CD4+CD25+Foxp3+ regulatory T cells (Tregs) were proposed in Pandiyan et al. (Nature Immunology, 2007) to suppress effector CD4+ T cells by consuming common gamma-chain cytokines, chiefly interleukin-2 (IL-2), thereby causing Bim-dependent 'cytokine deprivation' apoptosis (Bim being a proapoptotic member of the Bcl-2 protein family) of the responder cells. Later work established IL-2 consumption by Tregs as a real, quantifiable in vivo phenomenon and extended it to CD8+ T cells and natural killer (NK) cells, but simultaneously contested whether responder-cell apoptosis is actually required for suppression and showed Treg IL-2 capture is dispensable for control of CD4+ T cells. The surviving open problem is the in vivo necessity and quantitative share of cytokine deprivation as a suppressive mechanism: whether responder T cells made resistant to this death pathway escape Treg suppression in vivo is the subject of an unresolved published dispute, and no quantitative in vivo decomposition of Treg suppressor function exists that separates cytokine deprivation — including its apoptosis-dependent component (deprivation-induced death) and its non-lethal effects — from the other suppressive effectors.

Background

Regulatory T cells (Tregs) are a thymus-derived subset of CD4+ T lymphocytes marked by expression of the transcription factor Foxp3, which both specifies their lineage and drives their suppressive program; mice or humans lacking Foxp3 (the scurfy mouse, or human IPEX — immunodysregulation polyendocrinopathy enteropathy X-linked — syndrome) develop fatal systemic autoimmunity, which is why Foxp3-deficient phenotypes serve as a reference for what failure of Treg-mediated tolerance looks like. A long-standing question is the molecular mechanism(s) by which Tregs suppress other immune cells. Pandiyan, Zheng, Ishihara, Reed and Lenardo (Nature Immunology 8:1353–1362, 2007) reported that Tregs induce apoptosis of effector CD4+ T cells in vitro and in vivo (in a T cell transfer model of inflammatory bowel disease), that this death requires Bim, and that common gamma-chain cytokines (the interleukin family sharing the receptor subunit gamma-chain, such as IL-2, IL-4, IL-7, IL-15, and IL-21) block it. Bim is a proapoptotic 'BH3-only' member of the Bcl-2 protein family; when cytokines are withdrawn, declining activity of Akt (a key survival-signaling kinase inside T cells) allows Bim and related BH3-only proteins (e.g., Puma) to activate the mitochondrial apoptosis pathway — so-called cytokine-deprivation apoptosis, or 'death by neglect'. Because Tregs express high levels of CD25 (the alpha-chain of the IL-2 receptor) but cannot produce IL-2 themselves, the authors proposed that Tregs act as a local 'cytokine sink' that starves IL-2-producing effector T cells of the cytokine they need to survive, and concluded that cytokine deprivation–induced apoptosis is 'a prominent mechanism' of Treg suppression. Subsequent work gave this framework quantitative substance: mathematical and single-cell analyses showed that IL-2 in lymphoid tissue is a short-range resource shaped by competition between producer (effector) and consumer (Treg) cells (Busse et al., PNAS 2010; Oyler-Yaniv et al., Immunity 2017), and in vivo studies showed Tregs restrain CD8+ T cell effector pools by restricting IL-2 (Kastenmüller et al., Journal of Immunology 2011), control natural killer (NK) cells in autoimmune insulitis by depriving them of IL-2 (Sitrin et al., JEM 2013), and modulate IL-2 availability during influenza infection (León et al., Nature Communications 2014). However, the causal heart of the 2007 model was challenged: Szymczak-Workman, Delgoffe, Green and Vignali (Journal of Immunology 2011) showed Tregs suppress conventional T cells that are genetically resistant to apoptosis (Bim-deficient, Bim/Puma-double-deficient, or Bcl-2-overexpressing) in several in vitro and in vivo assays, prompting a published exchange with the authors (Pandiyan and Lenardo, Journal of Immunology 2012); and Chinen et al. (Nature Immunology 2016) showed, using Treg-specific IL-2 receptor gain- and loss-of-function genetics, that IL-2 capture by Tregs is dispensable for control of CD4+ T cells in vivo (though important for limiting CD8+ activation), while signaling via STAT5 (the transcription factor activated downstream of cytokine receptors such as the IL-2 receptor) inside the Treg is essential. Current reviews (e.g., Boyman and Sprent, Nature Reviews Immunology 2012; Wang et al., Signal Transduction and Targeted Therapy 2025) accordingly list cytokine consumption as one mechanism among several — the others including CTLA-4 (a Treg surface receptor that blocks T-cell costimulatory signals), adenosine generated by the CD39/CD73 ectoenzymes (cell-surface enzymes that convert extracellular ATP to the immunosuppressive molecule adenosine), IL-10, and TGF-β — and describe the relative in vivo importance of Treg suppressor mechanisms as still unsettled. The genetic-resistance test bearing on this dispute has itself been performed in part and remains contested: Pandiyan et al. (2007) reported that Bim-deficient responder T cells resist Treg-mediated suppression and that Tregs suppress inflammatory bowel disease by inducing apoptosis of colitogenic cells in a Bim-dependent way (the paper's Figures 6 and 8), whereas Szymczak-Workman et al. (2011) found suppression of apoptosis-resistant responders intact in in vivo homeostasis and colitis-recovery assays, and Pandiyan and Lenardo (2012) attributed the discrepancy to the timing of Treg transfer relative to responder activation — the later transfer having, in their words, 'missed the window at which the effector cells are sensitive to cytokine deprivation' — and to the absence of direct in vivo measurement of apoptotic-cell frequency. What remains open is the resolution of this dispute under its contested conditions, and a quantitative in vivo decomposition of Treg suppressor function between cytokine deprivation, including its apoptosis-dependent component, and the other suppressive effectors.

