Ex vivo expansion of regulatory T cell and T helper 2 cells using a hematosphere culture

Abstract

Regulatory T cells (T-reg) are important components of immune system required to understand the mechanistic details of cancer immunity and autoimmune diseases. However, reliable and efficient methods of regulatory T cell expansion have not been established yet. Here, we show that a human peripheral blood mononuclear cell (PBMNC) derived blood-born hematosphere (BBHS) culture without cytokine treatment increased T-reg and T helper 2 cell (Th2) populations in the cell suspension. We found that the isolated cells suspended around the hematospheres expressed T helper cell markers. Importantly, the Foxp3+/CD25+ T-reg and IL-4+/CD25+ Th2 cell populations in the cell suspension were greatly expanded during hematosphere culture. Through ELISA analysis of the supernatant, we showed that secretion of Th2-related cytokines such as IL-5, IL-10, and IL-13 also increased. Taken together, hematosphere culture is a good method for ex vivo expansion of T-reg and Th2, which can provide therapeutic benefits for the treatment of immune disorders.

Introduction

Regulatory T cells (T-reg) are widely involved in various autoimmune diseases and the immune system (Zou, 2006). They suppress the immune response and play a crucial role in inducing and maintaining peripheral tolerance (Weber et al., 2007). T-reg dysfunction is a common cause of autoimmune disease. For cancer patients, peripheral tolerance induced by T-regs inhibits anti-tumor immunity providing clinical value (deLeeuw et al., 2012). Therefore, many trials have been conducted to develop efficient methods for expanding T-regs. For example, Schmidt et al. reported that TGF-β treatment during in vitro differentiation results in the expansion of T-regs (Schmidt et al., 2016). Another group showed that rapamycin promotes the expansion of functional T-regs (Hippen et al., 2011). However, these expansion methods usually require cytokine cocktails or chemicals that are potentially toxic to cells, and which is not cost-effective, so there is currently no gold standard protocol for the ex vivo expansion of T-regs.

Whole mononuclear cells (MNCs) play a crucial function in the human immune system and are crucial for a variety of clinical and research applications. It is known that MNCs isolated from peripheral blood have robust hematosphere-forming capacities on a low attachment surface and high cell density. The hematosphere can mimic an in vivo niche by expressing critical components, such as the extracellular matrix and cytokines (Hur et al., 2011). These blood-borne hematospheres (BBHSs) increase the plasticity of monocytes and can differentiate into various cells depending on culture conditions (Hur et al., 2012). A previous study demonstrated that monocyte M2 polarization was increased in hematosphere cultures (Hur et al., 2013). Another report demonstrated that the secretion of anti-inflammatory cytokines from M2-polarized macrophages induces T helper 2 cell (Th2) and T-reg differentiation (Martinez and Gordon, 2014; Mantovani et al., 2004). Thus, these studies suggest that hematosphere culture can expand T-reg and Th2. Although, the single suspending cells (non-BBHS), which do not contain hematospheres, expressed T-cell markers such as CD3 (Hur et al., 2013), a detailed list of the cellular components of non-BBHS has not yet been reported.

In this study, we investigated the composition of non-BBHS and the cytokines secreted in the hematosphere culture. Throughout this analysis, we have established an optimal method for ex vivo expansion of T-reg cells that can be applied to clinical treatment.