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Supplementary Figures 1-8

Figure legends for Supplementary Figures 1-8

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

Rana et al. demonstrate that white adipose tissue-resident multipotent stromal cells (WAT-MSC) support IL-33– and LFA-1/ICAM-1–mediated proliferation and type-2 cytokine expression by ILC2. Inversely, ILC2-derived IL-4 and IL-13 promote WAT-MSC eotaxin production and eosinophil recruitment, further maintaining type-2 immune homeostasis., Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue–resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1–mediated proliferation and activation of LFA-1–expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT., Graphical Abstract

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

The microenvironment of lymphoid organs can aid healthy immune function through provision of both structural and molecular support. In mice, fibroblastic reticular cells (FRCs) create an essential T-cell support structure within lymph nodes, while human FRCs are largely unstudied. Here, we show that FRCs create a regulatory checkpoint in human peripheral T-cell activation through 4 mechanisms simultaneously utilised. Human tonsil and lymph node-derived FRCs constrained the proliferation of both naïve and pre-activated T cells, skewing their differentiation away from a central memory T-cell phenotype. FRCs acted unilaterally without requiring T-cell feedback, imposing suppression via indoleamine-2,3-dioxygenase, adenosine 2A Receptor, prostaglandin E2, and transforming growth factor beta receptor (TGFβR). Each mechanistic pathway was druggable, and a cocktail of inhibitors, targeting all 4 mechanisms, entirely reversed the suppressive effect of FRCs. T cells were not permanently anergised by FRCs, and studies using chimeric antigen receptor (CAR) T cells showed that immunotherapeutic T cells retained effector functions in the presence of FRCs. Since mice were not suitable as a proof-of-concept model, we instead developed a novel human tissue-based in situ assay. Human T cells stimulated using standard methods within fresh tonsil slices did not proliferate except in the presence of inhibitors described above. Collectively, we define a 4-part molecular mechanism by which FRCs regulate the T-cell response to strongly activating events in secondary lymphoid organs while permitting activated and CAR T cells to utilise effector functions. Our results define 4 feasible strategies, used alone or in combinations, to boost primary T-cell responses to infection or cancer by pharmacologically targeting FRCs.