expansion of tumor-infiltrating lymphocytes (til) from human

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  • RESEARCH ARTICLE Open Access

    Expansion of tumor-infiltrating lymphocytes(TIL) from human pancreatic tumorsMacLean Hall1, Hao Liu1, Mokenge Malafa2, Barbara Centeno2, Pamela J. Hodul2, Jos Pimiento2,Shari Pilon-Thomas1,3 and Amod A. Sarnaik1,3*

    Abstract

    Background: We evaluated whether tumor infiltrating lymphocytes (TIL) could be expanded from surgicallyresected tumors from pancreatic cancer patients.

    Methods: Tumors were resected from pancreatic cancer patients. Tumors were minced into fragments andcultured in media containing high dose interleukin-2 (IL-2) for up to 6 weeks. T cell phenotype, activation markers,and reactivity were measured.

    Results: TIL expansion was measured in 19 patient samples. The majority of these TIL were CD4+ T cells and werehighly activated. Purified CD8+ T cells produced IFN- in response to HLA-matched pancreatic tumor targets. PD-1blockade and 4-1BB stimulation were demonstrated as effective strategies to improve effective TIL yield, includingthe production of tumor-reactive pancreatic TIL.

    Conclusions: TIL expanded from pancreatic tumors are functional and able to respond to pancreatic tumorassociated antigens. PD-1 blockade, 41BB stimulation, and CD8+ T cell enrichment are effective strategies toimprove TIL yield and tumor reactivity. These results support the development of adoptive cell therapy strategiesusing TIL for the treatment of pancreatic cancer.

    Keywords: Pancreatic cancer, T cells, Adoptive cell therapy, Tumor infiltrating lymphocytes (TIL)

    BackgroundPancreatic adenocarcinoma is the fourth-leading causeof cancer-related mortality in the United States. Patientsdiagnosed with this disease face a 5-year survival rate ofless than 5 %, and the only available treatments, surgery,chemotherapy and chemoradiation, have shown limitedeffectiveness [1, 2]. Only a small fraction (20 %) of thesepatients are even eligible for surgery with curative intent,and most will develop recurrent disease within 2 yearsof definitive therapy [3]. Therefore, alternative treatmentstrategies are urgently needed.Recent successes in immunotherapy for the treatment

    of metastatic melanoma have resulted in its application toother types of cancer. Specifically, adoptive cell therapy

    (ACT) is a particularly promising approach that utilizesendogenous tumor-infiltrating lymphocytes (TIL), whichare expanded in vitro from a surgically resected tumorand then re-infused back into the patient. This therapy formetastatic melanoma patients is associated with a 20 %complete response lasting beyond 3 years [4]. In patientswith gastrointestinal (GI) tumors, infiltration of CD3+ Tcells is associated with a higher rate of progression freesurvival [5], and pancreatic adenocarcinomas containingboth CD4+ and CD8+ T cells correlated with an improvedprognosis and significantly greater 5-year survival forthese patients [68]. Therefore, there is evidence of a hostT cell immune response in patients with pancreatic adeno-carcinoma, supporting the potential application of ACTusing TIL for this cancer histology.Correspondingly, there is no shortage of studies demon-

    strating that the tumor microenvironment of pancreaticadenocarcinoma is inherently immunosuppressive, with avast array of mechanisms to escape immune surveillance.These include co-inhibitory ligands, such as PDL1 and

    * Correspondence: amod.sarnaik@moffitt.orgEqual contributors1Department of Immunology, H Lee Moffitt Cancer Center and ResearchInstitute, Tampa, FL, USA3Cutaneous Oncology Program, H Lee Moffitt Cancer Center and ResearchInstitute, 10920 N. McKinley Dr, Tampa, FL 33612, USAFull list of author information is available at the end of the article

    2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

    Hall et al. Journal for ImmunoTherapy of Cancer (2016) 4:61 DOI 10.1186/s40425-016-0164-7

    http://crossmark.crossref.org/dialog/?doi=10.1186/s40425-016-0164-7&domain=pdfmailto:amod.sarnaik@moffitt.orghttp://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/

  • PDL2, which directly interact with T cells to dampen theireffector response [912], regulatory T cells [1317], re-duced antigen presentation [18], and suppressive cyto-kines [1921]. Additionally, activated TIL upregulatecheckpoint molecules such as PD-1, which may serve todull the intensity of the inflammatory response and inducetolerance toward tumor antigens [22].Despite all of these factors, TIL expanded in vitro to

    large numbers have the potential to be reprogrammed aseffectors of a productive anti-tumor response [4]. In fact,it has recently been demonstrated that PD-1+ TIL repre-sent the repertoire of clonally expanded tumor-reactivecells in melanoma [23]. Identifying and targeting enriched,tumor-specific TIL subsets in vitro and in vivo is an activearea of research intended to augment TIL growth andfunction in order to improve the efficacy of ACT.In the present study, we successfully expanded TIL from

