Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of... Show moreBackground: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of cancer immunotherapies, a comprehensive understanding of local and systemic immune profiles in patients with PDAC is required. Here, our goal was to decipher the interplay between local and systemic immune profiles in treatment-naive patients with PDAC. Methods: The immune composition of PDAC, matched non-malignant pancreatic tissue, regional lymph nodes, spleen, portal vein blood, and peripheral blood samples (collected before and after surgery) from 11 patients with PDAC was assessed by measuring 41 immune cell markers by single-cell mass cytometry. Furthermore, the activation potential of tumor-infiltrating lymphocytes as determined by their ability to produce cytokines was investigated by flow cytometry. In addition, the spatial localization of tumor-infiltrating innate lymphocytes in the tumor microenvironment was confirmed by multispectral immunofluorescence. Results: We found that CD103(+)CD8(+) T cells with cytotoxic potential are infrequent in the PDAC immune microenvironment and lack the expression of activation markers and checkpoint blockade molecule programmed cell death protein-1 (PD-1). In contrast, PDAC tissues showed a remarkable increased relative frequency of B cells and regulatory T cells as compared with non-malignant pancreatic tissues. Besides, a previously unappreciated innate lymphocyte cell (ILC) population (CD127(-)CD103(+)CD39(+)CD45RO(+) ILC1-like) was discovered in PDAC tissues. Strikingly, the increased relative frequency of B cells and regulatory T cells in pancreatic cancer samples was reflected in matched portal vein blood samples but not in peripheral blood, suggesting a regional enrichment of immune cells that infiltrate the PDAC microenvironment. After surgery, decreased frequencies of myeloid dendritic cells were found in peripheral blood. Conclusions: Our work demonstrates an immunosuppressive landscape in PDAC tissues, generally deprived of cytotoxic T cells and enriched in regulatory T cells and B cells. The antitumor potential of ILC1-like cells in PDAC may be exploited in a therapeutic setting. Importantly, immune profiles detected in blood isolated from the portal vein reflected the immune cell composition of the PDAC microenvironment, suggesting that this anatomical location could be a source of tumor-associated immune cell subsets. Show less
Duurland, C.L.; Santegoets, S.J.; Abdulrahman, Z.; Loof, N.M.; Sturm, G.; Wesselink, T.H.; ... ; Burg, S.H. van der 2022
Background Expression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed... Show moreBackground Expression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown. Methods We examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies. Results Central and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3 epsilon. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)beta 1. Conclusion High levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells. Show less
Groeneveldt, C.; Kinderman, P.; Wollenberg, D.J.M. van den; Oever, R.L. van den; Middelburg, J.; Mustafa, D.A.M.; ... ; Montfoort, N. van 2020
Background T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological... Show moreBackground T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. Methods The mutantp53andK-rasinduced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)(+)breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. Results Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8(+)T cells, at the cost of FoxP3(+)Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8(+)T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. Conclusions Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors. Show less
Berg, J.H. van den; Heemskerk, B.; Rooij, N. van; Gomez-Eerland, R.; Michels, S.; Zon, M. van; ... ; Haanen, J.B.A.G. 2020
Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50... Show moreTreatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. Purpose Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. Experimental Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. Results Five out of 10 patients, who were all anti-PD-1 naive at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. Conclusion The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial. Show less
Santegoets, S.J.; Duurland, C.L.; Jordanova, E.J.; Ham, V.J. van; Ehsan, I.; Loof, N.M.; ... ; Burg, S.H. van der 2020
Background Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative... Show moreBackground Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking. Methods We analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16(+)patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses. Results We show that the tumor microenvironment of HPV16(+)OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163(+)cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163(+)cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFN gamma and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163(+)cDC2 positively correlated with the infiltration by Tbet(+)and tumor-specific T cells, and with prolonged survival. Conclusions These data suggest an important role for intratumoral CD163(+)cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects. Show less
Ruben, J.M.; Garcia-Romo, G.S.; Breman, E.; Kooij, S. van der; Redeker, A.; Arens, R.; Kooten, C. van 2018
