In adult X-CGD patients, regulatory T cells are expanded while activated T cells display a NOX2-independent ROS increase.

The X-linked chronic granulomatous disease (X-CGD), a rare genetic disease characterised by recurrent infections, is caused by mutations of NOX2. Significant proportions of X-CGD patients display signs of immune dysregulation. Regulatory T cells (Tregs) are CD4+T lymphocytes that expand in active inflammation and prevent autoimmune disorders. Here we asked whether X-CGD is associated to Treg dysfunctions in adult patients. To this aim, the frequency of Tregs was analysed through intracellular flow cytometry in a cohort of adult X-CGD patients, carriers and controls. We found that Tregs were significantly expanded and activated in blood of adult X-CGD patients, and this was associated with activation of conventional CD4+T cells (Tconvs). T cell activation was characterised by accumulation of intracellular ROS, not derived from NOX2 but likely produced by cellular metabolism. The higher TNF production by Tconvs in X-CGD patients might contribute to the expansion of Tregs through the TNFR2 receptor. In summary, our data indicate that Tregs expand in adult X-CGD in response to immune activation, and that the increase of NOX2-independent ROS content is a feature of activated T cells.

RNA Flow Cytometry for the Study of T Cell Metabolism.

T cells undergo activation and differentiation programs along a continuum of states that can be tracked through flow cytometry using a combination of surface and intracellular markers. Such dynamic behavior is the result of transcriptional and post-transcriptional events, initiated and sustained by the activation of specific transcription factors and by epigenetic remodeling. These signaling pathways are tightly integrated with metabolic routes in a bidirectional manner: on the one hand, T cell receptors and costimulatory molecules activate metabolic reprogramming; on the other hand, metabolites modify T cell transcriptional programs and functions. Flow cytometry represents an invaluable tool to analyze the integration of phenotypical, functional, metabolic and transcriptional features, at the single cell level in heterogeneous T cell populations, and from complex microenvironments, with potential clinical application in monitoring the efficacy of cancer immunotherapy. Here, we review the most recent advances in flow cytometry-based analysis of gene expression, in combination with indicators of mitochondrial activity, with the aim of revealing and characterizing major metabolic pathways in T cells.

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Assessment of lipid load in tumor-infiltrating Tregs by flow cytometry.

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, are defined as immunosuppressive cells able to modulate a variety of immune cells in order to avoid unwanted and excessive immune responses; however, in the tumor context, Treg function contribute to inhibit immune surveillance, thus promoting cancer progression. In tumor microenvironment, where the availability of metabolic resources is strongly limited, Tregs are expanded and may exploit different metabolic routes to achieve a metabolic advantage, prevailing over effector cells. In this context an important role of lipid metabolism has been described thanks to the possibility to evaluate the intracellular lipid content selectively in tumor-infiltrating Tregs (TUM-Tregs). Taking into account the heterogeneous and complex build of tumor mass, we set-up a combined protocol that optimizes tumor-infiltrating lymphocytes (TIL) extraction from the tissue through a Percoll density gradient, and assesses ex vivo the lipid load in whole TUM-Treg population, evaluating by flow cytometry the incorporation of an intensely fluorescent lipophilic fluorophore able to specifically stain neutral lipids. This method provides an important advantage compared to the traditional technique based on microscopy, whose lipid level evaluation is limited to a tissue section, and hence may not be representative of the entire population.

Immunometabolic Checkpoints of Treg Dynamics: Adaptation to Microenvironmental Opportunities and Challenges.

In the last decades, immunologists have started to consider intracellular metabolism in relation with the dynamics and functions of immune cells, especially when it became clear that microenvironmental alterations were associated with immune dysfunctions. Regulatory T cells (Tregs) are equipped with a variety of immunological and metabolic sensors, and encompass circulating as well as tissue-resident cells, being therefore particularly susceptible to microenvironmental cues. Moreover, Tregs undergo metabolic reprogramming over the course of an immune response, allowing the use of alternate substrates and engaging different metabolic pathways for energetic demands. The study of metabolic mechanisms supporting Treg dynamics has led to puzzling results, due to several limitations, including the heterogeneity of population in the same tissues and between different tissues, the difficulty in considering all the interconnected metabolic pathways during a cellular process, and the differences between in vitro and in vivo conditions. Therefore, Treg reliance on different metabolic routes (oxidation rather than glycolysis) has been a matter of controversy in recent years. Metabolic reprogramming and altered bioenergetics are now identified as hallmarks in cancer, and are employed by cancer cells to determine the availability of metabolites and molecules, thus affecting the fate of tumor-infiltrating immune cells. In particular, the tumor microenvironment forces a metabolic restriction and a plethora of synergistic intrinsic and extrinsic stresses, leading to an impaired anti-tumor immunity and favoring Treg generation, expansion, and suppressive function. This leads to the understanding that Tregs and conventional T cells have different capability to adapt to metabolic hurdles. Considering the role of Tregs in dictating the outcome of tumor-specific responses, it would be important to understand the specific Treg metabolic profile that provides an advantage at the tumor site, to finally identify new targets for therapy. In this review, we will report and discuss the major recent findings about the metabolic pathways required for Treg development, expansion, migration and functions, in relation to tissue-derived signals. We will focus on the adipose tissue and the liver, where Tregs are exposed to a variety of metabolites, and on the tumor microenvironment as the context where Tregs develop the ability to adapt to perturbations in nutrient accessibility.

Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming.

Mitochondria are key players in the regulation of T cell biology by dynamically responding to cell needs, but how these dynamics integrate in T cells is still poorly understood. We show here that the mitochondrial pro-fission protein Drp1 fosters migration and expansion of developing thymocytes both in vitro and in vivo. In addition, we find that Drp1 sustains in vitro clonal expansion and cMyc-dependent metabolic reprogramming upon activation, also regulating effector T cell numbers in vivo. Migration and extravasation defects are also exhibited in Drp1-deficient mature T cells, unveiling its crucial role in controlling both T cell recirculation in secondary lymphoid organs and accumulation at tumor sites. Moreover, the observed Drp1-dependent imbalance toward a memory-like phenotype favors T cell exhaustion in the tumor microenvironment. All of these findings support a crucial role for Drp1 in several processes during T cell development and in anti-tumor immune-surveillance.

Wnt3a Neutralization Enhances T-cell Responses through Indirect Mechanisms and Restrains Tumor Growth.

The Wnt/ß-catenin pathway regulates T-cell functions, including the repression of effector functions to the advantage of memory development via Tcf1. In a companion study, we demonstrate that, in human cancers, Wnt3a/ß-catenin signaling maintains tumor-infiltrating T cells in a partially exhausted status. Here, we have investigated the effects of Wnt3a neutralization in vivo in a mouse tumor model. Abundant Wnt3a was released, mostly by stromal cells, in the tumor microenvironment. We tested whether Wnt3a neutralization in vivo could rescue the effector capacity of tumor-infiltrating T cells, by administering an antibody to Wnt3a to tumor-bearing mice. This therapy restrained tumor growth and favored the expansion of tumor antigen-specific CD8+ effector memory T cells with increased expression of Tbet and IFNγ and reduced expression of Tcf1. However, the effect was not attributable to the interruption of T-cell-intrinsic ß-catenin signaling, because Wnt3a/ß-catenin activation correlated with enhanced, not reduced, T-cell effector functions both ex vivo and in vitro Adoptively transferred CD8+ T cells, not directly exposed to the anti-Wnt3a antibody but infiltrating previously Wnt3a-neutralized tumors, also showed improved functions. The rescue of T-cell response was thus secondary to T-cell-extrinsic changes that likely involved dendritic cells. Indeed, tumor-derived Wnt3a strongly suppressed dendritic cell maturation in vitro, and anti-Wnt3a treatment rescued dendritic cell activities in vivo Our results clarify the function of the Wnt3a/ß-catenin pathway in antitumor effector T cells and suggest that Wnt3a neutralization might be a promising immunotherapy for rescuing dendritic cell activities. Cancer Immunol Res; 6(8); 953-64. ©2018 AACR.

Fatty acid metabolism complements glycolysis in the selective regulatory T cell expansion during tumor growth.

The tumor microenvironment restrains conventional T cell (Tconv) activation while facilitating the expansion of Tregs. Here we showed that Tregs' advantage in the tumor milieu relies on supplemental energetic routes involving lipid metabolism. In murine models, tumor-infiltrating Tregs displayed intracellular lipid accumulation, which was attributable to an increased rate of fatty acid (FA) synthesis. Since the relative advantage in glucose uptake may fuel FA synthesis in intratumoral Tregs, we demonstrated that both glycolytic and oxidative metabolism contribute to Tregs' expansion. We corroborated our data in human tumors showing that Tregs displayed a gene signature oriented toward glycolysis and lipid synthesis. Our data support a model in which signals from the tumor microenvironment induce a circuitry of glycolysis, FA synthesis, and oxidation that confers a preferential proliferative advantage to Tregs, whose targeting might represent a strategy for cancer treatment.

Distinct Blood and Visceral Adipose Tissue Regulatory T Cell and Innate Lymphocyte Profiles Characterize Obesity and Colorectal Cancer.

Visceral adipose tissue (VAT) is a main site where metabolic and immunologic processes interplay to regulate, at local and systemic level, the inflammatory status and immune response. Obesity-associated inflammation and immune dysfunctions are inextricably linked to tumor but, in spite of intense efforts, the mechanisms underpinning this association remain elusive. In this report, we characterized the profile of VAT-associated and circulating innate lymphocyte and regulatory T (Treg) cell subsets underlying inflammatory conditions, such as obesity and colorectal cancer (CRC). Analysis of NK, NKT-like, γδ T, and Treg cell populations in VAT and blood of healthy lean subjects revealed that CD56hi NK and OX40+ Treg cells are more abundant in VAT with respect to blood. Conversely, CD56dim NK and total Treg cells are most present in the circulation, while γδ T lymphocytes are uniformly distributed in the two compartments. Interestingly, a reduced frequency of circulating activated Treg cells, and a concomitant preferential enrichment of OX40-expressing Treg cells in VAT, were selectively observed in obese (Ob) subjects, and directly correlated with body mass index. Likewise, CRC patients were characterized by a specific enrichment of VAT-associated NKT-like cells. In addition, Ob and CRC-affected individuals shared a significant reduction of the Vγ9Vδ2/γδ T cell ratio at systemic level. The alterations in the relative proportions of Treg and NKT-like cells in VAT were found to correlate with the content of pro- and anti-inflammatory polyunsaturated fatty acids (PUFA), respectively. Overall, these results provide evidence for distinct alterations of the immune cell repertoire in the periphery with respect to the VAT microenvironment that uniquely characterize or are shared by different inflammatory conditions, such as obesity and CRC, and suggest that VAT PUFA composition may represent one of the factors that contribute to shape the immune phenotypes.

