BackgroundPrimary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of... Show moreBackgroundPrimary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome.MethodHere, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeutic in vivo settings allowed for the identification of immunological factors driving immunotherapy resistance.ResultsComparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163(hi) macrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages. In vivo studies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163(hi) macrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy.ConclusionsIn this study, a small population of CD163(hi) tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163(hi) M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance. Show less
Regulatory T cells (T-regs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. T-regs are characterized by a high expression of CD25, which is a... Show moreRegulatory T cells (T-regs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. T-regs are characterized by a high expression of CD25, which is a potentially valuable target for T-reg depletion to alleviate immune suppression. The preclinical anti-CD25 (alpha CD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear T-regs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral T-reg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant T-reg-depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting alpha CD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident T-regs, leading to a high effector T cell (T-eff) to T-reg ratio. Moreover, a combination of alpha CD25-m2a and an alpha PD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for alpha CD25 as a cancer immunotherapy. Show less
Borst, L.; Sluijter, M.; Sturm, G.; Charoentong, P.; Santegoets, S.J.; Gulijk, M.; ... ; Hall, T. van 2021
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... Show moreThe 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. Naive 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-beta in vitro, although TGF-beta 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. Show less
Nejad, E.B.; Labrie, C.; Elsas, M.J. van; Kleinovink, J.W.; Mittrucker, H.W.; Franken, K.L.M.C.; ... ; Burg, S.H. van der 2021
Background High serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but... Show moreBackground High serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective. Methods IL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1.control. Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6ra(fl/fl)xLysM(cre+) mice. Results Our therapeutic vaccination protocol elicits a strong tumor-specific CD8(+) T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8(+) T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6ra(fl/fl)xLysM(cre+) but not cre-negative control mice, while no differences in the vaccine-induced CD8(+) T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages. Conclusion IL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections. Show less
Elsas, M.J. van; Hall, T. van; Burg, S.H. van der 2020
Cancer immunotherapies, including checkpoint inhibitors, adoptive T cell transfer and therapeutic cancer vaccines, have shown promising response rates in clinical trials. Unfortunately, there is an... Show moreCancer immunotherapies, including checkpoint inhibitors, adoptive T cell transfer and therapeutic cancer vaccines, have shown promising response rates in clinical trials. Unfortunately, there is an increasing number of patients in which initially regressing tumors start to regrow due to an immunotherapy-driven acquired resistance. Studies on the underlying mechanisms reveal that these can be similar to well-known tumor intrinsic and extrinsic primary resistance factors that precluded the majority of patients from responding to immunotherapy in the first place. Here, we discuss primary and secondary immune resistance and point at strategies to identify potential new mechanisms of immune evasion. Ultimately, this may lead to improved immunotherapy strategies with improved clinical outcomes. 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
