CD4 T cell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or ...oxidative metabolic programs in vitro, requirements and mechanisms that control T cell glucose uptake and metabolism in vivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metabolism and function of thymocytes and Teff. Resting T cells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease in vivo. Treg cells, in contrast, were enriched in vivo and appeared functionally unaffected and able to suppress Teff, irrespective of Glut1 expression. These data show a selective in vivo requirement for Glut1 in metabolic reprogramming of CD4 T cell activation and Teff expansion and survival.
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•CD4 T cells express multiple glucose transporters, including Gluts 1, 3, 6, and 8•Glut1 has nonredundant function in activated, but not resting, CD4 T cells•CD4 Th1 and Th17 selectively require Glut1 in vivo to regulate immunologic diseases•Targeting T cell glucose metabolism in vivo can selectively impact effector cells
T cells undergo distinct metabolic reprogramming events upon activation and differentiation to inflammatory effectors or regulatory cells. Macintyre et al. show that Glut1 is the only glucose transporter required to drive glycolysis for growth and expansion of effector, but not resting or regulatory, CD4 T cells and induce inflammatory diseases.
FOXP3+ regulatory T (Treg) cells are a broadly acting and potent anti-inflammatory population of CD4+ T cells essential for maintaining immune homeostasis and preventing debilitating autoimmunity. ...Based on chemokine receptor expression, we identified distinct populations of Treg cells in human blood expected to colocalize with different Th cell subsets. Although each population was functionally suppressive, they displayed unique patterns of pro- and anti-inflammatory cytokine production, differentially expressed lineage-specifying transcription factors, and responded differently to antigens associated with Th1 and Th17 responses. These results highlight a previously unappreciated degree of phenotypic and functional diversity in human Treg cells that allows subsets with unique specificities and immunomodulatory functions to be targeted to defined immune environments during different types of inflammatory responses.
Abstract Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS- bl -PEG polymersomes (PSs; ...watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8+ T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4+ or CD8+ T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4+ T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8+ T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4+ and CD8+ T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses.
Regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we ...reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.
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•EZH2 expression is elevated in tumor-infiltrating (TI) Tregs•Pharmacological inhibition of EZH2 destabilizes FOXP3 expression and slows tumor growth•Genetic disruption of Ezh2 function in Tregs leads to robust anti-tumor immunity•Blockade of EZH2 in Tregs reprograms TI-Tregs to gain pro-inflammatory activity
EZH2 plays an intrinsic role in neoplastic cells as an oncogene, prompting the development of EZH2 inhibitors for cancer therapy. Wang et al. show that disrupting EZH2 function also has immunomodulatory activities and, when blocked in Tregs, promotes potent cancer immunity.
Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms ...responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.
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•Mechanisms of inflammatory adverse events induced by checkpoint blockade•Colitis-associated differentiation of CD8 Trm cells to cytotoxic effector cells•Persistence and expansion of CTLA-4+ Treg cells•Inflammatory pathways provide opportunities for therapeutic intervention
Single-cell analyses of immune checkpoint blockade-associated colitis patient samples reveal enrichment of regulatory T cells in colitic lesions and nominate inflammatory pathways for potential therapeutic intervention.
Inefficient T-cell reconstitution from x-ray-induced immune damage reduces antitumor response. To understand the profile of T-cell reconstitution after irradiation will overcome the barrier of ...antitumor immunity. This study aimed to identify the recovery profile of T-cell subsets following x-ray irradiation and to highlight the role of cinnamon on efficient T-cell restoration postexposure in the antitumor response.
CD3(+), CD8(+), and CD4(+) T cells and Th1, Th2, Th17, and regulatory T (Treg) cells were evaluated at different time points after single low-dose total body irradiation (SLTBI) with or without cinnamon treatments. T-bet, GATA3, RORγt, and Foxp3 signaling specific for Th1, Th2, Th17, and Treg were also analyzed by RT-PCR assay. The effects of cinnamon on efficient T-cell subset reconstitution was confirmed in a lung melanoma model in irradiated mice.
Reconstitution of CD4(+) T cells was delayed more than that of CD8(+) T cells in T-cell restoration after SLTBI. The production of IFNγ by Th1 or Tc1 cells was sharply decreased and was accompanied by reduced T-bet mRNA, even when total T-cell numbers had recovered; the frequencies of Th17 and Treg cells and their specific transcription factors (RORγt and Foxp3, respectively) were obviously increased. Irradiation-induced inefficient T-cell reconstitution impaired the antitumor capacities in the lung melanoma model. Pretreatment with cinnamon in irradiated mice accelerated the generation of Th1 and reduced the differentiation of Treg cells by activating T-bet and limiting transcriptions of Foxp3. Improvement resulting from cinnamon pretreatment on the efficient T-cell recovery profile from SLTBI promoted antitumor immunity in the lung melanoma model.
T-cell reconstitution from SLTBI was characterized by impaired Th1 and elevated Th17 and Treg cells. Cinnamon effectively improved the imbalance of T-cell subsets by promoting the proliferation of Th1 and by suppressing expansions of Th17 and Tregs. The role of cinnamon in efficient T-cell reconstitution from SLTBI is effective in antitumor immunity.
Understanding the factors that delineate the efficacy of T cell responses towards pathogens is crucial for our ability to develop potent therapies against infectious diseases. Multidimensional ...evaluation of T cell functionality at the single-cell level enables exhaustive analysis of combinatorial functional properties, hence polyfunctionality. We have recently invented an algorithm that quantifies polyfunctionality, the Polyfunctionality Index (Larsen et al. PLoS One 2012). Here we demonstrate that quantitative assessment of T cell polyfunctionality correlates with T cell efficacy measured as the capacity to kill target cells in vitro and control infection in vivo.
