Leo Chen

Direct Expansion of Human Allospecific FoxP3+CD4+ Regulatory T Cells with Allogeneic B Cells for Therapeutic Application

Compelling evidence from animal studies has demonstrated that allospecific FoxP3+CD4+ regulatory T (Treg) cells expanded ex vivo can be used as effective therapeutic tools in the treatment of allograft rejection and graft-vs-host disease. Despite the promising results from animal studies, there remain major barriers to developing Treg cell-based immunotherapy in humans. Currently, no effective approach has been established for selective expansion of human allospecific Treg cells ex vivo. Additionally, the very low frequency of Treg cells present in human peripheral blood could pose a formidable challenge to obtaining a sufficient number of Treg cells from a single donor for ex vivo expansion for therapeutic utilization. Extending our recent finding that mouse B cells preferentially induce expansion of alloreactive Treg cells, we report herein that human Treg cells can be expanded ex vivo with allogeneic B cells. The expanded Treg cells express very high levels of FoxP3, maintain anergic phenotype, and are potent suppressors capable of inhibiting the alloproliferation of third-party responder T cells at very low Treg-to-T effector cell ratio in an alloantigen-specific manner. The alloantigen specificity demonstrated by B cell-expanded Treg cells is not determined by the HLA haplotypes of the Treg cells, but it is induced and determined by the haplotype of the B cells used to expand them. Our findings represent a significant advance in the development of Treg cell-based immunotherapy in humans and raise the possibility of using third-party Treg cells for therapeutic applications.

All-trans retinoic acid and rapamycin synergize with transforming growth factor-β1 to induce regulatory T cells but confer different migratory capacities

Tregs play important roles in maintaining immune homeostasis, and thus, therapies based on Treg are promising candidates for the treatment for a variety of immune‐mediated disorders. These therapies, however, face the significant challenge of obtaining adequate numbers of Tregs from peripheral blood that maintains suppressive function following extensive expansion. Inducing Tregs from non‐Tregs offers a viable alternative. Different methods to induce Tregs have been proposed and involve mainly treating cells with TGF‐β‐iTreg. However, use of TGF‐β alone is not sufficient to induce stable Tregs. ATRA or rapa has been shown to synergize with TGF‐β to induce stable Tregs. Whereas TGF‐β plus RA‐iTregs have been well‐described in the literature, the phenotype, function, and migratory characteristics of TGF‐β plus rapa‐iTreg have yet to be elucidated. Herein, we describe the phenotype and function of mouse rapa‐iTreg and reveal that these cells differ in their in vivo homing capacity when compared with mouse RA‐iTreg and mouse TGF‐β‐iTreg. This difference in migratory activity significantly affects the therapeutic capacity of each subset in a mouse model of colitis. We also describe the characteristics of iTreg generated in the presence of TGF‐β, RA, and rapa.

A Novel mTORC1-Dependent, Akt-Independent Pathway Differentiates the Gut Tropism of Regulatory and Conventional CD4 T Cells

The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4+ conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only ∼50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally occurring Treg or induced Treg in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4β7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4β7 is dependent on activation of a mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, because specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3 did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [mTORC]2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes.

Murine Tim-1 is excluded from the immunological synapse

The interaction between T cells and APCs bearing cognate antigen results in the formation of an immunological synapse (IS). During this process, many receptors and signaling proteins segregate to regions proximal to the synapse. This protein movement is thought to influence T cell function. However, some proteins are transported away from the IS, which is controlled in part by ERM family proteins. Tim-1 is a transmembrane protein with co-stimulatory functions that is found on many immune cells, including T cells. However, the expression pattern of Tim-1 on T cells upon activation by APCs has not been explored. Interestingly, in this study we demonstrate that the majority of Tim-1 on activated T cells is excluded from the IS. Tim-1 predominantly resides outside of the IS, and structure/function studies indicate that the cytoplasmic tail influences Tim-1 polarization. Specifically, a putative ERM binding motif (KRK 244-246) in the Tim-1 cytoplasmic tail appears necessary for proper Tim-1 localization. Furthermore, mutation of the KRK motif results in enhanced early tyrosine phosphorylation downstream of TCR/CD28 stimulation upon ectopic expression of Tim-1. Paradoxically however, the KRK motif is necessary for Tim-1 co-stimulation of NFAT/AP-1 activation and co-stimulation of cytokine production. This work reveals unexpected complexity underlying Tim-1 localization and suggests potentially novel mechanisms by which Tim-1 modulates T cell activity.