Abstract
Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.
Highlights
Produced primarily by activated T cells, IL-2 influences critical aspects of the immune response and homeostasis
To evaluate the signaling capacity of the muteins independently of their affinity for CD25, we evaluated the phospho STAT5 (pSTAT5) response in cells that are negative for CD25. pSTAT5 responses in these cells, shown here by pSTAT5 data from CD25– Tconv cells (Figure 2B) and natural killer (NK) cells (Supplementary Figure 2), are significantly weaker compared to the CD25+ gated cells, and these represent IL-2 mutein activities generated solely through IL2RB and IL2RG We note that the recombinant IL-2 showed stronger activity than the wildtype IL-2 in our molecular format, which may be due to aggregation and a resulting increase in avidity
Since we observed that foxp3 transcript levels diminished with decreased IL-2 activity, we evaluated whether IL-2 activity is required for continuous maintenance of demethylation in Treg-specific demethylated region (TSDR) by performing bisulfite sequencing analysis of genomic DNA from purified Treg cells stimulated with wildtype IL-2 of IL-2 muteins
Summary
Produced primarily by activated T cells, IL-2 influences critical aspects of the immune response and homeostasis. IL-2 serves dual opposing functions; it potently amplifies proliferative responses of effector T (Teff) and natural killer (NK) cells, while regulating immune homeostasis by driving regulatory T (Treg) cell proliferation, differentiation, and function [review by Abbas et al [1]]; and both axes have been leveraged to treat human diseases. The therapeutic dose range is narrow, as doses that induce more robust Treg cell responses simultaneously drive Teff and NK cells activity, which can reduce efficacy or lead to disease exacerbation and/or toxicity [3, 4, 7, 12]
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