Regulatory T cells with multiple suppressive and potentially pro-tumor activities accumulate in human colorectal cancer.

Tregs can contribute to tumor progression by suppressing antitumor immunity. Exceptionally, in human colorectal cancer (CRC), Tregs are thought to exert beneficial roles in controlling pro-tumor chronic inflammation. The goal of our study was to characterize CRC-infiltrating Tregs at multiple levels, by phenotypical, molecular and functional evaluation of Tregs from the tumor site, compared to non-tumoral mucosa and peripheral blood of CRC patients. The frequency of Tregs was higher in mucosa than in blood, and further significantly increased in tumor. Ex vivo, those Tregs suppressed the proliferation of tumor-infiltrating CD8(+) and CD4(+) T cells. A differential compartmentalization was detected between Helios(high) and Helios(low) Treg subsets (thymus-derived versus peripherally induced): while Helios(low) Tregs were enriched in both sites, only Helios(high) Tregs accumulated significantly and specifically in tumors, displayed a highly demethylated TSDR region and contained high proportions of cells expressing CD39 and OX40, markers of activation and suppression. Besides the suppression of T cells, Tregs may contribute to CRC progression also through releasing IL-17, or differentiating into Tfr cells that potentially antagonize a protective Tfh response, events that were both detected in tumor-associated Tregs. Overall, our data indicate that Treg accumulation may contribute through multiple mechanisms to CRC establishment and progression.

Divergent effects of type-I interferons on regulatory T cells.

Regulatory T cells (Treg) exert a dominant role in the protection of unwanted immune responses and in the resolution of inflammation. To ensure the proper mounting of protective immune responses, Treg should be finely modulated by microenvironmental signals, mostly conveyed by cytokines. Type-I interferons are pleiotropic cytokines, best known for their anti-viral activities but also playing relevant immunostimulatory as well as immunomodulatory functions. The impact of type-I interferons on Treg homeostasis and functions is quite controversial, as some studies indicate that interferons sustain Treg stability and suppression, while other reports describe a null or even negative role for interferons in Treg activities. Interferons may also establish alternative routes of suppression, through the induction of other suppressive populations, such as Tr1 and the recently discovered FoxA1+ Treg. Discrepant results about Treg behavior in vivo emerge also from data collected in patients with multiple sclerosis, chronic hepatitis C or cancer undergoing interferon therapy. Concurrent events, such as Treg-extrinsic interferon activities, desensitization to chronic interferon exposure, and changes in microenvironmental signals during the evolution of diseases, may contribute to depict such a complex scenario, in which short-term and long-term effects of interferon exposure may give rise to apparently opposite conclusions.

Human OX40 tunes the function of regulatory T cells in tumor and nontumor areas of hepatitis C virus-infected liver tissue.

UNLABELLED: Regulatory T cells (Tregs) can be considered as a mixed population of distinct subsets, endowed with a diverse extent and quality of adaptation to microenvironmental signals. Here, we uncovered an opposite distribution of Treg expansion, phenotype, and plasticity in different microenvironments in the same organ (liver) derived from patients with chronic hepatitis C: On the one side, cirrhotic and tumor fragments were moderately and highly infiltrated by Tregs, respectively, expressing OX40 and a T-bethigh IFN-γ- "T-helper (Th)1-suppressing" phenotype; on the other side, noncirrhotic liver specimens contained low frequencies of Tregs that expressed low levels of OX40 and highly produced interferon-gamma (IFN-γ; T-bet+IFN-γ+), thus becoming "Th1-like" cells. OX40-expressing and Th1-suppressing Tregs were enriched in the Helios-positive subset, carrying highly demethylated Treg cell-specific demethylated region that configures committed Tregs stably expressing forkhead box protein 3. OX40 ligand, mostly expressed by M2-like monocytes and macrophages, boosted OX40+ Treg proliferation and antagonized the differentiation of Th1-like Tregs. However, this signal is counteracted in noncirrhotic liver tissue (showing various levels of inflammation) by high availability of interleukin-12 and IFN-γ, ultimately leading to complete, full Th1-like Treg differentiation. CONCLUSION: Our data demonstrate that Tregs can finely adapt, or even subvert, their classical inhibitory machinery in distinct microenvironments within the same organ.