Manipulating regulatory T cells: is it the key to unlocking effective immunotherapy for pancreatic ductal adenocarcinoma?

The five-year survival rates for pancreatic ductal adenocarcinoma (PDAC) have scarcely improved over the last half-century. It is inherently resistant to FDA-approved immunotherapies, which have transformed the outlook for patients with other advanced solid tumours. Accumulating evidence relates this resistance to its hallmark immunosuppressive milieu, which instils progressive dysfunction among tumour-infiltrating effector T cells. This milieu is established at the inception of neoplasia by immunosuppressive cellular populations, including regulatory T cells (Tregs), which accumulate in parallel with the progression to malignant PDAC. Thus, the therapeutic manipulation of Tregs has captured significant scientific and commercial attention, bolstered by the discovery that an abundance of tumour-infiltrating Tregs correlates with a poor prognosis in PDAC patients. Herein, we propose a mechanism for the resistance of PDAC to anti-PD-1 and CTLA-4 immunotherapies and re-assess the rationale for pursuing Treg-targeted therapies in light of recent studies that profiled the immune landscape of patient-derived tumour samples. We evaluate strategies that are emerging to limit Treg-mediated immunosuppression for the treatment of PDAC, and signpost early-stage trials that provide preliminary evidence of clinical activity. In this context, we find a compelling argument for investment in the ongoing development of Treg-targeted immunotherapies for PDAC.


Introduction
Since 1863when Rudolf Virchow first observed leukocyte infiltrates decorating neoplastic tissuesresearch has uncovered a dynamic interplay between the immune system and pre-malignant cells, which governs their progressive transformation to invasive derivatives (1).In parallel, efforts to leverage the immune system to treat malignancy have a long history; in 1868, Wilhelm Busch reported tumour regression after intentionally infecting patients with Streptococcus pyogenes (2).Today, immunotherapy has revolutionised clinical oncology: immune checkpoint inhibitors (ICIs; specifically anti-PD-1, -CTLA-4, and -PD-L1 antibodies) provide unprecedented rates of durable anti-tumour responses in patients with several types of cancer (3).However, ICIs, including the combination of anti-CTLA-4 and anti-PD-L1 antibodies, have yielded limited responses in pancreatic ductal adenocarcinoma (PDAC); a malignancy of the exocrine pancreas that constitutes 95% of pancreatic cancer cases (4,5).Accordingly, PDAC carries a bleak prognosis: globally, the 5-year survival rate at the time of diagnosis is 9% (6).
Substantial research has sought to identify immunological mechanisms that render PDAC resistant to ICIs.Concomitantly, these studies have unearthed therapeutic targets that could feasibly be exploited to induce anti-tumour immunity in PDAC; indeed, strategies to restrain immunosuppressive regulatory T cells (T regs ), myeloid cells, and cancer-associated fibroblasts are currently under development (7,8).The manipulation of CD4 + T regs has gained considerable traction, stemming from the discovery that an abundance of intratumoral T regs correlates with a poor prognosis in PDAC patients (9).Herein, we propose a mechanism for the intrinsic resistance of PDAC to ICIs; discuss the rationale for pursuing T reg -targeted therapies in the context of PDAC; and evaluate emerging strate gies to limit T r e g -mediated immunosuppression.Overall, we argue that T reg -targeted immunotherapies offer a valuable opportunity to improve clinical outcomes in PDAC.
It is well established that the baseline density of tumourinfiltrating T eff cells is a critical determinant of therapeutic responses to ICIs (17,18).Thus, the immunologically 'cold' phenotype that characterises PDAC has often been attributed to the physical exclusion of T e ff cells from the tumour microenvironment (TME) (19,20).However, recent studies have challenged this paradigm, identifying heterogenous baseline infiltrates of CD4 + and CD8 + T eff cells that correlate with prolonged overall survival in PDAC patients (14,15,(21)(22)(23)(24)(25)(26).There is also evidence for ongoing anti-tumour immunity; Freed-Pastor et al. identified a population of HLA-DR + Ki67 + CD57 -CD8 + T cellsindicative of an activated, proliferative phenotypethat are present in the majority of patients (27).Altogether, these studies suggest that inducing T eff cell-mediated anti-tumour immunity in PDAC may not be as intractable as is widely considered (23).
In further support of this notion, a rare subset (~1.6.%) of PDAC patients with hypermutated mismatch repair deficient (dMMR) tumours exhibit marked therapeutic responses to anti-PD-1 antibodies (28).These tumours present a broad repertoire of neoantigens, which direct potent anti-tumour immune responses (29,30).Indeed, in this patient cohort, sequencing of the TCR Vb chain revealed that 94% of intratumoral T cell clonotypes were unique to tumours, implying the existence of a neoantigen-specific immune response (24).Overall, this highlights the importance of neoantigens as a substrate for T eff -mediated anti-tumour immunity indeed, on the basis of this principle, pembrolizumab and nivolumab (anti-PD-1) were granted FDA-approval in 2017 for the treatment of dMMR tumours, irrespective of their tissue of origin (31).In this context, it is notable that recent studies have challenged the claim that MMR-proficient PDAC harbours a limited repertoire of neoantigens.Freed-Pastor et al. investigated a cohort of 57 advanced PDAC patients and discovered that they all possessed neoepitopes with predicted ability to bind MHC class-I molecules (27).Accordingly, studies have consistently identified intratumoral neoantigen-reactive CD8 + T cells in PDAC patients, indicating that these neoantigens are capable of directing antitumour immunity (27,32,33).
Nevertheless, it is evident that this population of intratumoral neoantigen-reactive CD8 + T cells is not sufficient to drive therapeutic responses to FDA-approved ICIs in MMR-proficient PDAC.Indeed, multi-omic profiling of the PDAC immune landscape in resectable patients has revealed that 'dysfunctional' and 'senescent' phenotypesboth hypofunctional states, defined by the expression of multiple inhibitory receptors: TIGIT, LAG-3, TIM-3, and CD39dominate the intratumoral T eff cell repertoire, leaving few activated T cells that are thus unable to control the tumour (15, 25).In addition, a more pronounced exhaustion signature has been observed in CD8 + T cells from fine-needle biopsy samples of advanced, unresectable PDAC (14).
This progressive dysfunction of intratumoral T eff cells can be attributed to the profoundly immunosuppressive TME.It is established by the progressive infiltration of immunosuppressive cells: T regs , myeloid-derived suppressor cells, neutrophils, and tumour-associated macrophages (34).In the murine KC model, these populations dominate the immune landscape of pancreatic intraepithelial neoplasia (PanIN): precursor lesions that culminate in the development of PDAC (19).Other non-immune cellular populations also contribute to the immunosuppressive TME.For example, a subset of cancer-associated fibroblasts present antigenic peptides in association with MHC class-II molecules; however, they lack expression of classical co-stimulatory molecules and thus command CD4 + T cells to the T reg lineage (35).In summary, neoantigen-specific T eff responses are dampened by the gradual accumulation of immunosuppressive cells in the TME, which dictates the progression from PanIN to PDAC.Hence, the development of immunomodulatory therapies for PDAC must focus on surmounting the hallmark immunosuppressive TME (36).Importantly, the progressive nature of intratumoral T eff cell dysfunction promises to confer a broad window during which such therapies might be effective.

