Nicole E. Scharping

B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6.

Jackson et al. propose a role for B cell–intrinsic IFN-γ receptor signaling in spontaneous germinal center activation and autoantibody production.

Efficacy of PD-1 blockade is potentiated by metformin-induced reduction of tumor hypoxia

Low oxygen levels in tumors can act as a barrier to effective antitumor immunity. Mitigation of tumor hypoxia using a commonly prescribed type II diabetes drug, metformin, resulted in significant synergy with PD-1 blockade immunotherapy. Blockade of the coinhibitory checkpoint molecule PD-1 has emerged as an effective treatment for many cancers, resulting in remarkable responses. However, despite successes in the clinic, most patients do not respond to PD-1 blockade. Metabolic dysregulation is a common phenotype in cancer, but both patients and tumors are metabolically heterogeneous. We hypothesized that the deregulated oxidative energetics of tumor cells present a metabolic barrier to antitumor immunity through the generation of a hypoxic microenvironment and that normalization of tumor hypoxia might improve response to immunotherapy. We show that the murine tumor lines B16 and MC38 differed in their ability to consume oxygen and produce hypoxic environments, which correlated with their sensitivity to checkpoint blockade. Metformin, a broadly prescribed type II diabetes treatment, inhibited oxygen consumption in tumor cells in vitro and in vivo, resulting in reduced intratumoral hypoxia. Although metformin monotherapy had little therapeutic benefit in highly aggressive tumors, combination of metformin with PD-1 blockade resulted in improved intratumoral T-cell function and tumor clearance. Our data suggest tumor hypoxia acts as a barrier to immunotherapy and that remodeling the hypoxic tumor microenvironment has potential to convert patients resistant to immunotherapy into those that receive clinical benefit. Cancer Immunol Res; 5(1); 9–16. ©2016 AACR.

Tumor Microenvironment Metabolism: A New Checkpoint for Anti-Tumor Immunity.

When a T cell infiltrates a tumor, it is subjected to a variety of immunosuppressive and regulatory signals in the microenvironment. However, it is becoming increasingly clear that due to the proliferative and energetically-deregulated nature of tumor cells, T cells also operate at a metabolic disadvantage. The nutrient dearth of the tumor microenvironment (TME) creates “metabolic checkpoints” upon infiltrating T cells, impacting their ability to survive, proliferate and function effectively. In this review, we summarize the basics of tumor cell and T cell metabolism and discuss recent advances elucidating the individual metabolic checkpoints exerted on T cells that drive their dysfunction in the TME.

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells

Mice overexpressing B cell activating factor of the TNF family (BAFF) develop systemic autoimmunity characterized by class-switched anti-nuclear Abs. Transmembrane activator and CAML interactor (TACI) signals are critical for BAFF-mediated autoimmunity, but the B cell developmental subsets undergoing TACI-dependent activation in settings of excess BAFF remain unclear. We report that, although surface TACI expression is usually limited to mature B cells, excess BAFF promotes the expansion of TACI-expressing transitional B cells. TACI+ transitional cells from BAFF-transgenic mice are characterized by an activated, cycling phenotype, and the TACI+ cell subset is specifically enriched for autoreactivity, expresses activation-induced cytidine deaminase and T-bet, and exhibits evidence of somatic hypermutation. Consistent with a potential contribution to BAFF-mediated humoral autoimmunity, TACI+ transitional B cells from BAFF-transgenic mice spontaneously produce class-switched autoantibodies ex vivo. These combined findings highlight a novel mechanism through which BAFF promotes humoral autoimmunity via direct, TACI-dependent activation of transitional B cells.

Cutting Edge: BAFF Overexpression Reduces Atherosclerosis via TACI-Dependent B Cell Activation

Patients with systemic lupus erythematosus exhibit accelerated atherosclerosis, a chronic inflammatory disease of the arterial wall. The impact of B cells in atherosclerosis is controversial, with both protective and pathogenic roles described. For example, natural IgM binding conserved oxidized lipid epitopes protect against atherosclerosis, whereas anti-oxidized low-density lipoprotein (oxLDL) IgG likely promotes disease. Because BAFF promotes B cell class-switch recombination and humoral autoimmunity, we hypothesized that excess BAFF would accelerate atherosclerosis. In contrast, BAFF overexpression markedly reduced hypercholesterolemia and atherosclerosis in hyperlipidemic mice. BAFF-mediated atheroprotection required B cells and was associated with increased protective anti-oxLDL IgM. Surprisingly, high–titer anti-oxLDL IgM production and reduced atherosclerosis was dependent on the BAFF family receptor transmembrane activator and CAML interactor. In summary, we identified a novel role for B cell–specific, BAFF-dependent transmembrane activator and CAML interactor signals in atherosclerosis pathogenesis, of particular relevance to the use of BAFF-targeted therapies in systemic lupus erythematosus.

Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions

SUMMARY To fulfill bioenergetic demands of activation, T cells perform aerobic glycolysis, a process common to highly proliferative cells in which glucose is fermented into lactate rather than oxidized in mitochondria. However, the signaling events that initiate aerobic glycolysis in T cells remain unclear. We show T cell activation rapidly induces glycolysis independent of transcription, translation, CD28, and Akt and not involving increased glucose uptake or activity of glycolytic enzymes. Rather, TCR signaling promotes activation of pyruvate dehydrogenase kinase 1 (PDHK1), inhibiting mitochondrial import of pyruvate and facilitating breakdown into lactate. Inhibition of PDHK1 reveals this switch is required acutely for cytokine synthesis but dispensable for cytotoxicity. Functionally, cytokine synthesis is modulated via lactate dehydrogenase, which represses cytokine mRNA translation when aerobic glycolysis is disengaged. Our data provide mechanistic insight to metabolic contribution to effector T cell function and suggest that T cell function may be finely tuned through modulation of glycolytic activity.

B‐cell intrinsic TLR7 signals promote depletion of the marginal zone in a murine model of Wiskott–Aldrich syndrome

Patients with Wiskott–Aldrich syndrome (WAS) exhibit prominent defects in splenic marginal zone (MZ), resulting in abnormal T‐cell‐independent antibody responses and increased bacterial infections. B‐cell‐intrinsic deletion of the affected gene WAS protein (WASp) markedly reduces splenic MZ B cells, without impacting the rate of MZ B‐cell development, suggesting that abnormal B‐cell retention within the MZ accounts for MZ defects in WAS. Since WASp regulates integrin‐dependent actin cytoskeletal rearrangement, we previously hypothesized that defective B‐cell integrin function promotes MZ depletion. In contrast, we now report that B‐cell‐intrinsic deletion of the TLR signaling adaptor MyD88 is sufficient to restore the MZ in WAS. We further identify TLR7, an endosomal single‐stranded RNA (ssRNA) receptor, as the MyD88‐dependent receptor responsible for WAS MZ depletion. These findings implicate spontaneous activation of MZ B cells by ssRNA‐containing self‐ligands (likely derived from circulating apoptotic material) as the mechanism underlying MZ depletion in WAS. Together, these data suggest a previously unappreciated role for B‐cell intrinsic TLR signals in MZ homeostasis, of relevance to both pathogen responses and to the development of systemic autoimmunity.

4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to these therapies. The tumor microenvironment can impose metabolic restrictions on T cell function, creating a resistance mechanism to immunotherapy. We have previously shown tumor-infiltrating T cells succumb to progressive loss of metabolic sufficiency, characterized by repression of mitochondrial activity that cannot be rescued by PD-1 blockade. 4-1BB, a costimulatory molecule highly expressed on exhausted T cells, has been shown to influence metabolic function. We hypothesized that 4-1BB signaling might provide metabolic support to tumor-infiltrating T cells. 4-1BB costimulation of CD8+ T cells results in enhanced mitochondrial capacity (suggestive of fusion) and engages PGC1&agr;-mediated pathways via activation of p38-MAPK. 4-1BB treatment of mice improves metabolic sufficiency in endogenous and adoptive therapeutic CD8+ T cells. 4-1BB stimulation combined with PD-1 blockade results in robust antitumor immunity. Sequenced studies revealed the metabolic support afforded by 4-1BB agonism need not be continuous and that a short course of anti–4-1BB pretreatment was sufficient to provide a synergistic response. Our studies highlight metabolic reprogramming as the dominant effect of 4-1BB therapy and suggest that combinatorial strategies using 4-1BB agonism may help overcome the immunosuppressive metabolic landscape of the tumor microenvironment.

