Immunosuppressive γδ T cells foster pancreatic carcinogenesis

KEYWORDS:

• αβ T cells
• CD4⁺CD25⁺FOXP3⁺ TREG cells
• chemokine signaling
• galectin 9
• IL-10
• PD-L1

γδ T cells are a subset of CD3⁺CD4⁻CD8⁻lymphocytes expressing a peculiar T-cell receptor (TCR) that recognizes peptide as well as non-peptide antigens in an MHC-unrestricted fashion.¹ A priori, the diversity of γδ TCRs would be greater than that of conventional αβ TCRs and B-cell receptors (BCRs) combined, in both mice and humans. However, such a potential diversity is never fully realized. Indeed, γδ T cells develop under a very strict endogenous and microenvironmental control, which allows for the emergence of TCRs that are encoded by a single Vγ and Vδ gene and often exhibit limited, if any, junctional diversity.¹ Thus, at least in some aspects, γδ T cells are more similar to cells of the innate immune system like natural killer (NK) cells than to αβ T lymphocytes and B cells. Moreover, γδ T cells are much less abundant in the peripheral blood than αβ T cells, but are particularly enriched in epithelial tissues like the skin and intestinal tract, where they can account of up to 50% of CD3⁺ cells.¹

Classically, γδ T cells were regarded as immune effector cells responding to a few specific signals of viral or endogenous origin, encompassing (but not limited to) NKG2D ligands that are expressed by malignant precursors as a result of DNA damage or oncogenic stress.² Upon TCR ligation, some subsets of γδ T cells mediate cytotoxic effects through perforin 1 (PRF1) and granzyme B (GRZB), and support T_H1 helper responses by secreting interferon gamma (IFNγ) and tumor necrosis factor (TNF).^1,3 In specific preclinical models, interleukin 17 (IL-17)-producing γδ T cells also contribute to tumor-targeting immune responses elicited by chemotherapy or specific radiotherapeutic regimens.^4-6 These considerations have fostered the development of multiple strategies to promote tumor recognition and elimination by γδ T cells,⁷ including bispecific antibodies simultaneously targeting one Vγ chain and one tumor-associated antigen.^8,9 To the best of our knowledge, however, γδ T cell-activating approaches achieved limited, if any, clinical success so far.^1,10 Accumulating evidence suggests indeed that multiple γδ T-cell subsets favor, rather than counteract, tumor progression, mainly as they secrete potentially immunosuppressive cytokines like interleukin-10 (IL-10) or IL-17.^11,12 Recent data from George Miller's laboratory (from New York University School of Medicine, New York, US) lend further support to this notion as they demonstrate that γδ T cells foster pancreatic carcinogenesis by engaging immunological checkpoints on αβ T cells.¹³

Daley and collaborators observed that γδ T cells are virtually absent from the normal human pancreas but account for up to 75% CD3⁺ T lymphocytes infiltrating human pancreatic ductal adenocarcinomas (PDAs). γδ T cells outnumbered CD8⁺ αβ T cells in the microenvironment of human PDAs as they exhibited a considerable enrichment in effector memory (CD27⁻CD45RA⁻) activated (CD62L⁻) components. Moreover, Vγ9⁺ γδ T cells, which are known to mediate tumoricidal functions, were poorly represented among PDA-infiltrating γδ T cells, whereas they were abundant among peripheral blood mononuclear cell (PBMC)-derived CD3⁺ cells.¹³ Along similar lines, γδ T cells abundantly infiltrated mouse PDAs generated by the orthotopic implantation of malignant cells from Pdx1^Cre;Kras^G12D;Tp53^R172H(KPC) mice (which spontaneously develop invasive PDAs by 8 weeks of age) into syngeneic immunocompetent hosts. In this system, γδ T cells exhibited a peculiar phenotype (as compared to splenic γδ T cells), as they were characterized by the upregulation of FAS ligand (FASLG), killer cell lectin-like receptor subfamily B member 1C (KLRB1C, best known as NK1.1), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, best known as CD39), junction adhesion molecule like (JAML), and TNF receptor superfamily member 4 (TNFRSF4, best known as OX40), as well as by enriched FOXP3⁺ and Vγ4⁺ fractions. Moreover, PDA-infiltrating γδ T cells differed from their splenic counterparts relative to the upregulation of IL-10, interleukin 17A (IL-17A), NKG2D, multiple Toll-like receptors (namely, TLR4, TLR7, and TLR9), and chemokine (C–C motif) receptors (namely, CCR2, CCR5, and CCR6). Similar observations were obtained in 6-month-old Ptf1a^Cre;Kras^G12D (KC) mice, a model of pre-invasive PDA.¹³ Of note, KPC-derived cells orthotopically implanted into Ccr2^−/−, Ccr5^−/−, or Ccr6^−/− mice recruited limited amounts of γδ T cells as compared to KPC-derived PDAs established in wild-type (WT) mice, demonstrating the importance of chemokine signaling in this setting.¹³

To test whether γδ T cells would play an etiologic role in pancreatic carcinogenesis, Daley and collaborators crossed KC mice with Tcrd^−/−mice (which lack γδ T cells but contain normal amounts of αβ T lymphocytes). Tumor progression and cancer-associated fibrosis were considerably retarded in the pancreata of Ptf1a^Cre;Kras^G12D;Tcrd^−/−mice as compared with the pancreata of KC mice, and the Tcrd^−/−genotype was sufficient to confer a 1-year survival extension to KC mice. Along similar lines, 6-week-old KC mice receiving a γδ T cell-depleting antibody for eight consecutive weeks exhibited retarded tumor progression and limited fibrosis as compared to KC mice treated with a control antibody. Moreover, KPC-derived aggressive PDAs orthotopically established in Tcrd^−/−mice were unable to progress as rapidly as KPC-derived PDAs implanted into WT hosts. In this setting, the Tcrd^−/−genotype as well as the administration of a γδ T cell-depleting antibody considerably extended the survival of tumor-bearing mice.¹³

Upon excluding the possibility that γδ T cells would promote pancreatic carcinogenesis by a direct effect on malignant cells, the authors postulated that PDA-infiltrating γδ T cells might resemble CD4⁺CD25⁺FOXP3⁺ T_REG cells in their ability to mediate robust immunosuppressive effects. Indeed, KPC-derived PDAs established in Tcrd^−/−mice recruited superior amounts of CD4⁺ and CD8⁺αβ T cells as compared to KPC-derived PDAs growing in WT hosts, and PDA-infiltrating αβ T cells exhibited signs of activation and T_H1 polarization, including increased expression of IFNG, TNF, T-box 21 (TBX21, best known as T-bet), CD44, inducible T cell co-stimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA), GRZB, OX40, and programmed cell death 1 (PDCD1, best known as PD-1), as well as CD62L downregulation. Similar results were obtained by comparing PDAs developing in Ptf1a^Cre;Kras^G12D;Tcrd^−/−versus KC mice. Moreover, the co-depletion of CD4⁺ and CD8⁺ T lymphocytes with specific antibodies abolished the beneficial effects of the Tcrd^−/−genotype on pancreatic carcinogenesis, which de facto ascribed the ability of γδ T cells to foster tumor progression (at least in the pancreas) to the inhibition of αβ T cells.¹³

After excluding the possibility that such an activity would rely on soluble mediators, Daley and colleagues investigated the expression of immunosuppressive molecules on PDA-associated versus splenic γδ T cells, finding increased levels of galectin 9 (LGALS9) and CD274 (best known as PD-L1) on γδ T cells infiltrating invasive as well as pre-invasive PDAs. Notably, PDA-infiltrating γδ T cells expressed higher levels of LGALS9 and PD-L1 than PDA cells themselves, although LGALS9 and PD-L1 expression was comparable in PDA-associated γδ T cells, macrophages, and myeloid-derived suppressor cells (MDSCs). CCR2, CCR5, and CCR6 signaling was required not only for the recruitment of γδ T cells to developing PDAs, but also for normal LGALS9 and PD-L1 expression. These preclinical findings matched the observation that PDA patients had increased levels of LGALS9⁺ and PD-L1⁺ γδ T cells in the circulation and even more so in the tumor microenvironment as compared to healthy subjects.¹³

The PD-L1/PD-1 axis is well known for its prominent immunosuppressive effects, and no less than three PD-L1/PD-1 targeting monoclonal antibodies (namely, atezolizumab, pembrolizumab and nivolumab) are currently approved by the US Food and Drug Administration (FDA) and equivalent agencies worldwide for use in cancer patients.¹⁴ Corroborating a mechanistic involvement of the PD-L1/PD-1 in the ability of γδ T cells to foster pancreatic carcinogenesis, a PD-L1-targeting antibody prevented γδ T cells from inhibiting the acquisition of activation markers by αβ T cells in co-culture experiments. Moreover, KPC-derived PDAs implanted orthotopically in WT mice were sensitive to treatment with either a PD-L1-targeting or a LGALS9-targeting antibody as tumors exhibited increased infiltration by activated (CD44⁺CD62L⁻) αβ T cells, while neither of these two immunotherapeutic regimens incremented the beneficial effects on tumor progression provided by the Tcrd^−/−genotype.¹³

In summary, the findings by Daley and collaborators delineate a molecular mechanism through which PDA-infiltrating γδ T cells suppress anticancer immunosurveillance (Fig. 1). This said, whether tumor-infiltrating γδ T cells mediate similar effects in other settings remains to be determined. Indeed, a large meta-analysis of gene expression data from approximately 18,000 human tumors identified a γδ T cell-related genetic signature as the most consistent indicator of improved disease outcome across all types of cancer confounded.¹⁵ Thus, although the parallelism with CD4⁺CD25⁺FOXP3⁺ T_REG cells leaps to the eye, it may be too early to talk about γδ T_REG cells.

Figure 1. γδ T cells foster pancreatic cancer progression by engaging immunological checkpoints on αβ T cells. Developing pancreatic ductal adenocarcinomas (PDAs) recruit high levels of γδ T cells by secreting ligands for several chemokine (C–C motif) receptors, namely CCR2, CCR5 and CCR6. PDA-infiltrating γδ T cells not only release interleukin 10 (IL-10) and interleukin 17 (IL-17), but also express galectin 9 (LGALS9) and CD274 (best known as PD-L1), hence establishing a robust immunosuppressive microenvironment that foster disease progression (A). In the absence of γδ T cells, developing PDAs recruit increased amounts of αβ T cells that are not subjected to local inhibition via PD-1 and hence efficiently limit disease progression (B).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

ORCID

Laura Mondragón http://orcid.org/0000-0002-3257-045X