Introduction: Harnessing regulatory immune populations in cell-based therapies for transplantation is a promising tolerance-promoting approach. Innate lymphoid cells (ILCs) are a recently discovered family of lymphocytes that lack antigen specificity and instead rapidly respond to tissue signals and orchestrate immune responses through cytokines to maintain homeostasis¹. Recent studies have revealed that a subset of murine group 2 ILCs are able to produce IL-10 (referred to as ILC2₁₀) and limit inflammation in mouse models of graft-versus-host disease (GVHD), islet transplant and ischemia reperfusion injury, providing evidence that these cells have tolerance-promoting functions in a wide variety of inflammatory contexts^2,3. In this study, we examine the cell therapy potential of human ILC2₁₀ to limit allogeneic T cell responses using a pre-clinical model of GVHD, and examined mechanism by which ILC2₁₀ inhibit pathogenic T cell responses.
Methods: We developed methods to isolate and expand human ILC2s and ILC3s from blood in a manner that maintains expression of signature cytokines and transcription factors. Single cell RNAseq and flow cytometric analysis revealed ILC2s expanded using our approach expressed high levels of the regulatory cytokine IL-10, consistent with ILC2₁₀ identity. Ability to suppress allogeneic T cell responses was evaluated in a xenogeneic GVHD model, in which NOD-scid IL2Rγ^null (NSG) mice are given human PBMCs that cause multi-organ tissue pathology. Effects of a single infusion of expanded allo-ILC2₁₀ at the time of PBMC transfer or following xenogeneic GVHD onset was evaluated. Ability to regulate T cells in vivo and in vitro were assessed using flow cytometry, and secreted cytokines measured using cytometric bead array. Role of various secreted factors in ILC2₁₀-mediated regulation of T cells was evaluated in co-cultures assays where T cells were activated and cultured with allogeneic ILC2₁₀ in the presence or absence of neutralization antibodies.
Results: We show a single infusion of ex vivo expanded ILC2₁₀ decreases the severity of xenogeneic GVHD and prolongs survival of NSG mice. This infusion of ILC2₁₀ limited allogenic T cell proliferation and tissue infiltration while also reducing proportions of IFN-γ producing CD4+ T helper 1 cells and cytotoxic CD8+ T cells in vivo. In vitro co-cultures revealed direct ability of ILC2₁₀ to suppress CD4+ and CD8+ IFN-γ production. Neutralizing experiments demonstrate ILC2₁₀ regulation of T cells involves both IL-10 and IL-4.
Conclusion: Collectively, our findings support that human ILC2₁₀ may have applications in adoptive cell-based therapies for transplantation and demonstrate direct ability to suppress allogeneic T cell responses both in vitro and in vivo.