Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high... Show moreImmune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional sin-gle-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict re-sponses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4+ and CD8+ T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8+ T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for ther-apy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy. Show less
Pardieck, I.N.; Sluis, T.C. van der; Gracht, E.T.I. van der; Veerkamp, D.M.B.; Behr, F.M.; Duikeren, S. van; ... ; Arens, R. 2022
Vaccination regimens and the number of doses required for optimal immunity and protection are critical factors in the translation of vaccines. Here the authors show administration of a three dose... Show moreVaccination regimens and the number of doses required for optimal immunity and protection are critical factors in the translation of vaccines. Here the authors show administration of a three dose protocol of a single T cell epitope to the SARS-CoV-2 spike protein induces a robust CD8(+) T cell response and confers protection in a lethal murine challenge model of infection.Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8(+) T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8(+) T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8(+) effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8(+) T cell response leading to protection against otherwise lethal SARS-CoV-2 infection. Show less
Nejad, E.B.; Labrie, C.; Sluis, T.C. van der; Duikeren, S. van; Franken, K.L.M.C.; Roosenhoff, R.; ... ; Burg, S.H. van der 2020
High serum levels of interleukin-6 (IL-6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely... Show moreHigh serum levels of interleukin-6 (IL-6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)-associated tumor model expressing IL-6 to investigate the impact of tumor-expressed IL-6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor-produced IL-6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor-produced IL-6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T-cell-mediated killing. Furthermore, no overt differences in the frequencies of tumor-infiltrating subsets of T cells or CD11b(+)myeloid cells were observed. IL-6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II(+)myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL-6-mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL-6-mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer. Show less
Nejad, E.B.; Labrie, C.; Abdulrahman, Z.; Elsas, M.J. van; Rademaker, E.; Kleinovink, J.W.; ... ; Burg, S.H. van der 2020
Background Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the... Show moreBackground Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments. Methods We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients. Results Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-alpha and transforming growth factor-beta signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naive hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8(+)T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients. Conclusion An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities. Show less
Zom, G.G.; Willems, M.M.J.H.P.; Khan, S.; Sluis, T.C. van der; Kleinovink, J.W.; Camps, M.G.M.; ... ; Ossendorp, F.A. 2018
Ligands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of... Show moreLigands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV.\nNaïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur.\nSLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy.\nThese data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant.\nBACKGROUND\nMETHODS\nRESULTS\nCONCLUSION Show less
Zom, G.G.; Willems, M.M.J.H.P.; Khan, S.; Sluis, T.C. van der; Kleinovink, J.W.; Camps, M.G.M.; ... ; Ossendorp, F. 2018
We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated... Show moreWe aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis.Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage in flux, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis. Show less
Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear... Show more Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear that when different cancer therapies are combined they can potentially synergize. Understanding how individual cancer therapeutics work enables the development of the most optimal combinations of cancer therapies. In this thesis, we have used interesting combinations of chemotherapy, cancer vaccines and immune checkpoint blockade and obtained insights in the mechanisms involved in these combinations. For example, we combined chemotherapy with vaccination and observed that none of the tested chemotherapeutics had a negative effect on vaccination, while only a few displayed synergy with vaccination in tumor eradication. We found that vaccine-induced T cells produce Tumor Necrosis Factor-α which could sensitize tumor cells for chemotherapy-induced cell death. Furthermore, we have shown that different cancer therapies modulate intratumoral myeloid cells in a unique manner which can critically affect the therapeutic efficacy of the therapy. Understanding how different therapies affect the local tumor environment will help to improve clinical responses and decrease toxicity in patients. Show less
Welters, M.J.; Sluis, T.C. van der; Meir, H. van; Loof, N.M.; Ham, V.J. van; Duikeren, S. van; ... ; Burg, S.H. van der 2016
