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IN THE SPOTLIGHT

Regulatory T Cells Keep Pancreatic Cancer at Bay Berk Aykut 1 , Ruonan Chen 1 , and George Miller 1 , 2

1 S. Arthur Localio Laboratory and Department of Surgery, New York Uni-versity School of Medicine, New York, New York. 2 Department of Cell Biol-ogy, New York University School of Medicine, New York, New York. Corresponding Author: George Miller, New York University School of Medicine, 435 East 30th Street, Room 417, New York, NY 10016. Phone: 646-501-2208; Fax: 212-263-6840; E-mail: george.miller@nyumc.org Cancer Discov 2020;10:345–7 doi: 10.1158/2159-8290.CD-20-0002 ©2020 American Association for Cancer Research.

Summary: Although CD4 + FOXP3 + T regulatory (Treg) cells are well-known mediators of immunologic tolerance, their infl uences in the tumor microenviroment are incompletely understood. Writing in this issue of Cancer Discovery , Zhang and colleagues demonstrate that in pancreatic cancer, Treg cells promote the differentiation of tumor-restraining myofi broblastic cancer-associated fi broblasts, challenging the existing notion that Treg cells enable tumor progression.

See related article by Zhang et al., p. 422 (5).

It’s well known there’s always two sides, if no more. —George Eliot (1819–1880), Middlemarch

Anticancer treatments have been revolutionized by immu-notherapy, which modulates a host’s immune system and overcomes mechanisms of T-cell suppression. As such, immu-notherapy can result in durable tumor regression in a number of malignancies. However, the majority of pancreatic ductal adenocarcinomas (PDA) are resistant to current immune check-point inhibitors. Although in many cancers CD8 + T cells are directed toward CTL function and induce tumor clearance, in PDA their deletion or depletion does not accelerate oncogenesis ( 1 ). It is thought that multiple parallel mechanisms of immu-nosuppressive mechanisms weaken T-cell activation. There-fore, overcoming tumor microenvironment (TME)–induced immune suppression to improve responses to immunotherapy remains a major clinical challenge. In PDA, T regulatory (Treg) cells accumulate during the preinvasive stage of the disease ( 2 ). Historically, Treg cells are considered a homogeneously tumor-promoting population and various mechanisms of Treg cell–mediated suppression of CTL responses have been pro-posed, including competition for access to antigen-presenting dendritic cells (DC; ref. 3 ). Initial hopes for understanding the role of Treg cells in PDA came with the previously reported observation that Treg cell depletion resulted in attenuated tumor growth in an orthotopic syngeneic model of pancreatic cancer ( 4 ). Unlike CD8 + T cells, intratumoral Treg cells were found to engage in prolonged interactions with tumor-associ-ated CD11b + DCs and reduce their expression of costimulatory molecules. In line with these observations, depletion of Treg cells was found to turn tumor-infi ltrating T cells that are dis-pensable to outcome in PDA into indispensable mediators of tumor protection.

Within this framework comes the study in this issue by Zhang and colleagues that addresses the critical question of the interrelationship among the various stromal cells found in PDA and Treg cells ( 5 ). Using multiplex IHC of surgical tissue samples obtained from human PDA resections, the authors demonstrated that Treg cells are present in most tumor samples and correlate with macrophage and CD8 + T-cell infi ltration. Furthermore, they verifi ed by immunostaining and single-cell sequencing analysis that Treg cells are present in pancre-atic intraepithelial neoplasia (PanIN)–bearing Kras +LSL/G12D ; Ptf1a +/Cre (KC) as well as iKras* and iKras*;p53* mice. To understand the role of Treg cells in the formation of PanIN, they generated Kras +/LSL–G12D ; Ptf1a +/Cre ; Foxp3 tm3(DTR/GFP)Ayr

(KC;Foxp3 DTR ) mice in which the human diphtheria toxin receptor (DTR) is expressed under the control of the Foxp3locus. The most novel fi nding in this study is that the admin-istration of diphtheria toxin to deplete Treg cells resulted in immune cell infi ltration with acinar-ductal metaplasia (ADM) and dedifferentiation in KC;Foxp3 DTR pancreata. In addition, KC;Foxp3 DTR animals exhibited an increased pancreas-to-body ratio, which is a marker of tumor burden. These obser-vations together suggest that, in the presence of oncogenic Kras , depletion of Treg cells causes a deleterious infl ammation that coorchestrates PanIN formation. Although these fi nd-ings are ostensibly paradoxical to previous studies, Zhang and colleagues use an autochthonous model of slowly progressing PDA. As such, the observations from this study can be com-pared to a limited extent to transplantation models that are much more aggressive and harbor a distinct TME.

