try. However, the ligandþ FDC are likely to interact with TIGIT-expressing T cells in vivo, thereby preventing potent antitumorT-cell responses in FL. We were not able to provide direct prooffor this hypothesis, but further support comes from the in vitrocultures of FL T cells. When cultured in the absence of CD155þ

or CD112þ cells, CD8 TIGITþ FL T cells could regain their TCRsignaling capacity. Based on this, we cannot exclude the pos-sibility that culture of FL derived CD8 T cells over time alsoremoves other suppressive signals, as demonstrated by effec-

tiveness of TIL therapy in FL (57). Although the mechanismsunderpinning how TIGIT modulates T-cell–intrinsic signalingis poorly understood, studies in NK cells suggest that TIGITupon ligation recruits the inositol 5-phosphatase SHIP1 toattenuate signaling downstream of SLP76, leading to dephos-phorylation of ERK and subsequent inhibition of IFNg pro-duction (58, 59). This is in agreement with what we observed inFL T cells; that TIGIT expression correlated with highly reducedTCR-induced p-ERK that translated into reduced IFNg produc-tion in CD8 FL T cells, while phosphorylation of CD3z andSLP76 remained unaffected and similar to healthy controlT cells. Our hypothesis, that low TCR-induced p-ERK marksdysfunctional T cells in FL, is further supported by the current

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Figure 6.

Dysfunctional TCR distal signaling in FL CD8 TIGITþ T cells can be restored. Single cell suspensions from FL LN were assayed for TCR-induced signalingand analyzed by phospho-flow cytometry at day 0 and after 48-hour in vitro culture. The cryopreserved cell suspensions contained T cells and tumorcells, while FDC were not detectable in these cultures. Signaling was induced using a-CD3 and a-CD28 antibodies for 2 minutes, followed by avidincrosslinking for 1 or 4 minutes, and is shown as median fold change (FC) induction relative to unstimulated cells, using arcsinh transformed data. A,TCR-induced p-ERK (40) in TIGIT� and TIGITþ CD8 T cells from one representative FL sample at day 0. B, Levels of TCR-induced p-ERK (40) and p-SLP76 (10)in CD8 T cells from FL LN (n ¼ 6) at day 0. � , P < 0.05 by paired t test. C, Schematic overview of in vitro cultures. TCR signaling was induced insingle cell suspensions from FL LN at day 0 and after 2 days culture. D, TCR-induced signaling was measured in the same FL specimens (n ¼ 4) atday 0 and after 48 hours in vitro culture in the presence of low IL2. Bar graphs show mean � SEM. � , P < 0.05 by paired t-test. E and F, TCR-inducedp-ERK (40) was measured in TIGIT� and TIGITþ CD8 T cells from the same FL specimens at day 0 and after 48 h in vitro culture (in medium only). E, Histogramsshow one representative FL sample. F, Recovery of TCR-induced p-ERK by in vitro culture shown in TIGIT� and TIGITþ CD8 T cells from FL LN (n ¼ 4).�� , P < 0.01; �� , P < 0.0001 by paired t test.

Josefsson et al.





understanding that impaired activation of ERK is an indicatorof T-cell anergy. This is based on observations showing thatuncoupling of the ERK pathway is an important underlyingmechanism in antigenic unresponsiveness of T cells. Antigenrecognition under suboptimal conditions, such as lack ofcostimulation or upregulation of coinhibitory receptors, canact to disrupt TCR-induced p-ERK, hence resulting in poor T-cell effector function (60, 61).

While TIGIT can recruit SHIP1 to modulate cell function,PD-1 blocks signaling events downstream of the TCR byrecruiting the protein tyrosine phosphatases SHP1 and SHP2.These phosphatases can inhibit phosphorylation of signalingeffectors both proximal and distal to the TCR (62–64). In fact,we found low levels of TCR-induced p-ERK to be associatedwith TIGIT as well as PD-1 expression. This indicates that TIGITand PD-1 may both contribute to the dysfunctional TCR-induced signaling observed in FL, potentially by recruitmentof different phosphatases. In context with the finding thatTIGIT and PD-1 were the two major coinhibitory receptors,and often coexpressed by FL T cells, this provides a rationale forcoblockade of these receptors to improve T-cell activity andtumor killing. Although not yet explored in lymphoma,coblockade of TIGIT and PD-1 has generated promising resultsfrom preclinical studies in other cancer types. Combinedblockade of the two receptors led to complete responses intumor mouse models of breast and colorectal cancers, whileblocking only one receptor had little effect (27). Furthermore,coblockade of PD-1 and TIGIT led to increased IFNg produc-tion in CD8 TILs frommelanoma patients, and TIGIT blockadewas able to restore cytokine production in CD8 T cells fromAML patients (26, 65).

In conclusion, our results provide new insights into mechan-isms that may contribute to immune suppression in FL. In-depthmapping of coinhibitory receptor expression and functionalassessment in distinct T-cell subtypes will enhance our biologicalunderstanding for the complex regulation of antitumor T-cellresponses, and exploiting this further in relation to immune

checkpoint blockade is needed to further enhance the precisionof this therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.E. Josefsson, K. Huse, E.M. Inderberg, E.B. Smeland,R. Levy, J.M. Irish, J.H. MyklebustDevelopment of methodology: S.E. Josefsson, K. Huse, R. Levy, J.H. MyklebustAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.E. Josefsson, K. Huse, A. Kolstad, K. Beiske,B. �stenstad, R. LevyAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): S.E. Josefsson, K. Huse, K. Beiske, C.B. Steen,O.C. Lingjærde, E.B. Smeland, R. Levy, J.M. Irish, J.H. MyklebustWriting, review, and/or revision of the manuscript: S.E. Josefsson, K. Huse,A. Kolstad, K. Beiske, C.B. Steen, E. M. Inderberg, B. �stenstad, E.B. Smeland,R. Levy, J.M. Irish, J.H. MyklebustAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.E. Josefsson, K. Huse, A. Kolstad, R. LevyStudy supervision: K. Huse, J.H. MyklebustOther (application to regional ethics committee): A. KolstadOther (supply of reagents generated by herself): D. Pende

AcknowledgmentsWe thank Eva Kimby for critical review of the manuscript. This work

was supported by the Research Council of Norway (FRIMEDBIO 230817/F20; S.E. Josefsson) and Centre of Excellence (Centre for Cancer Biomedicine;J.H. Myklebust and E.B. Smeland), the Norwegian Cancer Society (162948;K.Huse, 163151; E.B. Smeland, and 162844; J.H.Myklebust), and AssociazioneItaliana per la Ricerca sul Cancro (AIRC IG-16764; D. Pende).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 11, 2017; revised October 10, 2017; accepted November 30,2017; published OnlineFirst December 7, 2017.

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2018;24:870-881. Published OnlineFirst December 7, 2017.Clin Cancer Res Sarah E. Josefsson, Kanutte Huse, Arne Kolstad, et al. Suppression of T-cell Receptor SignalingFollicular Lymphoma Tumors and Characterized by Reversible T Cells Expressing Checkpoint Receptor TIGIT Are Enriched in

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