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Senescent cells (SnCs) are implicated in the pathogenesis of age-related diseases including osteoarthritis (OA), in part via expression of a senescence-associated secretory phenotype (SASP) that includes immunologically relevant factors and cytokines. In a model of posttraumatic OA (PTOA), anterior cruciate ligament transection (ACLT) induced a type 17 immune response in the articular compartment and draining inguinal lymph nodes (LNs) that paralleled expression of the senescence marker p16INK4a (Cdkn2a) and p21 (Cdkn1a). Innate lymphoid cells, γδ+ T cells, and CD4+ T cells contributed to IL-17 expression. Intra-articular injection of IL-17-neutralizing antibody reduced joint degeneration and decreased expression of the senescence marker Cdkn1a. Local and systemic senolysis was required to attenuate tissue damage in aged animals and was associated with decreased IL-17 and increased IL-4 expression in the articular joint and draining LNs. In vitro, we found that Th17 cells induced senescence in fibroblasts and that SnCs skewed naive T cells toward Th17 or Th1, depending on the presence of TGF-β. The SASP profile of the inflammation-induced SnCs included altered Wnt signaling, tissue remodeling, and cell-cycle pathways not previously implicated in senescence. These findings provide molecular targets and mechanisms for senescence induction and therapeutic strategies to support tissue healing in an aged environment.

Impressive responses have been observed in patients treated with checkpoint inhibitory anti–programmed cell death-1 (PD-1) or anti–cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti–PD-1 and anti–CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.

Myeloid cells comprise a major component of the tumor microenvironment (TME) that promotes tumor growth and immune evasion. By employing a small-molecule inhibitor of glutamine metabolism, not only were we able to inhibit tumor growth, but we markedly inhibited the generation and recruitment of myeloid-derived suppressor cells (MDSCs). Targeting tumor glutamine metabolism led to a decrease in CSF3 and hence recruitment of MDSCs as well as immunogenic cell death, leading to an increase in inflammatory tumor-associated macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSCs themselves led to activation-induced cell death and conversion of MDSCs to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid-derived cells, leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced antitumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.

The metabolic characteristics of tumors present considerable hurdles to immune cell function and cancer immunotherapy. Using a glutamine antagonist, we metabolically dismantled the immunosuppressive microenvironment of tumors. We demonstrate that glutamine blockade in tumor-bearing mice suppresses oxidative and glycolytic metabolism of cancer cells, leading to decreased hypoxia, acidosis, and nutrient depletion. By contrast, effector T cells responded to glutamine antagonism by markedly up-regulating oxidative metabolism and adopting a long-lived, highly activated phenotype. These divergent changes in cellular metabolism and programming form the basis for potent antitumor responses. Glutamine antagonism therefore exposes a previously undefined difference in metabolic plasticity between cancer cells and effector T cells that can be exploited as a “metabolic checkpoint” for tumor immunotherapy.

Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). In this study, we characterized CD14+HLA-DR(-/lo) cells sorted from the tumors, draining lymph nodes, and peripheral blood of HNSCC patients. CD14+HLA-DR(-/lo) cells were phenotyped as CD11b+, CD33+, CD34+, arginase-I+, and ROS+. In all 3 compartments, they suppressed autologous, antigen-independent T cell proliferation in a differential manner. The abundance of MDSC correlated with stage, but did not correlate with previous treatment with radiation or subsites of HNSCC. Interestingly, MDSC from all 3 compartments showed high phosphorylated STAT3 levels that correlated with arginase-I expression levels and activity. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated MDSC’s suppressive function. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity. Analysis of the human arginase-I promoter region showed multiple STAT3-binding elements, and ChIP demonstrated that phosphorylated STAT3 binds to multiple sites in the arginase-I promoter. Finally, rescue of arginase-I activity after STAT3 blockade restored MDSC’s suppressive function. Taken together, these results demonstrate that the suppressive function of arginase-I in both infiltrating and circulating MDSC is a downstream target of activated STAT3.

Immune-mediated tissue regeneration driven by a biomaterial scaffold is emerging as an innovative regenerative strategy to repair damaged tissues. We investigated how biomaterial scaffolds shape the immune microenvironment in traumatic muscle wounds to improve tissue regeneration. The scaffolds induced a pro-regenerative response, characterized by an mTOR/Rictor-dependent T helper 2 pathway that guides interleukin-4–dependent macrophage polarization, which is critical for functional muscle recovery. Manipulating the adaptive immune system using biomaterials engineering may support the development of therapies that promote both systemic and local pro-regenerative immune responses, ultimately stimulating tissue repair.

Asymmetric division can generate effector and memory CD8 + T cell precursors. Powell and colleagues show asymmetric partitioning of mTORC1 activity upon CD8 + T cell division, which results in distinct metabolic programming of daughter T cells. The asymmetric partitioning of fate-determining proteins has been shown to contribute to the generation of CD8 + effector and memory T cell precursors. Here we demonstrate the asymmetric partitioning of mTORC1 activity after the activation of naive CD8 + T cells. This results in the generation of two daughter T cells, one of which shows increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell has relatively low mTORC1 activity and increased lipid metabolism, expresses increased amounts of anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between T cell antigen receptor (TCR)-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells.

Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios(+) and Helios(-) Treg, we profiled cell-surface markers of FoxP3(+) Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios(+) Treg and that a Helios(+) Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios(+) Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios(+) cells in culture. Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.

Polysaccharide A (PSA), an immunogenic capsular component of non-toxigenic Bacteroides fragilis (NTBF) strain NCTC 9343, is reported to promote mucosal immune development and suppress colitis. Contrastingly, enterotoxigenic Bacteroides fragilis (ETBF) is highly associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), rapidly inducing IL-17-dependent murine colitis and tumorigenesis. In specific-pathogen-free (SPF) C57BL/6 wild-type (WT) and multiple intestinal neoplasia (MinApc716+/−) mice, we show that sequential treatment of the NTBF strain, 9343, followed by the ETBF strain, 86-5443-2-2 (86), diminished colitis and tumorigenesis. Mice treated simultaneously with 9343 and 86 exhibited both severe colitis and tumorigenesis. Abrogated disease severity in sequentially treated mice was attributed to 9343 strain dominance and decreased IL-17A, but 86 colonization prior to or simultaneous with 9343 mitigated the anti-inflammatory effect of 9343. Remarkably, 9343-mediated protection was independent of PSA, as sequentially treated mice receiving ΔPSA 9343 exhibited similar protection. Further, SPF WT and Min mice colonized with PSA-competent or PSA-deficient 9343 exhibited similar IL-10, IL-17, and IFN-γ responses. Treatment of 86-colonized mice with 9343 failed to disrupt 86 pathogenesis. Our findings demonstrate that 9343 colonization, independent of PSA, offers prophylaxis against colitis-inducing 86 but may not be a valid therapy once colitis is established.

BackgroundCurrent therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy.Methods66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung ‘PM interface’ and ‘PM interior’ regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection.ResultsIHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS.ConclusionsOsteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy.