This study evaluated the effects of a novel immunotherapeutic, IRX-2, on T cell polarization in an in vitro model simulating the human tumor microenvironment. In this model, IRX-2 prevented the induction of inducible Treg (Tr1) and favored the differentiation of Teff.
We previously reported the increased frequency of Treg in the peripheral blood and within tumor-infiltrating lymphocytes (TIL) of HNC patients [14
]. The prognostic impact of Treg accumulations in HNC is unknown [14
], although in other human solid tumors, the increased Treg frequency among TIL has been variously linked to better or worse prognosis [17
]. In HNC, infiltrations of primary tumors with activated CD4+
T cells have been reported to correlate with improved overall survival [21
]. This suggests that therapies increasing Teff within TIL could be beneficial in HNC and that alterations in the tumor microenvironment could benefit antitumor immunity.
T cells are characterized by plasticity [12
T cells cultured in the absence of IRX-2 acquired the Treg phenotype and immunosuppressive cytokine profile. In contrast, in the presence of IRX-2 CD4+
T cells had a phenotype characteristic of Teff and produced IFN-γ rather than TGF-β1 and IL-10. Thus, IRX-2 favored CD4+
T cell differentiation into Teff rather than Tr1. Functionally, T cells cultured in the absence of IRX-2 were strongly suppressive, while T cells polarized by IRX-2 mediated significantly reduced suppression. IRX-2 did not completely prevent the induction of Tr1 but rather shifted the ratio of Treg/Teff towards Teff. Specifically, the average TGF-β1/IFN-γ ratio was 6:1 without IRX-2 vs. 1:1.2 with IRX-2. IRX-2 did not affect T cell proliferation or their viability, suggesting that it does not eliminate any T cells but rather expands Teff.
We and others have reported that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin promotes Treg expansion [22
], while activation of the Akt–mTor pathway antagonizes the induction of FOXP3+
]. A significant increase in the level of pAkt is observed when T cells are cultured in the presence of IRX-2. In human Treg, the restoration of Akt activity reduces Treg-mediated suppression [25
]. Thus, enhanced Akt phosphorylation by IRX-2, leading to activation of the Akt–mTOR pathway, is a likely mechanism responsible for shifting the balance toward Teff differentiation and reduction of FOXP3 expression in the co-culture system. Also, IRX-2-mediated Akt activation was shown to protect CD4+
T cells from tumor-induced apoptosis [7
The differentiation of Teff is known to be influenced by IFN-γ, which stabilizes T-bet expression [26
]. Also, IFN-γ facilitates the conversion of CD4+
cells into functional CD4+
Treg in humans and mice [27
]. Thus, its presence in the co-culture could influence differentiation of Teff as well as Treg. IRX-2 contains considerable levels of IFN-γ (e.g., 2.2 ng/mL). Thus, T-bet expression and thereby Teff induction in the presence of IRX-2 might be driven by IFN-γ. IRX-2 contains multiple naturally occurring cytokines, and it is possible that more than one cytokine contributes to a shift in the phenotype and function of cultured T cells.
IL-2 is known to promote T cell-mediated antitumor immunity in patients with cancer [28
]. It is also required for Treg expansion and activity [29
]. In renal cell carcinoma and melanoma patients treated with a high dose of rhIL-2, significant increases of Treg in the peripheral circulation were reported [13
]. In contrast, we did not observe increases in numbers or frequency of Treg in HNC patients treated with neoadjuvant IRX-2 in the phase II trial, although IRX-2 contained ~6 ng (i.e., 125 IU/mL) of IL-2. Furthermore, an increase in lymphocytic infiltrates in tumor samples obtained from HNC patients before and after the neoadjuvant IRX-2 treatment was associated with improved survival [Berinstein et al., submitted].
In vivo, Treg survival critically depends on the activation of the IL-2 receptor and STAT5 signaling [29
]. CD25- or CD122-deficient mice lack Treg and STAT5-deficient mice show dramatically decreased Treg frequency because Foxp3 expression is directly controlled by activation of STAT5 [31
]. It has also been suggested that the inhibition of Akt/mTOR pathway in the presence of IL-2 leads to persistent activation of STAT5 and Treg expansion [23
]. Limiting STAT5 phosphorylation decreases Treg suppressor function [32
]. IL-2-induced STAT5 activation provides an explanation for the increased Treg frequency in cancer patients treated with rhIL-2 [13
]. The observed lower pSTAT5 levels in T cells cultured with IRX-2 suggests that IRX-2 favors Teff differentiation rather than Treg expansion. Further, IRX-2 did not increase the Treg frequency or numbers in the peripheral circulation of patients treated with IRX-2 in the recent phase II clinical trial [Berinstein et al., submitted].
In tumor-bearing hosts, the induction of robust antitumor responses is desirable. The ability to manipulate the tumor microenvironment so that it favors the differentiation of Teff cells rather than Treg represents a promising immunotherapeutic strategy. While many current therapies aim at eliminating or silencing Treg, the alternative approach of protecting and augmenting functions of Teff is equally important. In this study, using an in vitro model simulating the tumor microenvironment, we show that IRX-2 promotes Teff differentiation and helper functions without inducing Treg. In the clinic, IRX-2 administered to the vicinity of tumor-draining LN in HNC patients exerted multiple effects on immune cells and prolonged survival [Berinstein et al., submitted]. Thus, IRX-2 might be a promising component of future cancer therapies.