Supplementary Figure from Low-Dose JAK3 Inhibition Improves Antitumor T-Cell Immunity and Immunotherapy Efficacy

Immunotherapy with anti‐PD1/PD‐L1 is effective in only a subgroup of patients with malignant pleural mesothelioma (MPM). We investigated the efficacy of a combination of anti‐PD1/PD‐L1 and dendritic cell (DC) therapy to optimally induce effective anti‐tumor immunity in MPM in both humans and mice. Data of nine MPM patients treated with DC therapy and sequential anti‐PD1 treatment were collected and analyzed for progression‐free survival (PFS) and overall survival (OS). Survival and T‐cell responses were monitored in AC29 mesothelioma‐bearing mice treated concurrently with the combination therapy; additionally, the role of the tumor‐draining lymph node (TDLN) was investigated. The combination therapy resulted in a median OS and PFS of 17.7 and 8.0 months, respectively. Grade 3 to 4 treatment‐related adverse events had not been reported. Survival of the mesothelioma‐bearing mice treated with the combination therapy was longer than that of untreated mice, and coincided with improved T‐cell activation in peripheral blood and less T‐cell exhaustion in end stage tumors. Comparable results were obtained when solely the TDLN was targeted. We concluded that this combination therapy is safe and shows promising OS and PFS. The murine data support that PD‐L1 treatment may reinvigorate the T‐cell responses induced by DC therapy, which may primarily be the result of TDLN targeting.

Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (T reg cells) in αPD-L1 therapy–resistant solid tumor–bearing mice. T reg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating T reg cells in human patients with skin cancer, and in patients with non–small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1–induced PD-1 + T reg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of T reg cells, resulting in therapy resistance, suggesting that T reg cell targeting is an important adjunct strategy to enhance therapeutic efficacy.

Immune checkpoint blockade (ICB) using anti-PD-1/PD-L1 and anti-CTLA-4 antibodies significantly enhances survival in metastatic melanoma patients and has recently been shown to prolong relapse-free survival in stage III and high-risk stage II melanoma patients (Eggermont et al., 2018; Luke et al., 2022; Robert et al., 2015). However, a significant proportion of patients does not respond to ICB prior to or following surgery, for reasons incompletely understood. We and others recently identified tumor-draining lymph nodes (TDLNs) to be critically involved in anti-PD-L1 treatment efficacy in preclinical tumor models (Dammeijer et al., 2020; Fransen et al., 2018).

Abstract Terminal T-cell exhaustion poses a significant barrier to effective anticancer immunotherapy efficacy, with current drugs aimed at reversing exhaustion being limited. Recent investigations into the molecular drivers of T-cell exhaustion have led to the identification of chronic IL2 receptor (IL2R)–STAT5 pathway signaling in mediating T-cell exhaustion. We targeted the key downstream IL2R-intermediate JAK 3 using a clinically relevant highly specific JAK3-inhibitor (JAK3i; PF-06651600) that potently inhibited STAT5-phosphorylation in vitro. Whereas pulsed high-dose JAK3i administration inhibited antitumor T-cell effector function, low-dose chronic JAK3i significantly improved T-cell responses and decreased tumor load in mouse models of solid cancer. Low-dose JAK3i combined with cellular and peptide vaccine strategies further decreased tumor load compared with both monotherapies alone. Collectively, these results identify JAK3 as a novel and promising target for combination immunotherapy.

BACKGROUND:Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma. METHODS:We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS. FINDINGS:Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS. INTERPRETATION:Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-β in vitro, although TGF-β signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

Background Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. Methods Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. Results Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. Conclusion These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1(+) T cells which closely associate with PD-L1(+) cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.

