Elsevier

Journal of Autoimmunity

Volume 44, August 2013, Pages 13-20
Journal of Autoimmunity

Differential targeting of IL-2 and T cell receptor signaling pathways selectively expands regulatory T cells while inhibiting conventional T cells

https://doi.org/10.1016/j.jaut.2013.06.009Get rights and content

Highlights

  • Conventional but not regulatory T cell proliferation requires PLCγ activation.

  • Cyclosporine A blocks conventional but not regulatory T cell proliferation.

  • Cyclosporine A combined with IL-2 prevents experimental autoimmune encephalomyelitis.

Abstract

Strategies to expand regulatory T cells hold therapeutic potential for ameliorating T cell-mediated autoimmunity. Recently, we reported that the requirements for T cell receptor signaling in conventional T cell and regulatory T cell proliferation are different. Using mutant mice that display defective T cell receptor-mediated phospholipase Cγ (PLCγ) activation, we hereby demonstrate that PLCγ activation is required for antigen-specific conventional T cell proliferation but not for IL-2-induced regulatory T cell proliferation. This led us to hypothesize that in conjunction with IL-2, pharmacological inhibition of T cell receptor-mediated PLCγ activation might offer a novel therapeutic strategy to expand regulatory T cells while simultaneously inhibiting conventional T cell proliferation. Indeed, using the calcineurin inhibitor Cyclosporine A to inhibit signaling downstream of PLCγ, we found that Cyclosporine A attenuated antigen-specific Tconv proliferation but permitted IL-2-induced regulatory T cell expansion in vitro and in vivo. Furthermore, the combination of Cyclosporine A and IL-2 was superior over either Cyclosporine A or IL-2 monotherapy in protection against the T cell-mediated demyelinating autoimmune disease mouse model, experimental autoimmune encephalomyelitis. Thus, a combination of TCR signaling inhibition and IL-2 might be a beneficial strategy in expanding regulatory T cells and inhibiting conventional T cell proliferation in autoimmune settings.

Introduction

Regulatory T cells (Treg)s are a subset of T cells with suppressive properties. They are crucial for protecting the host from autoimmunity by inhibiting the response of self-reactive T cells and for preventing immunopathology in over exuberant immune responses directed against foreign antigens [1], [2]. Because of their potent inhibitory function, therapies aimed at the selective expansion of Tregs hold therapeutic potential for ameliorating conventional T cell (Tconv)-mediated diseases [3], [4]. Indeed, previous studies in mice have demonstrated that Tregs can be selectively increased in vivo by pharmacologic intervention and that this expansion of Tregs leads to positive outcomes in a variety of Tconv-mediated diseases [5], [6], [7], [8], [9].

In order to devise strategies to increase Treg numbers in vivo, it is critical to understand the signaling mechanisms that lead to Treg proliferation. Several signaling pathways implicated in Treg proliferation include those driven by IL-2, co-stimulatory molecules, and the TCR [10], [11], [12], [13]. IL-2 is essential for the maintenance and proliferation of Tregs, as the acute neutralization of IL-2 collapses the homeostasis of Tregs [14] and the administration of IL-2 promotes Treg proliferation [5]. In addition to IL-2, Treg proliferation was believed to require the interaction of the TCR with MHC class II (MHCII) expressed on dendritic cells (DC)s. In support of this argument, adoptively transferred Tregs do not proliferate in MHCII-deficient hosts and the deletion of TCR signaling proteins in T cells leads to the decrease in Treg division and survival [10], [11], [15], [16]. However, we have recently shown that the provision of exogenous IL-2 induces the proliferation of Tregs adoptively transferred in MHCII-deficient hosts or when the Tregs lacked TCR signaling capacity [17]. This suggests that TCR signaling is dispensable for IL-2-induced Treg proliferation. As antigen-specific Tconv proliferation is entirely dependent on TCR stimulation, we sought to take advantage of the differential requirement of the TCR in the proliferation of these two T cell subsets. Thus, we tested whether the pharmacological inhibition of TCR signaling in combination with IL-2 could allow Tregs to selectively expand while simultaneously inhibiting the antigen-specific proliferation of Tconvs.

In the present manuscript, we demonstrate that costimulation but not TCR-activated phospholipase Cγ (PLCγ) is required for IL-2-induced Treg proliferation. Using the calcineurin inhibitor Cyclosporine A (CSA) to inhibit signaling pathways downstream of PLCγ, we show that in combination with IL-2, CSA increases Tregs while preventing antigen-specific Tconv proliferation. Moreover, CSA and IL-2 displayed an additive effect to protect against experimental allergic encephalomyelitis. Thus, a combination of TCR signaling inhibition and IL-2 might be beneficial to increase the Treg:Tconv ratio in treatment of autoimmunity.

Section snippets

Mice

C57BL/6 (B6), B6.SJL (CD45.1 congenic), and OT-II TCR transgenic mice were purchased from the National Cancer Institute or Taconic Farms (Germantown, NY). Mice expressing one loxp-flanked allele of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with either one allele of wild-type (WT) SLP-76 (cHet mice) or SLP-76 with a Y145F mutation (Y145F mice) were previously described [18], [19]. A Tamoxifen-inducible Cre was used for deletion of the loxp-flanked SLP-76 and a

Treg proliferation in vitro requires costimulation but not TCR signaling

One major signaling pathway downstream of the TCR occurs through PLCγ, which leads to Ca2+ flux and diacylglycerol-mediated signaling [23]. To test whether this TCR-activated pathway was required for Treg proliferation, we used Y145F mice that express a Tamoxifen-inducible Cre and one floxed and one Y145F mutant allele of SLP-76. T cells from Tamoxifen-treated Y145F mice exhibit defective PLCγ phosphorylation and Ca2+ flux [24]. Despite this defect, Tregs from Tamoxifen-treated cHet mice (one

Discussion

A deep understanding of the mechanisms that control Treg proliferation in vivo is necessary for enhancing clinical applications involving Tregs. TCR stimulation of Tregs has been considered necessary for Treg expansion, since Tregs adoptively transferred into MHCII-deficient hosts do not proliferate [15]. However, more recent studies have challenged this view by demonstrating that Tregs proliferate in the absence of MHCII in vitro if IL-2 is provided [20], [31]. This raised the possibility that

Conclusions

In summary, we took advantage of the differential requirement of TCR signaling in Tconv vs. Treg proliferation and showed that the combination of CSA and IL-2 expands Tregs, while simultaneously inhibiting antigen-specific Tconv proliferation. These effects translated to a beneficial and additive effect of IL-2 and CSA in EAE disease progression. Thus, a combination of TCR signaling inhibition and IL-2 might be a beneficial approach for expanding Tregs while simultaneously inhibiting Tconv

Acknowledgments

We thank the Kambayashi, Wu, Behrens, Nichols, and Koretzky lab members for helpful discussions. This work was supported by grants from the National Blood Foundation, the University of Pennsylvania internal funds, and the National Institutes of Health (K08HL086503, K08NS062138, R01HL111501).

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    These authors contributed equally to this work.

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