Cancer-associated fi broblasts (CAF) represent a heteroge-neous cell population. Among several other cell types, resi-dent tissue fi broblasts are considered predecessors ( 6 ). In the pancreas, CAFs are prevalent in the stroma of PanIN lesions and can deposit extensive amount of extracellular matrix, leading to a prominent desmoplastic reaction with a dense fi brotic stroma that is characteristic of pancreatic cancer ( 7 ). To investigate whether Treg cells altered the fi broblast population in PanIN lesions, Zhang and colleagues fl uores-cently labeled α-smooth muscle actin (SMA), a marker for myofi broblastic CAFs (myCAF) in KC and KC;Foxp3 DTR mice. CAFs are generally considered master regulators of various tumor-supportive processes ( 6 ). Nonetheless, in the context of PDA, the exact functions of CAFs continue to be debated. As such, depletion of CAFs results in poorly differentiated

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tumors, enhanced metastatic spread, and reduced survival (8). The current study goes beyond these previous findings and identifies Treg cells as a novel regulator of myCAFs. To investigate the extent to which myCAFs can induce immu-nologic reprogramming, the team analyzed the immune cell landscape of PanIN lesions after Treg cell depletion. They treated mice with caerulein and depleted Treg cells one week before sacrifice to allow for a similar severity of PanIN lesions. This treatment protocol allows the study of immune-cell infiltration independent of the PanIN lesion load. Impor-tantly, despite mostly unchanged T-cell numbers, depletion of Treg cells increased F4/80+ macrophages/DCs with an inhibitory ARG1+ phenotype. Subsequent analysis of the secretome revealed that the influx of immune-suppressive myeloid cells is dependent on CCR1. To test whether the influx of immunosuppressive myeloid cells upon Treg cell depletion can be abrogated by pharmacologic blockade of CCR1, the authors used the CCR1 inhibitor BX471. They found that CCR1 blockade reduced dysplasia and fibrosis despite the depletion of Treg cells. Of note, CCR1 blockade was recently shown to have a synergistic antitumoral effect when used in combination with anti–PD-L1 in a murine model of breast cancer (9).

CD4+ T cells play a crucial role in antitumor immunity. Typically, they recognize peptides presented by MHC II mol-ecules expressed on the surface of antigen-presenting cells such as macrophages or DCs. Such activation results in the differentiation of naïve CD4+ T cells into T helper cells (Th) through activation of specific transcription factors. In pancreatic cancer, Th1-polarized CD4+ T cells are thought to mediate tumor protection. Conversely, Th2-polarized CD4+ T cells are associated with tumor-permissive anergy (1). Zhang and colleagues thus analyzed Th1 and Th2 subsets in pan-creata of Treg cell–depleted mice. The team found that Treg

cell–depleted KC;Foxp3DTR had increased concentrations of Th2-associated and decreased concentrations of Th1-associ-ated cytokines. Finally, the authors showed that depletion of CD4+ T cells in KC;Foxp3DTR mice mitigated immune infiltra-tion as well as ADM and resulted in decreased fibrosis (Fig. 1).

These findings of Zhang and colleagues add to an emerging theme that challenges the previous notion where Treg cells are seen as critical mediators of immune suppression in PDA. As such, previous work demonstrated that, despite the osten-sible tolerogenic Treg phenotype induced by PDA-infiltrating DCs, CD4+ Treg cells have negligible tumor- promoting or immune-suppressive functions when compared with Tr1 cells (10). Not only are these new insights important to the under-standing of PDA, but the mechanisms uncovered in this study could have relevance for other tumor entities with a strong fibrotic component as well. Furthermore, could these discoveries shed light on the mystery of why so far in clinic, no Treg cell–targeted therapy has been proven to be effective? The information we learn from forthcoming studies inves-tigating this question will allow us to not only understand the biology of Treg cells better but improve upon the current clinical outcomes as well.

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

Published online March 3, 2020.

REFERENCES 1. Daley D, Mani VR, Mohan N, Akkad N, Ochi A, Heindel DW,

et al. Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance. Nat Med 2017;23:556–67.

Figure 1.  Depletion of Treg cells accelerates PDA by modulating intratumoral fibroblast differentiation. A, Treg cells secrete TGFβ and promote dif-ferentiation of fibroblasts into tumor-restraining smooth muscle actin–expressing cancer-associated fibroblasts (myCAF). B, Treg cell depletion results in reduced TGFβ levels and accumulation of tumor-promoting fibroblasts, which recruit protumorigenic myeloid cells via CCR1. Depletion of Treg cells also induces a pathologic Th2 response, which co-orchestrates pancreatic carcinogenesis. Mφ, macrophage.

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2020;10:345-347. Cancer Discov   Berk Aykut, Ruonan Chen and George Miller  Regulatory T Cells Keep Pancreatic Cancer at Bay

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