Abstract Effective antitumor immunity involves successful priming of tumor-specific T-cells by dendritic cells (DC) in the lymph node (LN), followed by trafficking, infiltration and sustained elimination of tumor cells by T-cells. Immunotherapies aim to facilitate or further invigorate these processes, but at which anatomic sites these therapies act and in which specific patients remains unknown. We and others have identified macrophages as key mediators of immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are capable of negating the effectiveness of multiple conventional- and immune-targeted anticancer therapies. Several current therapeutic strategies aim to deplete or reprogram macrophages; however, as these drugs act systemically, their precise mechanism and site of action remains unclear. Besides TAMs, macrophages in the LN have been identified as potent immune modulatory cells in diverse settings. Using a novel method that allows for the specific interrogation of LN-macrophages, we aimed to investigate the role of LN macrophages in regulating DC-induced anti-tumor immunity. To determine the immune modulatory functions of the tumor on the LN, we extensively characterized the immune contexture and phenotype of the tumor-draining lymph node (TDLN) compared to a distant non-tumor draining lymph node (non-TDLN) using multicolor flow- and histo-cytometry, in an orthotopic mouse model of peritoneal mesothelioma. In addition, the effects of systemic macrophage depletion using an CSF1R-kinase inhibitor or clodronate encapsulated liposomes (CEL) were evaluated. We developed a method to specifically deplete TDLN-macrophages while leaving TAMs and other tissue macrophages intact, by intrapleural (i.pl) injection of low-dose CEL. Using this model, we investigated the immune-regulatory properties of LN-macrophages following adoptive transfer of activated, tumor-loaded DCs in the TDLN by both flow- and histo-cytometry. Comparison of the TDLN and non-TDLN immune contexture revealed prominent shifts in immune cell frequencies and phenotypes, including a decrease in T-cell frequencies and a marked increase in CD169+ LN-macrophages in the TDLN. Systemic macrophage targeting using CSF1R-kinase inhibition or CEL effectively minimized LN-macrophage subsets as well as TAMs, therefore preventing the specific interrogation of LN-macrophage biology during tumor growth and in the context of immune activation. Conversely, titrating CEL doses down to 5% of the total dosing volume injected i.pl. allowed for the specific depletion of LN-macrophages while leaving systemic macrophages undisturbed. LN-macrophages limited the presence of migratory ex vivo activated and tumor-loaded DCs, as indicated by increased counts of CFSE+ DCs in the in the TDLN following adoptive transfer in CEL-pretreated mice. Depletion of TDLN-macrophages increased LN- and blood frequencies and activation status of DC-induced CD8+ T-cells and CD4+ T-helper cells, indicative of enhanced antitumor immunity. Furthermore, whereas DC-induced immune activation alone was insufficient to impede tumor growth and prolong survival, elimination of LN-macrophages prior to DC-administration enabled the successful impediment of tumor growth. Interestingly, interrogation of tumor-infiltrating T-cell (TIL) phenotype and distribution showed a robust increase in CD4+ T-helper TILs and a concurrent decrease in CD8+ TIL- exhaustion phenotype, as evidenced by diminished PD-1 and LAG-3 expression. Altogether, these results indicate an important immune inhibitory role for LN-macrophages in settings of immune activation in the solid tumor setting. These findings further increase our knowledge of where and how macrophages exert their immune-inhibitory functions and allow for better insight into the underpinnings of immunotherapy resistance. Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Depletion of macrophages in the tumor-draining lymph node enhances dendritic cell-induced antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A165.

Antagonistic antibodies to programmed cell death protein 1 (PD1) and its ligand PD-L1 have revolutionized the treatment of multiple metastasized cancers including melanoma and non-small cell lung cancer. Despite these advances, a majority of patients do not achieve durable responses to these therapies. Further insights into the mode of action and potential biomarkers predicting clinical response are therefore warranted. Although the focus has been on biomarker identification in the tumor or peripheral blood, the role of PD-L1 in the tumor draining lymph node (TDLN) has not yet been investigated. As the TDLN is crucial for orchestrating antitumor immune responses, we assessed the role of PD-L1 in the TDLN on survival and anti-tumor immunity. To assess the extent of PD-L1 expression in different tissues during tumor growth and inflammation, we measured PD-L1 levels on multiple cell subsets in the tumor, TDLN and non-TDLN on baseline and following injection of activated dendritic cells (DCs) by multicolor flow cytometry. We exploited the intraperitoneal (i.p.) localization of mesothelioma tumors by injecting a range of anti-PD-L1 antibody concentrations intrapleurally (i.pl). This allowed us to investigate the role of PD-L1 in the TDLN while leaving tumoral PD-L1 intact. When sole targeting of PD-L1 expressed in the TDLN was achieved, we injected advanced tumor-bearing mice with low-dose anti-PD-L1 i.pl and compared the effects on survival and antitumor immune responses to systemic administration of the antibody alone or in combination with DC-induced immune activation. Besides the well-documented expression of PD-L1 by cells in the TME, we detected significant levels of PD-L1 in the TDLN, mainly on macrophages and dendritic cells. Furthermore, surface PD-L1 expression doubled on these cells following adoptive transfer of inflammatory bone-marrow derived DCs. Injecting a near hundredfold lower dose of 2.5µg of anti-PD-L1 antibody i.pl. blocked PD-L1 in the TDLN and prevented translocation of the antibody to other sites. In the advanced disease setting, anti-PD-L1 monotherapy or adoptive DC-transfer only marginally improved survival (median survival of 24 days in untreated mice compared to 25 days for both monotherapies). A single i.pl. injection of low-dose antibody prior to DC-administration was as effective in prolonging survival as compared to repeated high-dose systemic injection of the antibody combined with adoptive DC-transfer (median survival of 35 and 35.5 days, respectively). When investigating the effects on antitumor immune responses, we found the increase in T-cell proliferation to be dependent on systemic anti-PD-L1 administration, whereas activation of T-cells indicated by CD69-positivity, was largely dependent on TDLN-localized PD-L1. Until now, dissecting the spatial roles of PD-L1 in immune regulation has proven difficult. By using a model allowing for separate dosing of PD-L1 blocking antibodies to different anatomic compartments, we identify PD-L1 to be of major importance in the TDLN. Our findings aid in the understanding of how antitumor immunity is regulated and provide a new perspective on biomarker and therapeutic target identification. Citation Format: Floris F. Dammeijer, Mandy van Gulijk, Melanie M. Lukkes, Menno van Nimwegen, Rudi W. Hendriks, Thorbald T. van Hall, Heleen H. Vroman, Joachim J.G.J.V. Aerts. Specifically targeting PD-L1 in the tumor-draining lymph node unmasks its spatiotemporal role in perturbing antitumor immunity and survival [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A164.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with ex vivo antigen-loaded DCs. Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care.