Stromal cells are important bone marrow (BM) niche components that regulate immune cell homeostasis through the production of cytokines such as interleukin 15 (IL-15). Although stromal-derived IL-15 is known to support lymphocyte survival, it remains unclear which stromal cell subsets are capable of IL-15 transpresentation, and how they influence specific lymphocyte populations. By using conditional IL-15 receptor alpha (IL-15Rα) deletion models, we demonstrate that IL-15Rα expression by BM stromal cells is essential for the maintenance of multiple IL-15-dependent lymphocyte populations. Deletion of IL-15Rα in Lepr+ or IL-7+ stromal cells selectively reduced central memory CD8+ T cells in the BM, whereas deletion of IL-15Rα in Osx+ stromal cells resulted in a marked loss of natural killer T (NKT) cells and tissue-resident memory CD8+ T cells. Surprisingly, endothelial-specific IL-15Rα deletion did not affect lymphocyte maintenance in the BM, but specifically impaired natural killer (NK) maturation and survival in the periphery, uncovering a role of endothelial IL-15 in mature NK cell maintenance. Together, our findings establish that transpresentation of IL-15 by distinct BM stromal cell subsets creates functionally specialized BM niches to support specific lymphocyte populations.

Systemic application of Toll-like receptor 7/8 (TLR7/8) agonists against cancer is severely limited due to uncontrolled immune activation. In this study, a platinum(IV)-based prodrug strategy is developed for the systemic administration of a TLR7/8 agonist, selectively activated in the malignant tissue simultaneously with the immunogenic cell death inducer oxaliplatin. Two oxaliplatin(IV)-based complexes are synthesized comprising the TLR7/8 agonist gardiquimod: Ox-Gardi-PEG, containing polyethylene glycol as the second axial ligand, and Ox-Gardi-Mal, a maleimide-bearing derivative to exploit the tumor-targeting effects of serum albumin. In vitro, cytotoxicity and immune pathway-inducing potency of Ox-Gardi-PEG and Ox-Gardi-Mal are diminished under standard cell culture conditions compared to free oxaliplatin and gardiquimod, respectively, and markedly enhanced under reducing conditions, underscoring the activation-by-reduction concept. In vivo, Ox-Gardi-Mal shows superior and TLR7/8 signaling-dependent anticancer efficacy and prolongs overall survival of cancer-bearing mice, while mitigating hematotoxic effects associated with oxaliplatin. Therapy significantly elevates expression of MHC-I on antigen-presenting immune cell subsets, increases the frequency of activated plasmacytoid dendritic cells and tumor-infiltrating CD8+ T cells, as well as depleted primarily immunosuppressive M2 macrophages. These results demonstrate that tumor-targeted oxaliplatin(IV)-based prodrugs carrying TLR7/8 agonists offer a potent dual-release strategy for improved immunochemotherapy, while minimizing excessive immune responses associated with systemic TLR7/8 activation.

Inverse correlation between metastatic load and immune infiltration

Number of resident and migratory cDCs is unchanged in the tumor-draining lymph nodes of metastases-bearing TYK2-/- host mice

Immunophenotyping of established metastases by flow cytometry in TYK2-/- and TYK2Δhem host mice

Basophils develop in the bone marrow and are associated with allergic inflammation but also contribute to protective immune responses against pathogens. Upon activation, they produce large amounts of cytokines, including interleukin (IL)-4 and IL-13. However, their physiological functions during homeostasis remain poorly defined. Here, using single-cell RNA sequencing and cytokine knock-in reporter mice, we identify basophils as the immune cell population with the highest IL-15 expression among immature and mature bone marrow leukocytes. Conditional deletion of IL-15 in basophils using Mcpt8-Cre mice results in a decrease of central-memory and CD69+ tissue-resident memory CD8+ T cells during steady-state conditions. In contrast, the homeostatic maintenance of other IL-15-dependent immune cell lineages, such as NK and NKT cells, was unaffected. Together, these findings identify basophils as a key source of IL-15 in the bone marrow that selectively supports memory CD8+ T cell maintenance.

