Cell communication within tissues is mediated by multiple paracrine signals including growth factors, which control cell survival and proliferation. Cells and the growth factors they produce and receive constitute a circuit with specific properties that ensure homeostasis. Here, we used computational and experimental approaches to characterize the features of cell circuits based on growth factor exchange between macrophages and fibroblasts, two cell types found in most mammalian tissues. We found that the macrophage-fibroblast cell circuit is stable and robust to perturbations. Analytical screening of all possible two-cell circuit topologies revealed the circuit features sufficient for stability, including environmental constraint and negative-feedback regulation. Moreover, we found that cell-cell contact is essential for the stability of the macrophage-fibroblast circuit. These findings illustrate principles of cell circuit design and provide a quantitative perspective on cell interactions.

Summary The differentiation of CD4+ helper T cell subsets with diverse effector functions is accompanied by changes in metabolism required to meet their bioenergetic demands. We find follicular B helper T (Tfh) cells exhibited less proliferation, glycolysis, and mitochondrial respiration, accompanied by reduced mTOR kinase activity compared to T helper 1 (Th1) cells in response to acute viral infection. IL-2-mediated activation of the Akt kinase and mTORc1 signaling was both necessary and sufficient to shift differentiation away from Tfh cells, instead promoting that of Th1 cells. These findings were not the result of generalized signaling attenuation in Tfh cells, as they retained the ability to flux calcium and activate NFAT transcription factor-dependent cytokine production. These data identify the interleukin-2 (IL-2)-mTORc1 axis as a critical orchestrator of the reciprocal balance between Tfh and Th1 cell fates and their respective metabolic activities following acute viral infection.

The maintenance of organismal homeostasis requires partitioning and transport of biochemical molecules between organ systems, their composite cells, and subcellular organelles. Although transcriptional programming undeniably defines the functional state of cells and tissues, underlying biochemical networks are intricately intertwined with transcriptional, translational, and post-translational regulation. Studies of the metabolic regulation of immunity have elegantly illustrated this phenomenon. The cells of the immune system interface with a diverse set of environmental conditions. Circulating immune cells perfuse peripheral organs in the blood and lymph, patrolling for pathogen invasion. Resident immune cells remain in tissues and play more newly appreciated roles in tissue homeostasis and immunity. Each of these cell populations interacts with unique and dynamic tissue environments, which vary greatly in biochemical composition. Furthermore, the effector response of immune cells to a diverse set of activating cues requires unique cellular adaptations to supply the requisite biochemical landscape. In this review, we examine the role of spatial partitioning of metabolic processes in immune function. We focus on studies of lymphocyte metabolism, with reference to the greater immunometabolism literature when appropriate to illustrate this concept.

Multiple myeloma is the most common indication for high-dose chemotherapy and autologous stem cell transplantation (ASCT), and lenalidomide maintenance post-transplant is now standard. Although lenalidomide doubles progression-free survival, almost all patients eventually relapse. Post-transplant immunotherapy to improve outcomes after ASCT therefore has great merit but first requires delineation of the dynamics of immune reconstitution. We evaluated lymphocyte composition and function after ASCT to guide optimal timing of immunotherapy and to identify potential markers of relapse. Regulatory T cells (Tregs) decline as CD8+ T cells expand during early lymphocyte recovery after ASCT, markedly reducing the Treg:CD8+ effector T-cell ratio. These CD8+ T cells can respond to autologous dendritic cells presenting tumor antigen in vitro as early as day +12 post-transplant, becoming antigen-specific cytolytic T-lymphocyte effectors and thereby demonstrating preservation of cellular reactivity. CD4+ and CD8+ T cells express the negative regulatory molecules, CTLA-4, PD-1, LAG-3, and TIM-3, before and after ASCT. A subpopulation of exhausted/senescent CD8+ T cells, however, down-regulates CD28 and up-regulates CD57 and PD-1, characterizing immune impairment and relapse after ASCT. Relapsing patients have higher numbers of these cells at +3 months after transplant, but before detection of clinical disease, indicating their applicability in identifying patients at higher risk of relapse. PD-1 blockade also revives the proliferation and cytokine secretion of the hyporesponsive, exhausted/senescent CD8+ T cells in vitro. Collectively, these results identify T cell exhaustion/senescence as a distinguishing feature of relapse and support early introduction of immunotherapy to stimulate antitumor immunity after ASCT.

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging their acquired effector function. Metabolic reprograming from the naïve-state is required throughout these phases of activation1. In CD4 T cells, T cell receptor (TCR) ligation, along with co-stimulatory and cytokine signals induce a glycolytic anabolic program required for biomass generation, rapid proliferation, and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodeling) and function are coordinately orchestrated by signal transduction and transcriptional remodeling; however, it remains unclear whether these processes are independently regulated by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support T helper 1 (Th1) cell differentiation and effector function, biochemically uncoupling these processes. We find that the TCA cycle is required for terminal Th1 cell effector function through succinate dehydrogenase (SDH; Complex II), yet the activity of SDH suppresses Th1 cell proliferation and histone acetylation. In contrast, we show that Complex I of the electron transport chain (ETC), the malate-aspartate shuttle, and citrate export from the mitochondria are required to maintain aspartate synthesis necessary for Th cell proliferation. Furthermore, we find that mitochondrial citrate export and malate-aspartate shuttle promote histone acetylation and specifically regulate the expression of a set of genes involved in T cell activation, proliferation, and metabolic programming. Combining genetic, pharmacological, and metabolomics approaches, we demonstrate that T helper cell differentiation and terminal effector function can be biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, citrate export, and Complex I supply the substrates needed for proliferation and epigenetic remodeling during early T cell activation, while Complex II consumes the substrates of these pathways, antagonizing differentiation and enforcing terminal effector function. Our data suggest that transcriptional programming works in concert with a parallel biochemical network to enforce cell state.

CD8+ tissue-resident memory T cells (TRM cells) are poised at the portals of infection and provide long-term protective immunity. Despite their critical roles, the precise mechanics governing TRM cell reactivation in situ are unknown. Using a TCR-transgenic Nur77-GFP reporter to distinguish “antigen-specific” from “bystander” reactivation, we demonstrate that lung CD8+ TRM cells are reactivated more quickly, yet less efficiently, than their counterparts in the draining LNs (TLN cells). Global profiling of reactivated memory T cells revealed tissue-defined and temporally regulated recall response programs. Unlike the reactivation of CD8+ TLN cells, which is strictly dependent on CD11c+XCR1+ APCs, numerous antigen-presenting partners, both hematopoietic and non-hematopoietic, were sufficient to reactivate lung CD8+ TRM cells, but the quality of TRM cell functional responses depended on the identity of the APCs. Together, this work uncovers fundamental differences in the activation kinetics, mechanics, and effector responses between CD8+ memory T cells in peripheral vs. lymphoid organs, revealing a novel tissue-specific paradigm for the reactivation of memory CD8+ T cells.

CD4 + effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff actually adopt in frontline tissues in vivo , we applied single-cell transcriptome and chromatin analysis on colonic Teff cells, in germ-free or conventional mice, or after challenge with a range of phenotypically biasing microbes. Subsets were marked by expression of interferon-signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic T H subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T H markers distributed in a polarized continuum, which was also functionally validated. Clones derived from single progenitors gave rise to both IFN-γ and IL17-producing cells. Most transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activity of AP1 and IRF transcription factor families, not the canonical subset master regulators T-bet, GATA3, RORγ.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1–3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβ receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15–diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFβ-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.