    19 patients with pancreatic adenocarcinoma in the presenceof high dose IL-2. The majority of these TIL were CD4+ Tcells and were highly activated. Both PD-1 blockadeand 4-1BB agonism led to improved TIL yields. Enrich-ment of CD8+ T cells was an effective strategy to measuretumor-reactive pancreatic TIL. These results support theuse of TIL expanded from pancreatic tumors in ACTstrategies for patients with pancreatic adenocarcinoma.

    ResultsPancreatic TIL are predominantly CD4In order to establish the feasibility of isolating andexpanding TIL from pancreatic adenocarcinomas, weadapted our previous experience with melanoma to es-tablish pancreatic TIL cultures [24]. Tumors were surgi-cally resected from 20 different patients with pancreaticcancer at the Moffitt Cancer Center and used to set uptumor fragment cultures for the isolation and propaga-tion of TIL. Of note, the volume of tumor received inthe laboratory after pathological analysis was consider-ably smaller compared to those typically obtained toderive melanoma TIL. This resulted in an average of14.3 tumor fragments from resected pancreatic cancerspecimens from which TIL were initially propagated,compared to over 48 fragments from a typical melanomaresection. Despite this, TIL were successfully isolatedfrom at least one plated fragment in 19 of the 20 patienttumors (95 %) in the presence of high dose IL-2(6000 IU/mL) after 3 to 6 weeks of culture (Table 1 andAdditional file 1: Table S1) and were predominantlyCD4+ T cells. TIL yield varied between patient sampleswith an average yield of 1.79x107 TIL from an average ofjust over 14 fragments per patient. Four samples of the 17(23.5 %) measured gave rise to at least 25 million TIL, theminimum number required for initiation of a clinical scalerapid expansion protocol (REP). Extrapolated to the 48fragments typically set up during a clinical scale expansion,

    the average yield increased to over 83 million TIL, withnine of 17 (52.9 %) samples meeting the REP initiationthreshold. The majority of these expanded TIL were CD4+

    (66.1 21.0 %), while CD8+ T cells comprised a mean of25.6 17.0 % (Fig. 1a). The remaining analyzed CD3+ TILnot within these single positive gates were predominantlydouble negative cells. Importantly, pancreatic TIL werealso capable of REP, the second phase of pre-infusion TILgrowth. Three patient samples were subjected to the full,two week REP which resulted in an average fold expansionof 964 (Additional file 1: Table S2), which compared simi-larly to our experience in melanoma TIL where ~1000 foldexpansion is observed during the REP. Twelve additionalsamples underwent an 8 day mini-REP and exhibited a 93-fold expansion on average over that time (n = 12, data notshown). This data demonstrate that generating sufficientTIL for adoptive cell therapy from pancreatic adenocarcin-oma fragments is possible given the procurement of ad-equately sized specimens needed to yield the high numbersof TIL required to initiate the REP.

    Pancreatic TIL have an activated phenotypeTo characterize the phenotype of these expanded pan-creatic TIL, we examined the expression of markers in-volved in the activation, differentiation and function ofT cells. Since the majority of TIL were CD4+, we investi-gated the presence of regulatory T cells (Tregs) by intracel-lular staining for FOXP3 on the CD4+CD25+ population.We found that the frequency of CD4+CD25+FOXP3+ Tregsis quite low (~4 %) in expanded pancreatic TIL as aproportion of the total CD3+ population (Fig. 1b). TILwere nearly entirely CD45RO+ CCR7, characteristic ofantigen-experienced effector T cells (data not shown). Ap-proximately 50 % of CD8+ cells and 40 % of CD4+ cellsexpressed CD69 (Fig. 1c i), indicating an efficient and sus-tained T cell stimulation [25]. TIL also exhibited a lowlevel of CD27 expression (Fig. 1c ii), a marker known tobe down-regulated as T cells approach terminal differenti-ation [26]. We further characterized the phenotype ofthese cultured TIL and found that more than 40 % ofCD8+ cells expressed the co-stimulatory marker CD28(Fig. 1c iii) and more than 20 % cells expressed GITR(Fig. 1c iv), a receptor crucial for the proliferation of acti-vated CD8+ T cells [27]. Additionally, less than 5 % of TILwere 4-1BB+, a co-stimulatory marker that is expressed onactivated T cells after TCR engagement (Fig. 1c v) [28]. IL

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