Takatoshi Chinen and Alexander Y Rudensky are at Memorial Sloan Kettering Cancer Center, New York, NY. Arun K Kannan is in the immunology discovery division of Biogen in Cambridge MA.
Therapies that aim to harness the immune system to target and disable cancer cells have stoked tremendous hope and in have shown promise. One success story is ipilimumab (Yervoy; Bristol-Myers Squibb) which was approved for the treatment of metastatic melanoma in 2011. Ipilimumab is a monoclonal antibody that acts by disrupting endogenous mechanisms within T cells that otherwise reduce their level of activation. In clinical trials, a significant increase in survival time could be attributed to ipilimumab for those whose disease was unresponsive to standard chemotherapy. Nevertheless, trial participants suffered complications including gastrointestinal, hepatic and skin inflammation. These observations reinforce the call for a better understanding of immune reactions induced by immunotherapy, and more generally, how a proper balance between immune activation and inhibition is achieved (Nature Med. 2017).
What are the signaling mechanisms that immune cells use to mediate system-wide immune repression?
Regulatory T cells (Tregs) play an important role in restraining the immune system by maintaining peripheral tolerance, preventing autoimmune disorders and limiting chronic inflammation (Vignali 2008). Accordingly, quantitative deficits in Treg cells in humans and experimental mouse models leads to multiple organ-specific autoimmune diseases. This is consistent with the notion that the balance between Tregs and effector T cells (i.e. CD4+ helper and CD8+ cytotoxic) is dysregulated in autoimmune and inflammatory disorders. A major challenge is to develop a comprehensive account of the mechanisms through which Tregs repress immune responses and the relative importance of each in varying contexts.
Tregs have been shown to act through mechanisms that involve direct cell-contact, such as modulating antigen presenting cell (APC) competency, as well as by modulating the extracellular milieu of effector T cells (Figure 1). Signalling by IL-2 has been the focus of much research as it is essential for the development, homeostasis and function of both Tregs and effector T cells (Klatzmann 2015) (Figure 1c).
Here we describe a study by Chinen et al. (Chinen 2016) who dissected the IL-2 signalling pathway in Tregs in order to establish the means by which its components impact effector T cell suppression. The authors suggested at least three (not necessarily mutually exclusive) ways in which the IL-2 receptor might mediate its effects: Facilitate homing of Tregs towards self-reactive T cells which actually produce IL-2 ligand; IL-2 sequestration and deprivations; and intracellular signalling to support maintenance, proliferation and function via JAK-STAT5, PI3K-Akt or Ras-ERK pathways (Li 2016) (Figure 2).
In Tregs, IL-2 binds to the extracellular domain of IL-2R which is comprised of three polypeptide chains: An -chain, a -chain and a common -chain (Figure 2). Cytosolic membrane signals are transmitted via JAK kinase which in turn phosphorylates the cytoplasmic mediator Signal Transducer and Activator of Transcription 5 (STAT5), consisting of a heterodimer of STAT5A and STAT5B. A master regulator of Treg cells is the forkhead box 3 (Foxp3) gene, a member of the forkhead/winged-helix family of transcription factors (Rudensky 2011). The consensus view of Foxp3 function is to amplify and stabilize features of Treg cells that are previously conferred by T cell receptor (TCR) and cytokine signaling (Figure 2).
In order to definitively establish the role of IL-2R in Treg cell function in vivo, the authors employed a transgenic mouse that disabled il2ra or il2rb only within cells that express Foxp3, that is, the Treg lineage. As expected, deletion of either receptor chain resulted in an early onset of fatal autoimmune inflammatory disease and lymphoproliferation. These phenotypes were accompanied by a drastic reduction in tyrosine-phosphorylation of STAT5 (Figure 2). Accordingly, ablation of stat5a and stat5b in peripheral Fox3p-expressing cells mimicked the autoimmune phenotype observed in IL-2R mutants. Finally, constitutive expression of stat5b in an il2ra-deficient strain (i.e. unable to sequester IL-2) was sufficient to rescue the systemic inflammation and early fatal disease. These observations are consistent with a requirement for cell-intrinsic IL-2R signalling in Tregs.
In contrast to the rescue of early inflammatory disease, constitutive activation of stat5b only marginally restrained expansion of CD8+ T cells in later weeks (3-12) and these mice eventually succumb to disease, suggesting a STAT5 signalling-independent mechanism of suppression. Indeed, application of IL-2-neutralizing antibody in il2rb-deficient mice with constitutive expression of stat5b (i.e. stat5bCA) at 5 - 7 days of age completely suppressed CD8+ T cell activation and proliferation. These observations are consistent with the notion that Tregs suppress CD8+ T cells via IL-2 depletion.
Next, Chinen et al. turned their attention to the question of what mechanisms downstream of STAT5 signaling potentiated Treg suppression. Interestingly, the authors demonstrated that constitutive stat5b expression did not increase suppression by augmenting the number of Tregs but rather, it enhanced the suppressive potential of each individual cell. Moreover, previous work showed that large increases in STAT5 did not translate into increased genome binding of FOX3P (Arvey 2014).
To gain more insight into the qualitative differences conferred by persistent STAT5 signalling, the authors compared gene expression in Tregs with constitutive stat5b expression relative to controls. Amongst ~11 000 genes analyzed, there were 342 genes up-regulated and 314 down-regulated. Analysis of signaling pathways was performed to better understand the cellular functions potentiated by activated STAT5: BiNGO was used to identify Gene Ontology (GO) terms enriched in the gene expression data (10 589 genes total). The authors chose to display the enriched gene sets using an Enrichment Map in which gene sets are represented by nodes in a network and shared genes via edges connecting nodes. The visual nature of the Enrichment Map aids in the interpretability of enrichment analyses by providing a visual ‘landscape’ of the gene sets and reducing complexity by clustering redundant gene sets.
The authors chose to display the enriched gene sets using an Enrichment Map in which gene sets are represented by nodes in a network and shared genes via edges connecting nodes.
The pathway enrichment analysis and resulting Enrichment Map enabled authors to quickly narrow in on gene set products involved in cell-cell and cell-extracellular matrix interactions, cell adhesion and cell locomotion (Figure 3). This finding led authors to speculate that STAT5 could potentiate interactions with target cells. Indeed, this notion is supported by previous imaging studies in vivo demonstrating Tregs in stable contact with dendritic cells (DC) and the fact that Treg-DC interactions have been shown to modulate their ability of DCs to potentiate effector T cell activation (Figure 1). To this end, Chinen et al. demonstrated that constitutive expression of stat5b in Fox3P-expressing cells promoted the formation of conjugates between Tregs and DCs (Figure 4). Thus, the Enrichment Map helped to unify two bodies of knowledge: The ability of Tregs to modulate T cell activation via DCs and IL-2R signaling via STAT5.
… the Enrichment Map helped to unify two bodies of knowledge: The ability of Tregs to modulate T cell activation via DCs and IL-2R signaling via STAT5.
Here, a pathway enrichment analysis coupled to an Enrichment Map presented groups of altered pathways drawn from the underlying patterns of gene expression. This approach provided a link between cytokine signalling in Tregs and their ability to interact with DCs that represents a plausible mechanistic basis for how Tregs negatively regulate system-wide repression of immune activation. This detailed understanding provides the basis of rational approaches towards optimal design of Treg-cell based therapies for a variety of autoimmune and inflammatory disorders. Conversely, this knowledge may contribute to efforts to counteract the potential side-effects of activating immunotherapies.