What is the phenotype of T regs in PDAC?
To date, strategies targeting myeloid-derived suppressor cells or cancer-associated fibroblasts for the treatment of PDAC have generally failed to demonstrate therapeutic promise in clinical trials (37-40).However, one promising strategywhich has gained substantial traction in the context of PDACis combatting T reg -mediated immunosuppression.This originated from the discovery that an abundance of intratumoral T regs correlates with a poorer prognosis in PDAC patients (9).Accordingly, the depletion of T regs has been shown to delay tumour growth in orthotopically transplanted murine PDAC, albeit with conflicting results from other murine models (41,42).However, recent single-cell analyses have uncovered extensive diversity among intratumoral T regs ; in this context, it is important to re-evaluate the rationale for the development of T regtargeted therapies.

Effector T regs are highly immunosuppressive
Classically, CD4 + T regs have been defined according to expression of FOXP3, considered a lineage-specifying transcription factor (TF), or the interleukin (IL)-2 receptor a chain (CD25).In a seminal study, Hiraoka et al. discovered that the prevalence of FOXP3 + T regs increases during the progression from PanIN to advanced PDACat this latter stage, they constitute 35% (± 11%) of the total intratumoral CD4 + population (9,15).Further, it is estimated that 54% (± 19%) of intratumoral T regs are effector T regs (eT regs ; CD45RA - FOXP3 hi CD25 hi ) (15).These cells express high levels of TIGIT, CTLA-4, ICOS, CD39, and HLA-DR, which are indicative of functional activation and potent immunosuppressive capacity (14,15).This activated state has been attributed to sustained TCR stimulation, provided by the plethora of self-and quasi-selfantigens present in the inflammatory TME (43).However, a stable eT reg phenotype is also dependent on the expression of Helios, a member of the Ikaros TF family.Indeed, intratumoral Helios + T regs exhibit significantly higher expression of FOXP3, compared to Helios -T regs (44).
Intratumoral eT regs potently suppress CD8 + T cell-mediated immunity via the expression of co-inhibitory molecules e.g., CTLA-4, which prevents the functional maturation of dendritic cells (41); secretion of immunosuppressive cytokines e.g., IL-10, IL-35, and TGF-b; sequestration of IL-2, which hampers IL-2-dependent T cell activation; and the secretion of granzymes to lyse target CD8 + cells (45).In support of their immunosuppressive capacity in situ, spatial analyses reveal that 90% of T regs reside in close proximity to a CD8 + T cell in the PDAC TME (15).

