Robert A. Amezquita

Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.

IL-7-Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8+ T Cell Longevity

Memory T cells are critical for long-term immunity against reinfection and require interleukin-7 (IL-7), but the mechanisms by which IL-7 controls memory T cell survival, particularly metabolic fitness, remain elusive. We discover that IL-7 induces expression of the glycerol channel aquaporin 9 (AQP9) in virus-specific memory CD8+ T cells, but not naive cells, and that AQP9 is vitally required for their long-term survival. AQP9 deficiency impairs glycerol import into memory CD8+ T cells for fatty acid esterification and triglyceride (TAG) synthesis and storage. These defects can be rescued by ectopic expression of TAG synthases, which restores lipid stores and memory T cell survival. Finally, we find that TAG synthesis is a central component of IL-7-mediated survival of human and mouse memory CD8+T cells. This study uncovers the metabolic mechanisms by which IL-7 tailors the metabolism of memory T cells to promote their longevity and fast response to rechallenge.

Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8+ T Cell Terminal Differentiation and Loss of Multipotency

&NA; Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long‐term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ T cells during viral infection revealed increased H3K27me3 deposition at numerous pro‐memory and pro‐survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro‐memory and pro‐effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory T cell maturation. Abundant H3K27me3 deposition at pro‐memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro‐memory genes in effector T cells. Graphical Abstract Figure. No caption available. HighlightsH3K27me3 is more abundant at certain pro‐memory genes in TE CD8+ T cellsPRC2 is required for CD8+ T cell clonal expansion and TE differentiationH3K27me3 is deposited during late effector CD8+ T cell differentiationFOXO1 regulates H3K27me3 deposition at certain pro‐memory loci in TE cells &NA; Cytotoxic CD8+ T cells either terminally differentiate and die or form a rapidly responding population of memory T cells after pathogen clearance. Gray et al. define a temporal model for how effector T cells lose memory cell potential through selective epigenetic silencing of pro‐memory genes.

Autoreactive T Cells from Patients with Myasthenia Gravis Are Characterized by Elevated IL-17, IFN-γ, and GM-CSF and Diminished IL-10 Production

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature, Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus, we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4+ T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6+ memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17, in response to AChR, was also restricted to the CCR6+ memory T cell compartment in the MG cohort, indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression, indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells, because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations.

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell–cell interactions of circulating leukocytes with IL-1– or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17–induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17–activated PCs, but not ECs, induce pertussis toxin–sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1β, and IL-8. Furthermore, IL-17–activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17–treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17–activated PCs can modulate neutrophil functions within the perivascular tissue space.

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response

Follicular regulatory T cells promote the germinal center response after viral infection through provision of IL-10. Probing the functions of follicular regulatory T cells CD4+ follicular regulatory T (Tfr) cells express key molecules that are associated with regulatory T cell and follicular helper T cell functions. Although it is known that Tfr cells produce interleukin-10 (IL-10), it has been unclear whether IL-10 production by these cells regulates germinal center (GC) responses in vivo. By specifically ablating IL-10 expression in murine Tfr cells, Laidlaw et al. demonstrate that Tfr cell–derived IL-10 does support GC responses in the context of acute viral infection. They found dendritic cells and B cells in the GCs to be IL-10–responsive and showed that IL-10 promoted GC B cells to adopt a dark zone phenotype. CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell–derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction.

Myeloid-targeted immunotherapies act in synergy to induce inflammation and antitumor immunity

Eliciting effective antitumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF-1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or cosecreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset cosecreting TNF-&agr;, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, and on TNF-&agr; and IFN-&ggr;. Together, this study demonstrates the potential for targeting TAMs to convert a “cold” into an “inflamed” tumor microenvironment capable of eliciting protective T cell responses.

NK Cell Responses Redefine Immunological Memory

Immunological memory has traditionally been regarded as a unique trait of the adaptive immune system. Nevertheless, there is evidence of immunological memory in lower organisms and invertebrates, which lack an adaptive immune system. Despite their innate ability to rapidly produce effector cytokines and kill virally infected or transformed cells, NK cells also exhibit adaptive characteristics such as clonal expansion, longevity, self-renewal, and robust recall responses to antigenic or nonantigenic stimuli. In this review, we highlight the intracellular and extracellular requirements for memory NK cell generation and describe the emerging evidence for memory precursor NK cells and their derivation.

Immunology: The chronicles of T-cell exhaustion

&NA; T cells of the immune system often fail to target cancer cells because they enter a dysfunctional state known as exhaustion. Molecular analysis of T‐cell exhaustion provides insights into the clinical use of these cells.

ZEB1, ZEB2, and the miR-200 family form a counterregulatory network to regulate CD8+ T cell fates

Long-term immunity depends partly on the establishment of memory CD8+ T cells. We identified a counterregulatory network between the homologous transcription factors ZEB1 and ZEB2 and the miR-200 microRNA family, which modulates effector CD8+ T cell fates. Unexpectedly, Zeb1 and Zeb2 had reciprocal expression patterns and were functionally uncoupled in CD8+ T cells. ZEB2 promoted terminal differentiation, whereas ZEB1 was critical for memory T cell survival and function. Interestingly, the transforming growth factor &bgr; (TGF-&bgr;) and miR-200 family members, which counterregulate the coordinated expression of Zeb1 and Zeb2 during the epithelial-to-mesenchymal transition, inversely regulated Zeb1 and Zeb2 expression in CD8+ T cells. TGF-&bgr; induced and sustained Zeb1 expression in maturing memory CD8+ T cells. Meanwhile, both TGF-&bgr; and miR-200 family members selectively inhibited Zeb2. Additionally, the miR-200 family was necessary for optimal memory CD8+ T cell formation. These data outline a previously unknown genetic pathway in CD8+ T cells that controls effector and memory cell fate decisions.