Colorectal cancer liver metastasis (CRLM) is a major clinical problem. The regulators of immunosurveillance of CRLM could hold potential for developing therapeutic strategies to prevent or treat metastasis. In this study, using a murine colorectal cancer organoid-based transplantation model, we identified TYK2 as a key factor controlling CRLM. Evaluation of the effects of Tyk2 deletion in different subsets of immune cells and in colorectal cancer cells demonstrated that TYK2 was not required in cancer cells, macrophages, NK cells, T cells, or Kupffer cells. Instead, TYK2 controlled CRLM via a dendritic cell-dependent mechanism that relied on MHC-I-mediated cross-presentation of antigens to CD8+ T cells. Analysis of single-cell RNA sequencing data from primary colorectal cancer and CRLM revealed that TYK2 was predominantly expressed in a dendritic cell population destined to present antigens in tumor-draining lymph nodes. Treatment with the TYK2 inhibitor deucravacitinib, which is approved by the FDA for treating plaque psoriasis and is under clinical investigation for other autoimmune diseases, promoted CRLM. Together, these data demonstrate that TYK2 controls CRLM immunosurveillance, which should be carefully considered when treating patients with TYK2 inhibitors.Significance: TYK2 restricts the metastasis of colorectal tumors to the liver by supporting dendritic cell-dependent induction of antitumor CD8+ T cells, which could impact the use of TYK2 inhibitors in patients.

mRNA expression of cytokines, chemokines, costimulatory molecules and immune checkpoints in healthy and metastases-bearing livers of TYK2flox/flox and TYK2Δhem host mice

Altered immunophenotype of micrometastatic lesions in TYK2Δhem host mice

Expression of TYK2 in human CRC-derived LAMP3+ CCR7+ cDCs

Tyk2 deletion in AKP organoids has no effect on CRLM

TYK2 deficiency in host mice promotes liver metastasis of AKP organoids seeded via the portal vein

Evaluation of TYK2 deletion in conditional mouse models

CD8+ T cell differentiation has been associated with changes in the expression of long noncoding RNAs (lncRNAs). Yet, which and how lncRNAs regulate CD8+ T cell responses following infection in vivo remains incompletely understood. We performed deep RNA-seq to map the lncRNA expression landscape of CD8+ T cell subsets during infection and generated lncRNA knockout mouse models to evaluate the in vivo relevance of six lncRNAs. We identified Rroid2 to regulate effector CD8+ T cell function and effector-to-memory differentiation. Rroid2-deficient mice displayed increased CD44dim Foxp3+ regulatory T cells while the development of other immune cells, such as natural killer cells, was not affected. In CD8+ T cells, Rroid2 deficiency resulted in a fine-tuned downregulation of transcription factors Id2 and T-bet and impaired KLRG1+ and KLRG1- effector CD8+ T cell proliferation and cytotoxicity as well as effector-to-memory CD8+ T cell differentiation. The human orthologue of Rroid2, LINC01814, is also upstream of the transcriptional regulator ID2 and is highly expressed in human memory CD8+ T cells. Taken together, Rroid2 represents a key regulatory layer that controls CD8+ T cell differentiation.

Bone marrow (BM) mesenchymal stromal cells (MSC) provide microenvironmental niches that support hematopoietic stem cells and regulate hematopoiesis. Whether functional heterogeneity among BM MSCs contributes to the development and survival of distinct immune cell lineages remains incompletely understood. Here, we use an Il15 knockin reporter and multiple conditional deletion mouse models to show distinct differences in IL-15 expression between BM MSC subtypes. Conditional deletion of Il15 in Osx+ stromal cells results in decreased natural killer (NK) cell precursors, memory CD8+ T cells and NKT cells but not mature NK cells. Lepr+ stromal cells support the survival of mature NK cells and memory CD8+ T cells in the BM of older mice, while endothelial cells support mature NK cells and memory CD8+ T cells in the blood but not in the BM. Thus, our data suggest that MSC subtypes differentially regulate the development and survival of IL-15-dependent immune cell lineages in the BM.