FOXP3 + RORgt + T regs provide mitogenic signalling
FOXP3 + T regs exhibit extensive heterogeneity in PDAC.Strikingly, studies have discovered populations of FOXP3 + T regs that, in addition to IL-10, secrete high levels of pro-inflammatory cytokines.For example, Chellappa et al. identified T regs that coexpress FOXP3 and RORgt: a factor that specifies the type-17 Thelper cell lineage (T H 17) (46).These cells retained markers associated with FOXP3 + T regs e.g., CTLA-4, CD39, and ICOS, indicating an ability to robustly suppress anti-tumour immunity.However, through the simultaneous production of IL-17, these FOXP3 + RORgt + cells provide mitogenic signalling to transformed pancreatic epithelial cells, which upregulate the IL-17 receptor (47).Moreover, studies have identified populations of FOXP3 -T reg -like cells that share expression of molecules classically associated with immunosuppressive T reg functions (e.g., IL-10, CCR8, TIGIT, ICOS, CTLA-4) (48, 49).Barilla et al. demonstrated that the gene expression profile of one such population, termed T r 1 cells (CD49b, CD73, and AHR), was associated with decreased overall survival in PDAC patients (49).Furthermore, Whiteside et al. suggest that intratumoral T eff cells may adopt this FOXP3 -T reg -like phenotype following the ablation of FOXP3 + cells (48).
This profound heterogeneity likely explains conflicting reports regarding the overall contribution of T regs in the pathophysiology of PDAC.One notable study reported an increased prevalence of T regs in tumours of long-term PDAC survivors (24).Moreover, depletion of T regs prior to the development of PanIN in KC mice has been shown to accelerate malignant progression (42).Conceivably, the use of different experimental systems, including varied methods for detecting and defining intratumoral T regs , might accentuate specific T reg -associated functions and thereby explain these conflicting reports.Moreover, studies have suggested that, as part of normal immune homeostasis, intratumoral T regs accompany CD8 + T cell infiltrates (21,42,49), which may further obscure any relationship between the prevalence of intratumoral T regs and a poor prognosis.Nevertheless, harnessing the therapeutic manipulation of T regs will require a targeted approach, based on a detailed understanding of the heterogeneous functions ascribed to T regs , and their spatiotemporal dynamics in the PDAC TME (Figure 1).In addition, such an approach will reduce the systemic side-effects associated with T reg -targeted immunotherapies.

Apoptotic T regs are paradoxically immunosuppressive
This hypothesis is fortified by the discovery that apoptotic T regs , defined by increased expression of Ki67 and cleaved caspase-3, exert immunosuppressive effects in the oxidative TME.They release large quantities of ATP, which is converted into adenosine via CD39 and CD73ectoenzymes that are expressed by T regs and remain catalytically active after cell-death (50).Through the A 2A receptor, accumulating extracellular adenosine inhibits T eff cell proliferation; induces immunosuppressive dendritic cells; and stabilises surviving T regs (51).Thus, CD39 and CD73 expression correlate with a poor prognosis in patients with various solid tumours (52, 53).Importantly, this paracrine signalling pathway is likely to be operating in human PDAC, as intratumoral T regs express high levels of CD39.
4 What are the strategies to manipulate T regs for the treatment of PDAC?
The manipulation of T regs has captured significant attention from both scientific and commercial communities as a novel approach to the treatment of PDAC.The earliest attempts depleted T regs by targeting CD25 with antibodies, daclizumab, or the IL-2-diphtheria toxin fusion protein, ONTAK (54, 55).However, IL-2 signalling via CD25 promotes the survival of activated T eff cells, conferring a limited therapeutic window to CD25-targeted interventions.Nevertheless, these efforts provided proof-of-concept for the therapeutic manipulation of T regs .Today, numerous T reg -targeted therapies are under development for the treatment of advanced solid tumours, including PDAC (Table 1).