We employed the polyfunctionality index on two datasets selected for their unique ability to evaluate the polyfunctional imprint on T cell efficacy. 1) HIV-specific CD8+ T cells and 2) Leishmania major-specific CD4+ T cells were analysed for their capacity to secrete multiple effector molecules, kill target cells and control infection. Briefly, employing the Polyfunctionality Index algorithm we determined the parameter estimates resulting in optimal correlation between T cell polyfunctionality and T cell efficacy.
T cell polyfunctionality is correlated with T cell efficacy measured as 1) target killing (r=0.807, P<0.0001) and 2) lesion size upon challenge with Leishmania major (r=-0.50, P=0.004). Contrary to an approach relying on the Polyfunctionality Index algorithm, quantitative evaluation of T cell polyfunctionality traditionally ignores the gradual contribution of more or less polyfunctional T cells. Indeed, comparing both approaches we show that optimal description of T cell efficacy is obtained when gradually integrating all levels of polyfunctionality in accordance with the Polyfunctionality Index.
Our study presents a generalizable methodology to objectively evaluate the impact of polyfunctionality on T cell efficacy. We show that T cell polyfunctionality is a superior correlate of T cell efficacy both in vitro and in vivo as compared with response size. Therefore, future immunotherapies should aim to increase T cell polyfunctionality.
Severe immune suppression is frequent in late-stage tumor patients and promotes tumor immune evasion and subsequent tumor progression. Regulatory T cells (Treg) are major suppressors of anti-tumor ...immune responses. Therefore, targeting of Treg has become a key goal of anti-tumor therapy. Several preclinical and clinical observations suggest that Treg can be depleted by cyclophosphamide. Over a period of 3 months, we investigated the effect of metronomic low-dose cyclophosphamide on Treg numbers, suppressive capacity and proliferation on endogenous anti-tumor T-cell responses and on their correlation to clinical outcome in 12 patients with treatment-refractory metastasized breast cancer who received single-agent 50 mg cyclophosphamide p.o. daily. Cyclophosphamide treatment initially caused a significant reduction in circulating Treg by more than 40% (
P
= 0.002). However, Treg numbers completely recovered during the treatment due to increased proliferative activity and maintained their suppressive capacity. Treg depletion coincided with a strong increase in breast tumor–reactive T cells (
P
= 0.03) that remained at high levels during the whole period. Numbers of tumor-reactive T cells but not of Treg correlated with disease stabilization (
P
= 0.03) and overall survival (
P
= 0.027). We conclude that metronomic low-dose cyclophosphamide only transiently reduces Treg but induces stable tumor-specific T-cell responses, which correlate with improved clinical outcome in advanced-stage breast cancer patients.
The strong link between T‐cell metabolism and effector functions is well characterized in the murine system but hardly investigated in human T cells. Therefore, we analyzed glycolytic and ...mitochondrial activity in correlation to function in activated human CD4 and CD8 T cells. Glycolysis was barely detectable upon stimulation but accelerated beyond 24 h, whereas mitochondrial activity was elevated immediately in both T‐cell populations. Glucose deprivation or mitochondrial restriction reduced proliferation, had only a transient impact on “on‐blast formation” and no impact on viability, IFN‐γ, IL‐2, IL‐4, and IL‐10 production, whereas TNF was reduced. Similar results were obtained in bulk T cells and T‐cell subsets. Elevated respiration under glucose restriction demonstrated metabolic flexibility. Administration of the glycolytic inhibitor 2‐deoxy‐glucose suppressed both glycolysis and respiration and exerted a strong impact on cytokine production that persisted for IFN‐γ after removal of 2‐deoxy‐glucose. Taken together, glycolytic or mitochondrial restriction alone compromised proliferation of human T cells, but barely affected their effector functions. In contrast, effector functions were severely affected by 2‐deoxy‐glucose treatment.
Lytic CD8+ T cells at the psoriasis plaque’s epidermis secrete IL‐17, IL‐21 and IL‐22 and depends on autocrine TNFalpha production.
IL‐17‐producing CD4+ T lymphocytes (Th17) are currently considered ...relevant participants in the pathogenesis of psoriasis skin lesions. However, little is known about the potential role of IL‐17‐producing CD8+ T cells, which are also present at the psoriatic plaque. We have addressed the functional characterization of this CD8+ subtype of T lymphocytes from psoriasis patients. Our results show that CD8+IL‐17+ cells from psoriasis‐inflamed skin tissue produce TNF‐α and IFN‐γ (Th1‐related cytokines) as well as IL‐17, IL‐21, and IL‐22 (Th17‐related cytokines) efficiently. A significant up‐regulation of the RORC transcription factor is also observed. These cells are refractory to Tregs but show a proliferative response to anti‐CD3/CD28 stimulation that is enhanced by IL‐12 and IL‐15. Blocking of TNF‐α activity inhibits TCR‐mediated activation and IL‐17 production. CD8+IL‐17+ T cells are cytotoxic cells that display TCR/CD3‐mediated cytotoxic abilities to kill target cells. Thus, CD8+IL‐17+ T cells share some key features with Th17 cells and exhibit remarkable differential abilities attributable to the CD8+ lineage of T lymphocytes, adding new insights into the functional resources of IL‐17‐producing cells from human epidermis that could be of potential interest to our understanding of the pathogenesis of psoriasis.
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