Lukas Rindlisbacher’s research while affiliated with University of Zurich and other places

Group 1 innate lymphoid cells (ILCs) encompass NK cells and ILC1s, which have non-redundant roles in host protection against pathogens and cancer. Despite their circulating nature, NK cells can establish residency in selected tissues during ontogeny, forming a distinct functional subset. The mechanisms that initiate, maintain, and regulate the conversion of NK cells into tissue-resident NK (trNK) cells are currently not well understood. Here, we identify autocrine transforming growth factor-β (TGF-β) as a cell-autonomous driver for NK cell tissue residency across multiple glandular tissues during development. Cell-intrinsic production of TGF-β was continuously required for the maintenance of trNK cells and synergized with Hobit to enhance cytotoxic function. Whereas autocrine TGF-β was redundant in tumors, our study revealed that NK cell–derived TGF-β allowed the expansion of cytotoxic trNK cells during local infection with murine cytomegalovirus (MCMV) and contributed to viral control in the salivary gland. Collectively, our findings reveal tissue-specific regulation of trNK cell differentiation and function by autocrine TGF-β1, which is relevant for antiviral immunity.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting TIM-3 for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab-treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment-resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ T cells (Tc) enhanced Tc activation, proliferation and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3-treatment-mediated GVL effects are Tc-induced. In contrast to anti-PD-1 and anti-CTLA-4-treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host-disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We deciphered the connection between oncogenic mutations found in AML and TIM-3 ligands expression and identify anti-TIM-3-treatment as a strategy to enhance GVL effects via metabolic and transcriptional Tc-reprogramming, without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Abs in patients with AML relapse post-allo-HCT.

Background In multiple sclerosis (MS), B cells are considered main triggers of the disease, likely as the result of complex interaction between genetic and environmental risk factors. Studies on monozygotic twins discordant for MS offer a unique way to reduce this complexity and reveal discrepant subsets. Methods In this study, we analyzed B cell subsets in blood samples of monozygotic twins with and without MS using publicly available data. We verified functional characteristics by exploring the role of therapy and performed separate analyses in unrelated individuals. Findings The frequencies of CXCR3⁺ memory B cells were reduced in the blood of genetically identical twins with MS compared to their unaffected twin siblings. Natalizumab (anti-VLA-4 antibody) was the only treatment regimen under which these frequencies were reversed. The CNS-homing features of CXCR3⁺ memory B cells were supported by elevated CXCL10 levels in MS cerebrospinal fluid and their in vitro propensity to develop into antibody-secreting cells. Conclusions Circulating CXCR3⁺ memory B cells are affected by non-heritable cues in people who develop MS. This underlines the requirement of environmental risk factors such as Epstein-Barr virus in triggering these B cells. We propose that after CXCL10-mediated entry into the CNS, CXCR3⁺ memory B cells mature into antibody-secreting cells to drive MS. Funding This work was supported by Nationaal MS Fonds (OZ2021-016), Stichting MS Research (19-1057 MS, 20-490f MS, and 21-1142 MS), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program grant agreement no. 882424, and the Swiss National Science Foundation (733 310030_170320, 310030_188450, and CRSII5_183478).

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

Introduction: The inhibition of Bruton's tyrosine kinase (BTK) as a therapeutic strategy has dramatically improved the management of patients with chronic lymphocytic leukemia (CLL) and other B-cell-Non Hodgkin lymphomas (B-NHL). However, therapy resistance and treatment toxicity persist as significant clinical challenges. It has been previously reported that BTK inhibition not only targets the overactivated BCR pathway in CLL and other B-NHL, but also coincides with profound alterations in the tumor microenvironment including immune cells. Therefore, it is vital to understand these tumor-immune cell interactions during BTK inhibition to enhance response and reduce toxicity, advancing personalized care for CLL and B-NHL patients in the future. Methods: We developed a high-parametric analysis pipeline based on full spectrum cytometry, a novel technology allowing for the simultaneous assessment of 40 markers at single-cell level. In order to comprehensively assess the phosphoproteome relevant to BTK inhibition we established a signaling panel covering >30 proteins including pBTK, pSyk, pPLCg, pS6 and pAMPKa. Additionally, we designed 3 panels with up to 40 markers characterizing T cell activation and exhaustion, cellular trafficking, inhibitory immune checkpoints and cytokine production. Using our scalable and reproducible bioinformatics pipeline, we analyzed a cohort of 160 samples, longitudinally collected from 57 patients (CLL, MCL, MZL) before BTKi treatment, directly after treatment initiation, at leukocyte peak count and during therapy up to 6 months after therapy initiation. Results: Given that T cells are a crucial arm in governing anti-lymphoma immune response, we sought to elucidate the changes associated with BTK inhibition in the T cell compartment of the patients at high resolution (figure A). We observed certain changes in the fraction of T cell subtypes, notably a decrease in central memory stem T cells and an increase in γδ T cells at leukocyte peak and during later stages of therapy. Our analysis uncovered substantial changes in the T cell marker expression over the course of BTK inhibition. This includes a reversal of exhausted phenotype marked by downregulation of PD1, CD39 and CD38 and a skewed differentiation of CD4 T cells towards Th1 phenotype illustrated by a pronounced upregulation of the transcription factor T-bet. In addition, we detected restored proliferative and cytotoxic capacity in the CD8 and CD4 T cell compartments characterized by upregulation of GRZB, CD226, CD28, CD25 and Ki67. By leveraging our platform, we successfully captured the signalosome of malignant B cells at a high resolution. As anticipated, we detected significant heterogeneity in the malignant B cell signatures across individual patients. Interestingly, even at the initial sampling timepoint following treatment start, we observed substantially altered B cell signatures encompassing both the phosphoproteome and inhibitory immune checkpoints (figure B). Our preliminary data suggest that the dynamic changes induced by BTK inhibition extend beyond the signaling nodes associated with the BCR pathway and also involve the NFkB and p53 pathways. Conclusion: In this study, we established a platform for deep and longitudinal interrogation of the tumor and immune cell compartments to capture the dynamic and patient-specific changes occurring during BTK inhibition. Our preliminary data obtained from a large patient cohort of 57 B-NHL patients reveals a profound remodeling of not only the BCR signaling pathway in tumor cells, but also of the T cell compartment. Our data will be useful for a better understanding of treatment response and toxicity in the context of BTK inhibition.