Re-engineering next-generation ICIs
Allison and colleagues originally attributed the anti-tumour activity of anti-CTLA-4 monoclonal antibodies (mAbs) to the reinvigoration of dysfunctional T eff cells (56).However, accumulating evidence suggests that anti-CTLA-4 mAbs can preferentially deplete CTLA-4 hi T regs in vivo by antibodydependent cellular cytotoxicity (ADCC) (57)(58)(59)(60).Thus, in spite of the failure of prior clinical trials (4, 61), this novel mechanistic insight provides a rationale for the continued development of anti-CTLA-4 mAbs to treat PDAC.Clearly, however, this will necessitate re-engineering of existing anti-CTLA-4 mAbs; specifically, the fragment crystallisable (F c ) domain to enhance affinity for activatory F c g receptors and decrease affinity for inhibitory receptors, thereby promoting ADCC.This approach can be optimised with consideration of the relative abundance and distribution of specific F c gRs on local effector cells; indeed, the engineering of anti-CTLA-4 mAbs in this manner has been shown to increase therapeutic activity in tumour-bearing mice (59,62).Therefore, it is important that studies have identified intratumoral populations of F c gRIIIA (CD16)-expressing natural killer and myeloid cells in human PDAC (14)(15)(16).Moreover, Agenus  recently initiated a phase I/II trial to investigate botensilimaban F c -engineered anti-CTLA-4 mAb with enhanced affinity for F c gRIIIAin metastatic PDAC patients (NCT05630183).Further testament to the widespread interest in strategies to selectively deplete intratumoral T regs , there is renewed attention on the development of anti-CD25 mAbs.For example, Solomon et al. developed an anti-CD25 mAb (RG6292) that selectively depletes CD25 hi T regs , whilst preserving CD25-STAT5 signalling required for T eff cell-mediated anti-tumour immunity (63).Indeed, a phase I trial of RG6292, conducted in patients with advanced/metastatic solid tumours, indicated a manageable safety profile and preliminary antitumour activity (64).However, multi-omic analysis of patientderived tumour samples obtained during treatment with RG6292 is required to confirm this proposed mechanism of action in vivo.

Exploiting novel immune checkpoints
Since the discovery of CTLA-4 and PD-1, studies have identified a plethora of immune checkpointsboth inhibitory (e.g., TIGIT, LAG-3, TIM-3) and co-stimulatory (e.g., ICOS, OX40, GITR, 4-1BB) that might be exploited therapeutically to augment anti-tumour immunity.In PDAC, TIGIT and ICOS are expressed at high levels on intratumoral eT regs (14,15).TIGIT is also expressed, albeit at lower levels, by dysfunctional T eff cells, whereas ICOS is induced upon the activation of intratumoral T eff cells (14,15,27).Therefore, anti-TIGIT and agonistic ICOS mAbs might have a dual mechanism of action: the re-invigoration of dysfunctional T eff cells and selective depletion of activated T regs (65).However, achieving the optimal balance between these mechanisms will require F c engineering to effectively engage specific F c receptors (66).
Tiragolumab (IgG1k anti-TIGIT) has demonstrated tolerability and preliminary anti-tumour activity in patients with advanced solid tumours (67,68).Consequently, two early-stage trials are investigating anti-TIGIT mAbs, incorporated into combinatorial regimens, for the treatment of metastatic PDAC (NCT03193190, NCT05419479).By contrast, a phase I/II trial, investigating vopratelimab (IgG1k agonistic ICOS) for the treatment of advanced solid tumours, including three PDAC patients, reported limited efficacy (69).However, on-treatment emergence of ICOS hi CD4 + T eff cells was associated with therapeutic responses, suggesting that vopratelimab might indeed re-invigorate dysfunctional T eff cells in patients through ICOS activation.More generally, this illustrates that multi-omic analyses of on-treatment patient-derived samples during clinical trials may further advance our understanding of the PDAC immune landscape.

De-stabilising activated T regs
The development of strategies for selectively drugging T regs has been the subject of considerable research.One potential target is Helios; in PDAC patients, Helios + T regs are significantly enriched in the TME (70).Moreover, T reg -intrinsic deletion of Helios has been shown to enhance anti-tumour immunity in tumour-bearing mice (71).Interestingly, Helios-deficient T regs acquire a T eff phenotype including the production of pro-inflammatory cytokines (e.g., IFNg), which is attributed to downregulation of FOXP3 and derepression of T H 1/T H 2 lineage determinants (43).In the absence of the stabilising influence of Helios, it appears that the inflammatory TME promotes the trans-differentiation of T regs into activated T eff cells.Intriguingly, this novel T eff population is equipped with an inherently self-reactive TCR repertoire, which might be expected to direct a potent immune response against 'quasi-self' tumour antigens.
Transcription factors are traditionally considered difficult to drug.However, several recent studies have described smallmolecules that selectively enhance the proteasomal degradation of Helios (72,73).Future in vivo studies must determine whether these small-molecules can selectively destabilise activated intratumoral eT regs ; one clinical trial is currently evaluating this approach in advanced solid tumours (NCT03891953).