Problem Statement

Determine, in vivo, the necessity and quantitative contribution of Treg-mediated cytokine deprivation as a mechanism of immune suppression and tolerance, as left open by Pandiyan et al. (2007) and sharpened by the subsequent published dispute. Concretely: (i) do responder T cells rendered resistant to cytokine-deprivation apoptosis — for example by combined deficiency of the proapoptotic Bcl-2 family effectors that execute it (Bim together with related BH3-only proteins such as Puma, or enforced Bcl-2 overexpression) — escape Treg-mediated suppression in physiological settings? A result that settles the published dispute must address its contested conditions: the timing of Treg availability relative to responder activation (Pandiyan and Lenardo 2012 contested the 2011 in vivo assays because Tregs were transferred only after disease onset, missing the window of cytokine-deprivation sensitivity), direct measurement of responder apoptosis in vivo, and controls distinguishing escape from Treg suppression from the autoimmune and lymphoproliferative disease intrinsic to apoptosis-resistant strains such as Bim-deficient and Bcl-2-transgenic mice (Pandiyan, Zheng and Lenardo, Frontiers in Immunology 2011). (ii) In a defined tissue and inflammatory context (including infections where survival cytokines are abundant), what fraction of Treg suppressor function is attributable to depriving responder cells of common gamma-chain cytokines, versus Treg-intrinsic IL-2 receptor/STAT5-dependent functions (STAT5 being the transcription factor activated by IL-2 receptor signaling, shown by Chinen et al. 2016 to be separable and essential) and other suppressive effectors such as CTLA-4 (a Treg receptor that dampens T-cell costimulation), adenosine, IL-10, and TGF-β? The deprivation-attributable fraction must separate the apoptosis-dependent component (deprivation-induced death of responder cells) from non-lethal deprivation effects (impaired proliferation and cytokine production through interruption of IL-2 feedback), and must be reported against a stated suppressor-function readout that defines the denominator of the fraction. A complete answer establishes whether — and, in defined in vivo settings, how much — responder-cell cytokine deprivation, including its apoptosis-dependent component, is a load-bearing mechanism of Treg-mediated tolerance in vivo.

References

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