Introduction: Resistance to therapy remains a major challenge for Mantle Cell Lymphoma (MCL) patients, despite a diverse and expanding treatment landscape. There is thus a major need to better understand the mechanistic underpinnings of therapy resistance in order to optimize treatment selection. Given the advent of immunotherapies in recent years, there has been increasing evidence that not only tumor-intrinsic factors affect therapy resistance, but that tumor-immune cell interactions are an additional major contributor. In the context of MCL, such interactions have been poorly characterized, but hold the promise of identifying novel treatment approaches and better understanding resistance mechanisms to commonly applied therapies such as BTK inhibition. Methods: This study aimed to investigate the single-cell landscape of leukemic MCL using single-cell profiling of 26 patients and 10 healthy donors. In addition, we performed in vitro drug testing on a subset of patient samples (n=12; 6 treatments) to understand the effects of drugs on both tumor and immune cells in MCL . To comprehensively characterize the phenotypic and functional features of immune and tumor cells within a single-cell landscape, we employed high-dimensional full-spectrum cytometry. Therefore, we developed 5 high-dimensional panels covering markers to deeply interrogate T and NK cells, immune evasion and immune checkpoints, cytokines and cytotoxicity, cellular trafficking and intracellular signaling/phosphoproteome (covering >120 proteins). We combined this with single-cell RNA sequencing and a scalable and reproducible bioinformatics pipeline. To assess the disease specificity of the immune landscape, we also obtained single-cell data from other B-NHL including MZL and CLL (n=60 patients). Results: Our data indicate profound changes in the immune landscape, in particular in the T cell compartment of MCL patients. In this regard, multi-omics factor analysis (MOFA) confirmed a highly distinct T cell landscape in MCL patients, characterized by higher effector marker expression in conventional CD4+ and CD8+ T cells in addition to higher fraction of effector and effector memory T cells compared to healthy donors (Fig. 1A and B). Moreover, our analysis revealed a pronounced expansion of highly suppressive regulatory T cells (~5% of T cells) (Fig.1B). The emergence of this effector Treg (eTreg) subset in MCL patients might limit efficient anti-tumor immunity of CD8+ and conventional CD4+ T cells. Consistent with this, we observed a high fraction of several dysfunctional CD8+ T cells subsets. Notably, we found a remodeling of this eTreg subset in patients undergoing ibrutinib treatment leading to a less pronounced suppressive phenotype and concomitantly observed an increase of proteins crucial for cytotoxic function such as Granzyme B and Interferon-γ in effector T cells. We further sought to link tumor cell signatures with the healthy immune cell signatures in order to assess potential interactions and mechanisms of immune escape. As such, the tumor cell compartment (MCL cells) displayed high-expression of inhibitory immune checkpoint ligands such as PVR, HLA-G, CD70 and PD-L1 and antiphagocytic proteins such as CD47 and CD24, which may contribute to the dysfunctional T cell compartment. In line with this, our analysis revealed that high expression of the immune checkpoint ligand CD70 on malignant B cells was inversely associated with an activation module in CD4+ T cells, providing a potential axis contributing to immune escape, which may be leveraged for future therapeutic use. Finally, we identified tumor and immune cell signatures associated with in vitro drug response that may not only be useful for improving treatment selection, but also offer novel biological insights into potential mechanisms of treatment resistance. Conclusion: This study provides valuable insights into the single-cell landscape of leukemic MCL, offering crucial understanding of how tumor-immune cell interactions may contribute to disease progression and treatment resistance. The findings hold potential for therapeutic exploitation to eventually improve patient outcomes in Mantle Cell Lymphoma.

Metabolic-associated fatty liver disease (MAFLD) is a spectrum of clinical manifestations ranging from benign steatosis to cirrhosis. A key event in the pathophysiology of MAFLD is the development of nonalcoholic steatohepatitis (NASH), which can potentially lead to fibrosis and hepatocellular carcinoma, but the triggers of MAFLD-associated inflammation are not well understood. We have observed that lipid accumulation in hepatocytes induces expression of ligands specific to the activating immune receptor NKG2D. Tissue-resident innate-like T cells, most notably γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A licenses hepatocytes to produce chemokines that recruit proinflammatory cells into the liver, which causes NASH and fibrosis. NKG2D-deficient mice did not develop fibrosis in dietary models of NASH and had a decreased incidence of hepatic tumors. The frequency of IL-17A ⁺ γδ T cells in the blood of patients with MAFLD correlated directly with liver pathology. Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in the context of MAFLD.

Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192).

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. Implications MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.