Humanized mice are valuable preclinical models for immuno-oncology research because they allow modeling of human immune cells and human tumors in vivo. Myeloid cells are highly abundant in many tumors and have been associated with tumor progression, metastasis, and therapy resistance. Next-generation humanized mice have been generated to improve the development, diversity, and function of human myeloid cells. In this study, we analyzed human immune cell development and myeloid cell composition in NSG-Quad and MISTRG-6 mice. NSG-Quad mice supported the development of tissue-resident and tumor-infiltrating human macrophages at levels almost comparable to those of MISTRG-6 mice. However, the development of human CD4+ and CD8+ T cells was impaired in the blood and spleen but not in the tumor of NSG-Quad mice. In a subset of NSG-Quad mice, human monocytes exhibited increased cellular granularity and elevated expression of activation and checkpoint molecules, consistent with a monocyte hyperactivation syndrome. Our study provides a comprehensive comparative analysis of the frequency and characteristics of circulating, tissue-resident, and tumor-infiltrating myeloid cell populations in NSG-Quad and MISTRG-6 mice, which is key to accurately design and interpret human tumor xenograft studies, particularly with regard to faithful reconstruction of the human tumor-immune microenvironment and preclinical testing.

Tumor-associated stromal (TAS) cells within the tumor microenvironment (TME) exhibit marked transcriptional heterogeneity. However, the spatial organization of TAS subpopulations and their clinical relevance in human colorectal cancer (CRC) are incompletely understood. Using single-cell RNA sequencing (scRNA-seq) and multiplex imaging, we identified multiple TAS subpopulations with distinct intratumoral and peritumoral abundance, and found that CRC patients enriched in FAP+ TAS cells and CD73+ tumor cells had the lowest disease-free survival (DFS). NGFR+ stromal cells localized predominantly within tertiary lymphoid structures (TLS), which could be further classified into NGFR+ organized TLS, NGFR+ unorganized TLS and NGFR- TLS based on the spatial distribution of NGFR+ stromal cells. CRC patients enriched for NGFR+ organized TLS exhibited improved DFS. SCENIC analysis revealed HAND2, IKZF2 and SOX10 as transcriptional regulators of human NGFR+ antigen-presenting TAS cells, and they frequently expressed enteric glial cell markers SOX10, PLP1, CDH19 and NCAM1. In summary, our study demonstrates that the frequency and spatial distribution of TAS subpopulations, particularly NGFR+ TAS organization within TLS, varies between CRC patients and correlates with DFS and distinct changes in the TME.

BackgroundPatient-derived colorectal cancer (CRC) organoids (PDOs) solely consisting of malignant cells led to major advances in the understanding of cancer treatments. Yet, a major limitation is the absence of cells from the tumor microenvironment, thereby prohibiting potential investigation of treatment responses on immune and structural cells. Currently there are sparse reports describing the interaction of PDOs, cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in complex primary co-culture assay systems.MethodsPrimary PDOs and patient matched CAF cultures were generated from surgical resections. Co-culture systems of PDOs, CAFs and monocytic myeloid cells were set up to recapitulate features seen in patient tumors. Single-cell transcriptomics and flow cytometry was used to show effects of culture systems on TAM populations in the co-culture assays under chemotherapeutic and oncolytic viral treatment.ResultsIn contrast to co-cultures of tumor cells and monocytes, CAF/monocyte co-cultures and CAF/monocyte/tumor cell triple cultures resulted in a partial differentiation into macrophages and a phenotypic switch, characterized by the expression of major immunosuppressive markers comparable to TAMs in CRC. Oxaliplatin and 5-fluorouracil, the standard-of-care chemotherapy for CRC, induced polarization of macrophages to a pro-inflammatory phenotype comparable to the immunogenic effects of treatment with an oncolytic virus. Monitoring phagocytosis as a functional proxy to macrophage activation and subsequent onset of an immune response, revealed that chemotherapy-induced cell death, but not virus-mediated cell death, is necessary to induce phagocytosis of CRC cells. Moreover, CAFs enhanced the phagocytic activity in chemotherapy treated CRC triple cultures.ConclusionsPrimary CAF-containing triple cultures successfully model TAM-like phenotypesex vivoand allow the assessment of their functional and phenotypic changes in response to treatments following a precision medicine approach.

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl−/− mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl−/− tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment.