Yenkel Grinberg-Bleyer

IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells

Regulatory T cells (T reg cells) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). Interleukin 2 (IL-2), a cytokine which promotes T reg cell survival and function, may thus have therapeutic efficacy in T1D. We show that 5 d of low-dose IL-2 administration starting at the time of T1D onset can reverse established disease in NOD (nonobese diabetic) mice, with long-lasting effects. Low-dose IL-2 increases the number of T reg cells in the pancreas and induces expression of T reg cell–associated proteins including Foxp3, CD25, CTLA-4, ICOS (inducible T cell costimulator), and GITR (glucocorticoid-induced TNF receptor) in these cells. Treatment also suppresses interferon γ production by pancreas-infiltrating T cells. Transcriptome analyses show that low-dose IL-2 exerts much greater influence on gene expression of T reg cells than effector T cells (T eff cells), suggesting that nonspecific activation of pathogenic T eff cells is less likely. We provide the first preclinical data showing that low-dose IL-2 can reverse established T1D, suggesting that this treatment merits evaluation in patients with T1D.

Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs

CD4+CD25+Foxp3+ Tregs play a major role in prevention of autoimmune diseases. The suppressive effect of Tregs on effector T cells (Teffs), the cells that can mediate autoimmunity, has been extensively studied. However, the in vivo impact of Teff activation on Tregs during autoimmunity has not been explored. In this study, we have shown that CD4+ Teff activation strongly boosts the expansion and suppressive activity of Tregs. This helper function of CD4+ T cells, which we believe to be novel, was observed in the pancreas and draining lymph nodes in mouse recipients of islet-specific Teffs and Tregs. Its physiological impact was assessed in autoimmune diabetes. When islet-specific Teffs were transferred alone, they induced diabetes. Paradoxically, when the same Teffs were cotransferred with islet-specific Tregs, they induced disease protection by boosting Treg expansion and suppressive function. RNA microarray analyses suggested that TNF family members were involved in the Teff-mediated Treg boost. In vivo experiments showed that this Treg boost was partially dependent on TNF but not on IL-2. This feedback regulatory loop between Teffs and Tregs may be critical to preventing or limiting the development of autoimmune diseases.

Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered with regulatory T cells for GVHD prevention

Recipient-specific regulatory T cells (rsTreg) can prevent graft-versus-host disease (GVHD) by inhibiting donor T-cell expansion after hematopoietic stem cell transplantation (HSCT) in mice. Importantly, in adult humans, because of thymus involution, immune reconstitution during the first months after HSCT relies on the peripheral expansion of donor T cells initially present in the graft. Therefore, we developed a mouse model of HSCT that excludes thymic output to study the effect of rsTreg on immune reconstitution derived from postthymic mature T cells present within the graft. We showed that GVHD prevention with rsTreg was associated with improvement of the limited immune reconstitution compared with GVHD mice in terms of cell numbers, activation phenotype, and cytokine production. We further demonstrated a preserved in vivo immune function using vaccinia infection and third-party skin-graft rejection models, suggesting that rsTreg immunosuppression was relatively specific of GVHD. Finally, we showed that rsTreg extensively proliferated during the first 2 weeks and then declined. In turn, donor Treg proliferated from day 15 on. Taken together, these results suggest that rsTreg GVHD prevention is associated with improved early immune reconstitution in a model that more closely approximates the biology of allogeneic HSCT in human adults.

Effector T Cells Boost Regulatory T Cell Expansion by IL-2, TNF, OX40, and Plasmacytoid Dendritic Cells Depending on the Immune Context

CD4+CD25+Foxp3+ regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4+ T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell–dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell–mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell–dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

Cutting Edge: NF-κB p65 and c-Rel Control Epidermal Development and Immune Homeostasis in the Skin

Psoriasis is an inflammatory skin disease in which activated immune cells and the proinflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced proinflammatory gene expression, and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with its partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis.

κB-Ras Proteins Regulate Both NF-κB-Dependent Inflammation and Ral-Dependent Proliferation

The transformation of cells generally involves multiple genetic lesions that undermine control of both cell death and proliferation. We now report that κB-Ras proteins act as regulators of NF-κB and Ral pathways, which control inflammation/cell death and proliferation, respectively. Cells lacking κB-Ras therefore not only show increased NF-κB activity, which results in increased expression of inflammatory mediators, but also exhibit elevated Ral activity, which leads to enhanced anchorage-independent proliferation (AIP). κB-Ras deficiency consequently leads to significantly increased tumor growth that can be dampened by inhibiting either Ral or NF-κB pathways, revealing the unique tumor-suppressive potential of κB-Ras proteins. Remarkably, numerous human tumors show reduced levels of κB-Ras, and increasing the level of κB-Ras in these tumor cells impairs their ability to undergo AIP, thereby implicating κB-Ras proteins in human disease.

The Alternative NF-κB Pathway in Regulatory T Cell Homeostasis and Suppressive Function

CD4+Foxp3+ regulatory T cells (Tregs) are essential regulators of immune responses. Perturbation of Treg homeostasis or function can lead to uncontrolled inflammation and autoimmunity. Therefore, understanding the molecular mechanisms involved in Treg biology remains an active area of investigation. It has been shown previously that the NF-κB family of transcription factors, in particular, the canonical pathway subunits, c-Rel and p65, are crucial for the development, maintenance, and function of Tregs. However, the role of the alternative NF-κB pathway components, p100 and RelB, in Treg biology remains unclear. In this article, we show that conditional deletion of the p100 gene, nfkb2, in Tregs, resulted in massive inflammation because of impaired suppressive function of nfkb2-deficient Tregs. Surprisingly, mice lacking RelB in Tregs did not exhibit the same phenotype. Instead, deletion of both relb and nfkb2 rescued the inflammatory phenotype, demonstrating an essential role for p100 as an inhibitor of RelB in Tregs. Our data therefore illustrate a new role for the alternative NF-κB signaling pathway in Tregs that has implications for the understanding of molecular pathways driving tolerance and immunity.

Abstract B060: The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response

Cancers use a wide variety of mechanisms to dampen tumor immune response. Among them, CD4+Foxp3+ regulatory T cells (Tregs) are largely described to inhibit the function of effector cells. In several murine models of cancer, Treg depletion suppresses tumor growth. Interestingly, it has been proposed that anti-CTLA-4 therapies exert their anti-tumor effects by targeting Tregs. Therefore, precisely understanding the mechanisms governing Treg homeostasis may be a valuable strategy to enhance immune responses against cancer. We and other have previously shown that transcription factor NF-κB plays a major role in Treg development; we are now analyzing the role of different NF-κB subunits in Tregs by using conditional knock-out mice. We found that both p65 (RelA) and c-Rel subunits of NF-κB in Tregs, were required for both development of thymic Treg progenitors and subsequent differentiation into Foxp3-expressing mature Tregs. Ablation of NF-κB also prevented in vitro differentiation of inducible Tregs. Furthermore, specific deletion of either p65 or c-Rel subunits in natural Tregs using FoxP3-cre led to the development of a lethal autoimmune syndrome by 8 to 15 weeks of age. Ablation of both subunits further shortened the lifespan of animals to around 3 weeks of age. This was associated with profound changes in the molecular signature of NF-κB deficient Tregs, which down-regulated Treg-associated genes while acquiring an effector-like phenotype. Also, tamoxifen-induced acute deletion of NF-κB in mature Tregs reduced their survival and expression of Treg-associated markers. These data highlight a crucial role for NF-κB in maintaining Treg identity in vivo. We next explored the role of NF-κB in Treg homeostasis during tumor growth. Strikingly, melanoma growth was drastically reduced in mice lacking c-Rel, but not p65, in Tregs. This was associated with an increased polyclonal and antigen-specific effector T cell activation. Besides, CD8 T cell depletion abolished the protection against tumor growth. We are now proceeding to test the effect of specific c-Rel inhibitors on tumor growth. We believe our work reveals a central molecular mechanism in the maintenance of Treg identity and thus highlights novel therapeutic target for the treatment of cancer. Citation Format: Yenkel Grinberg-Bleyer, Hyunju Ho, Roland Schmid, Ulf Klein, Sankar Ghosh. The canonical NF-κB pathway is crucial for Treg homeostasis during the antitumor response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B060.