Interleukin-2 (IL-2), the first cytokine that was molecularly cloned, was shown to be a T cell growth factor essential for the proliferation of T cells and the generation of effector and memory cells. On the basis of this activity, the earliest therapeutic application of IL-2 was to boost immune responses in cancer patients. Therefore, it was a surprise that genetic deletion of the cytokine or its receptor led not only to the expected immune deficiency but also to systemic autoimmunity and lymphoproliferation. Subsequent studies established that IL-2 is essential for the maintenance of Foxp3 regulatory T cells (T cells), and in its absence, there is a profound deficiency of T cells and resulting autoimmunity. We now know that IL-2 promotes the generation, survival, and functional activity of T cells and thus has dual and opposing functions: maintaining T cells to control immune responses and stimulating conventional T cells to promote immune responses. It is well documented that certain IL-2 conformations result in selective targeting of T cells by increasing reliance on CD25 binding while compromising CD122 binding. Recent therapeutic strategies have emerged to use IL-2, monoclonal antibodies to IL-2, or IL-2 variants to boost T cell numbers and function to treat autoimmune diseases while dealing with the continuing challenges to minimize the generation of effector and memory cells, natural killer cells, and other innate lymphoid populations.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism . Previous studies showed that IL-2 is highly flexible 8,9 and exists in different conformations that favor either the high-affinity trimeric IL-2R or intermediate-affinity dimeric IL-2R, resulting in the activation of different immune cells 9 . This plasticity has complicated the use of the approved drug Proleukin at high doses to treat metastatic melanoma and renal cell carcinoma 10 , due to the role of IL-2 as an essential growth factor for T regs [11][12][13] . Moreover, adverse effects of high-dose IL-2 therapy have greatly limited its use 14,15 . Several studies have shown that low-dose IL-2 therapy preferentially activates T regs due to the constitutive high expression of IL-2Rα 16 and other cell-intrinsic factors that increase signal transduction sensitivity 17 . Treatment of mice and humans with low doses of IL-2 has been shown to ameliorate autoimmune diseases and graft-versus-host disease (GvHD) as well as delaying organ allograft rejection [18][19][20][21][22] . However, IL-2 therapy has some limitations, including difficulty in predicting the efficacious dose, off-target effects on different cell populations and a short in vivo half-life 23,24 . Thus, attempts have been made to engineer or modify the IL-2 structure to improve its therapeutic potential by modulating its ability to selectively target either T effs or T regs [25][26][27][28][29][30][31] . Selective antibodies against IL-2 can alter its conformation by binding a number of potential epitopes, thereby modifying the binding interaction of IL-2 to any of the IL-2R subunits and resulting in selective expansion of T regs or T eff cell subsets 32,33 . For example, it has been demonstrated that a rat anti-mouse IL-2 monoclonal antibody (JES6-1) can be administered in complex with wild-type mouse IL-2 and used to preferentially enhance T reg populations 26 . Binding of JES6-1 to IL-2 alters its conformation to lower the affinity of mIL-2 for CD25, such that CD25 high T regs compete favorably for IL-2 binding and expansion against T effs 33 . The therapeutic potential of IL-2 to selectively activate the tolerogenic immune response, combined with the imperative to develop a human T reg -selective IL-2 compound, led us to develop a mechanism-based screening strategy to identify human antibodies against human IL-2 that exhibit an in vivo T reg potentiation profile when complexed with hIL-2. This class of monoclonal antibody, exemplified by F5111.2, blocked IL-2Rβ binding and reduced IL-2Rα binding to IL-2, and, when administered in complex with hIL-2, preferentially promoted T reg expansion and was effective in models of autoimmune disease including type 1 diabetes and experimental autoimmune encephalomyelitis (EAE) in addition to GvHD. ResultsSelective IL-2 stimulation in T regs . To directly compare T reg and T eff sensitivity to IL-2, the pSTAT5 signaling response of T regs was analyzed in a mixed population of peripheral blood mononuclear cells (PBMCs).

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Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γ]). IL-2Rα, which is highly expressed on regulatory T (T) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward T cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating T expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

In this issue of Immunity, Spangler et al. and Mitra et al. demonstrate how structural changes in the IL-2 molecule alter interactions with the IL-2 receptor, leading to differential cellular targeting and biochemical responses and selective immune consequences.