AI-18 B cell IFN-γ receptor signalling promotes autoimmune germinal centresvia cell-intrinsic induction of BCL-6

Background Dysregulated germinal centrecenter (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although type 1 interferons (IFNs) are most frequently associated with lupus pathogenesis, type 2 interferon (IFN-γ) has also been shown to promote SLE. However, the respective impacts of these cytokines in promoting B cell activation during humoral autoimmunity have not been addressed. Materials and methods We recently developed a chimeric murine lupus model in which Wiskott-Aldrich syndrome protein (WAS)-deficient B cells promote spontaneous humoral autoimmunity (Jackson, et al. J Immunol 2014). An important advantage of the WAS chimaera model is that dysregulated immune responses are limited to the B cell compartment, allowing genetic manipulation in a B cell-intrinsic fashion. In the current study, we contrast the impact B cell-intrinsic type 1 IFN vs. IFN-γ signals on autoimmune GC formation and the pathogenesis of SLE. Results Although type 1 IFN prominently enhanced B cell responses in vitro, B cell-intrinsic IFNAR deletion exerted surprisingly minimal impacts on class-switched autoantibody titers and spontaneous GC formation in vivo. This finding suggested that other cytokines promote B cell activation in the WAS chimaera model. Notably, B cells directly initiated CD4+ T cell activation and T follicular helper cell formation via MHC Class II (MHC-II)-dependent antigen presentation. In addition, activated T cells exhibited prominent IFN-γ production that was lost following B cell-intrinsic MHC-II deletion, suggesting a direct role for IFN-γ in promoting autoimmune GC formation. Strikingly, B cell-intrinsic deletion of the IFN-γ receptor was sufficient to abrogate spontaneous GCs, class-switched autoantibodies and systemic autoimmunity. Mechanistically, although IFN-γ receptor signals increased B cell T-bet expression, B cell-intrinsic deletion of T-bet exerted an isolated impact on class-switch recombination to pathogenic IgG2c autoantibody subclasses without impacting GC development. Rather, in both murine and human B cells, IFN-γ synergized with BCR, TLR and/or CD40 activation signals to promote cell-intrinsic BCL-6 expression. Finally, IFN-γ driven BCL-6 expression in B cells was blocked using clinically-relevant Janus kinase inhibitors, ruxolitinib and tofacitinib. Conclusions Our study demonstrates that B cell-intrinsic IFN-γ receptor signals promote lupus pathogenesis via formation of spontaneous, autoimmune GCs. In addition, we have uncovered a novel cell-intrinsic program whereby IFN-γ, together with BCR-, TLR- and/or CD40 signals, orchestrates B cell expression of the GC master transcription regulator BCL-6. Our combined findings suggest that this IFN-γ signalling program may be a potential therapeutic target in SLE. Acknowledgements This work was supported by the National Institutes of Health under award numbers: R01HL075453 (DJR), R01AI084457 (DJR), R01AI071163 (DJR), DP3DK097672 (DJR) and K08AI112993 (SWJ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support provided by the Benaroya Family Gift Fund (DJR); by the ACR REF Rheumatology Scientist Development Award (SWJ); and by the Arnold Lee Smith Endowed Professorship for Research Faculty Development (SWJ).

Metabolic insufficiency underlies intratumoral cytotoxic T cell dysfunction

T cells have the remarkable ability to recognize and remove abnormal cells with precision, a feature that is very desirable for the treatment of cancer. However, while T cells specific for tumor antigens are primed and can infiltrate tumors, they are quickly rendered dysfunctional, through both cell intrinsic and cell extrinsic mechanisms. One way that tumors cripple T cell function is through the generation of an immunosuppressive microenvironment that is chronically inflamed, hypoxic, and nutrient poor. T cell activation and subsequent generation of effector function is bioenergetically demanding, requiring large amounts of metabolic intermediates to effectively proliferate, produce cytokines, and lyse target cells. We hypothesized that T cell dysfunction in cancer is due, in part, to metabolic insufficiency caused by chronic activation in metabolically dearth conditions. Using single-cell metabolic assays and extracellular flux analysis, we show that CD8+ cytotoxic T cells that infiltrate tumors demonstrate a progressive loss of mitochondrial function and mass, concomitant with upregulation of markers that correlate with T cell exhaustion. This mitochondrial dysfunction occurs independently of coinhibitory molecule signaling and specifically in the tumor microenvironment. This results in a failure to generate an adequate pool of ATP and in inability to effectively translate effector gene transcripts. This in stark contrast to T cells responding to an acute viral infection, where activated effector T cells demonstrate increased mitochondrial mass and ATP reserve. Further, artificial induction of mitochondrial dysfunction in T cells results in upregulation of coinhibitory molecules and an ‘exhausted-like’ phenotype, suggesting that metabolic insufficiency underlies the dysfunctional phenotype in cancer. Taken together, our data support a model in which tumor-infiltrating T cells have metabolic needs that cannot be met, resulting in failed effector function and tumor growth. Our studies also suggest that modulation or reprogramming of the altered metabolism of intratumoral T cells represents a potential strategy to reinvigorate dysfunctional T cells for the immunotherapeutic treatment of cancer.