Targeting chemokine receptors
The origin of intratumoral FOXP3 + T regs is unclearthey may differentiate locally from T eff cells or be recruited from the circulation.For the latter, targeting chemokine signalling axes (e.g., CCL2-CCR4; CCL5-CCR5) that can recruit T regs into the PDAC TME is of interest.However, this strategy has proved disappointing thus far; clinical trials investigating mogamulizumab (IgG1 anti-CCR4) reported offtarget depletion of T H 2/T H 17 cells, reflecting heterogeneous expression of CCR4 (74,75).
It is notable, therefore, that intratumoral eT regs uniquely express CCR8 (76).However, functional blockade of CCR8 does not affect T reg recruitment; they acquire CCR8 expression in the TME, perhaps suggesting that this axis mediates retention of intratumoral T regs (77).Nevertheless, CCR8 constitutes a target for the selective depletion of intratumoral eT regs in PDAC.Preclinical studies have demonstrated that anti-CCR8 mAbs profoundly suppress tumour growth in tumour-bearing mice (76,78).Further, this response coincided with the expansion of intratumoral CD4 + T eff cells and the preservation of systemic T reg populations, which may mitigate the risk of autoimmune-related adverse events.Currently, eight early-stage trials are investigating anti-CCR8 mAbs for the treatment of advanced solid tumours (NCT04895709, NCT06131398, NCT05635643, NCT05537740, NCT05007782, NCT05518045, NCT05101070, NCT05935098).

Combatting immunosuppressive adenosine
A p o p t o t i c T r e g s c o n v e r t A T P t o a d e n o s i n e , a n immunosuppressive metabolite, via ectoenzymes that remain catalytically active after cell-death.This raises the paradoxical possibility that the therapeutic depletion of T regs might not limit T reg -cell-mediated immunosuppression.This discovery provided impetus to the development of immunotherapies that target the adenosinergic pathway: CD39, CD73, and the A 2A /A 2B receptors.It is hoped that these therapies will synergise with T reg -targeted approaches, or other immunotherapeutic modalities, to induce potent anti-tumour immunity.To date, however, attempts to target this pathway with anti-CD73 mAbs have demonstrated no clinical benefit for PDAC patients; a phase-II trial investigating the combination of anti-CD73, anti-PD-L1, and chemotherapy revealed comparable efficacy to chemotherapy alone (79).

Conclusions and future perspectives
The manipulation of intratumoral T regs may prove a valuable addition to our currently limited armamentarium for the treatment of PDAC.This therapeutic strategy has the potential to reinvigorate anti-tumour immunity by reprogramming the immunosuppressive milieu that is first established in premalignant lesions.This notion is supported by promising earlystage clinical trials of T reg -targeted immunotherapies (68,80).Moreover, data from trials investigating anti-CCR8 mAbs and

FIGURE 1
FIGURE 1Phenotype of effector T reg cells in human PDAC.Effector T regscharacterised by the expression of FOXP3, CD25, TIGIT, CTLA-4, ICOS, CD39, and CCR8are activated by sustained TCR stimulation with abundant self-and quasi-self-antigens and stabilised by expression of the Helios transcription factor.These cells exhibit potent immunosuppressive capacity within the PDAC TME, where they exist in close proximity to CD8 + T lymphocytes.Specifically, they express co-inhibitory molecules (e.g., CTLA-4, TIGIT, ICOS); convert ATP to immunosuppressive adenosine via ectoenzymes that remain catalytically active after cell-death (CD39 and CD73); secrete immunosuppressive cytokines (e.g., IL-10, IL-35, TGF-b) and granzymes that lyse CD8 + T eff cells; and sequester IL-2 that is required for T eff cell activation.

TABLE 1 Continued
† human epidermal growth factor receptor 2; ‡ vascular endothelial growth factor.All information was obtained from the NIH clinical trials database (https://clinicaltrials.gov) or from the publicised development pipelines of pharmaceutical companies.The rows highlighted blue denote drugs that are under evaluation in clinical trials that include PDAC patients.