Improved therapeutic index of an acidic pH-selective antibody.

Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Regulatory T cells produce profibrotic cytokines in the skin of patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Pathologic conversion of regulatory T (Treg) cells into inflammatory cytokine-producing cells is thought to be an important step in the progression of autoimmunity, but whether loss of normal Treg cell function contributes to SSc is unknown. OBJECTIVE: We sought to determine whether Treg cells in the blood and skin of patients with SSc acquired abnormal production of effector cytokines. METHODS: Peripheral blood and skin biopsy specimens were collected from control subjects and patients with limited or diffuse SSc. Flow cytometry was used to evaluate expression of cell-surface proteins and the cytokine production profile of forkhead box protein 3-positive Treg cells compared with forkhead box protein 3-negative conventional T cells. RESULTS: Treg cells in the blood of patients with SSc had a normal phenotype and did not produce T-effector cytokines. In contrast, Treg cells from skin affected by SSc produced significant amounts of IL-4 and IL-13. Although Treg cells in the blood of patients with SSc did not make TH2 cytokines, they contained a significantly higher proportion of skin-homing cells expressing TH2 cell-associated chemokine receptors. Evidence that IL-33 caused the differentiation of skin Treg cells into TH2-like cells, combined with high tissue-localized expression of this cytokine in patients with SSc and expression of the ST2 chain of the IL-33 receptor on skin-localized Treg cells, suggests that IL-33 might be an important stimulator of tissue-localized loss of normal Treg cell function. CONCLUSION: These data are the first evidence for the presence of TH2-like Treg cells in human autoimmunity and show that Treg cell plasticity can be tissue specific. Localized dysfunction of Treg cells is a previously unknown factor that might contribute to fibrosis in patients with SSc.

The role of FOXP3 in regulating immune responses.

Regulatory T cells (Tregs) act in trans to control immune responses. The suppressive function of Tregs relies heavily on high and stable expression of the transcription factor FOXP3, which, together with other transcription factors, activates anti-inflammatory genes and represses proinflammatory genes. FOXP3 is required to shape the unique signaling mechanisms in Tregs, creating a positive-feedback pathway to further enhance its own expression. In addition, FOXP3 is thought to switch on a complex transcriptional network that leads to the stabilization of the Treg phenotype. Emerging data reveal that FOXP3 achieves this function in concert with several other transcription factors, many of which are associated with lineages of conventional T cells. In this review, we will discuss the structural features of FOXP3 and how it functions by interacting with other transcription factors. We will also summarize the role of FOXP3 in establishing the unique signaling cascades in Tregs. Finally, we will dissect the cooperative roles of FOXP3 and other T-cell lineage-defining transcription factors and discuss how these networks not only control the ability to Tregs to suppress different types of immune responses, but also enable Treg plasticity.

Helios+ and Helios- cells coexist within the natural FOXP3+ T regulatory cell subset in humans.

FOXP3-expressing T regulatory cells (Tregs) can be divided into two distinct subsets: naturally occurring Tregs (nTregs) that develop in the thymus, and induced Tregs (iTregs) that differentiate in peripheral tissues upon exposure to Ag in a tolerogenic environment. Recently it has been proposed that expression of Helios, an Ikaros family transcription factor, may specifically identify nTregs, allowing specific tracking of Tregs from different origins in health and disease. Surprisingly, we found that Helios(-) cells can be readily identified within naive (CD45RA(+)CD31(+)CCR7(+)CD62L(+)) FOXP3(+) Tregs, a finding inconsistent with the notion that lack of Helios expression identifies Ag-experienced iTregs that should express memory markers. To investigate the phenotype and function of naive Helios(+) and Helios(-) Tregs within the nTreg population, we isolated single-cell clones from each subset. We found that both Helios(+) and Helios(-) nTreg clones have a similar suppressive capacity, as well as expression of FOXP3 and cell surface proteins, including CD39 and CTLA-4. Helios(-) nTregs, however, produced significantly more CCL3 and IFN-γ compared with Helios(+) nTregs. Despite increased cytokine/chemokine production, Helios(-) FOXP3(+) nTreg clones were demethylated at the FOXP3 Treg-specific demethylated region, indicative of Treg lineage stability. When cultured under Th1-polarizing conditions, Helios(+) and Helios(-) nTreg clones had an equal ability to produce IFN-γ. Collectively, these data show that a lack of Helios expression does not exclusively identify human iTregs, and, to our knowledge, the data provide the first evidence for the coexistence of Helios(+) and Helios(-) nTregs in human peripheral blood.

T regulatory cell therapy in transplantation: stability, localization and functional specialization.

PURPOSE OF REVIEW: There is great hope that cellular therapy with regulatory T cells (Tregs) will be an effective way to induce alloantigen specific tolerance, ultimately allowing for reduction or elimination of nonspecific immunosuppression. In the past, considerable effort was focused on defining the optimal ways to isolate and expand Tregs from peripheral or cord blood. Now that expansion of therapeutically relevant numbers of Tregs is feasible, we need to consider what is going to happen to the cells when they are transferred in vivo. RECENT FINDINGS: For optimal function, Tregs must be able to traffic to the correct location(s) and, despite the presence of immunosuppressive therapy, live long enough to transfer their regulatory function to recipient T cells. Within the Treg pool, there are also functionally specialized subsets, identified by chemokine receptor expression and/or cytokine production, which control their trafficking and relative ability to suppress different types of T helper cells, respectively. Recent findings imply that the plasticity of appropriately obtained populations of Tregs may not be of as great concern as previously suggested. Experimental data have also provided evidence as to how one might design adjunctive treatment that best supports the viability and function of Tregs after transfer. SUMMARY: Knowledge of how Tregs work in transplantation comes from studies that do not recapitulate how these cells will be used in humans. There is a need to develop better preclinical models to study how the in-vivo function of human Tregs can be optimized to ensure they can meet the challenge of inducing transplantation tolerance.