Michal Kuczma

Foxp3-Deficient Regulatory T Cells Do Not Revert into Conventional Effector CD4+ T Cells but Constitute a Unique Cell Subset

Homeostasis in the immune system is maintained by specialized regulatory CD4+ T cells (Treg) expressing transcription factor Foxp3. According to the current paradigm, high-affinity interactions between TCRs and class II MHC-peptide complexes in thymus “instruct” developing thymocytes to up-regulate Foxp3 and become Treg cells. However, the loss or down-regulation of Foxp3 does not disrupt the development of Treg cells but abrogates their suppressor function. In this study, we show that Foxp3-deficient Treg cells in scurfy mice harboring a null mutation of the Foxp3 gene retained cellular features of Treg cells including in vitro anergy, impaired production of inflammatory cytokines, and dependence on exogenous IL-2 for proliferation and homeostatic expansion. Foxp3-deficient Treg cells expressed a low level of activation markers, did not expand relative to other CD4+ T cells, and produced IL-4 and immunomodulatory cytokines IL-10 and TGF-β when stimulated. Global gene expression profiling revealed significant similarities between Treg cells expressing and lacking Foxp3. These results argue that Foxp3 deficiency alone does not convert Treg cells into conventional effector CD4+ T cells but rather these cells constitute a distinct cell subset with unique features.

TCR Repertoire and Foxp3 Expression Define Functionally Distinct Subsets of CD4+ Regulatory T Cells

Despite extensive research efforts to characterize peripheral regulatory T (Treg) cells expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and Ag specificities remain ambiguous. In this study, we identify and define two subsets of peripheral Treg cells differing in Foxp3 expression level and TCR repertoires. Treg cells expressing a high level of Foxp3 and TCRs not used by naive CD4+ T cells present a stable suppressor phenotype and dominate the peripheral Treg population in unmanipulated mice. The second Treg subset, expressing a lower level of Foxp3 and using TCRs shared with naive CD4+ T cells constitutes a small fraction of all Treg cells in unmanipulated mice and enriches Treg population with the same Ag specificities as expressed by activated/effector T cells. This Treg subset undergoes extensive expansion during response to Ag when it becomes a major population of Ag-specific Treg cells. Thus, Treg cells expressing TCRs shared with naive CD4+ T cells have a flexible phenotype and may down-regulate Foxp3 expression which may restore immune balance at the conclusion of immune response or convert these cells to effector T cells producing inflammatory cytokines.

MBD2 regulates TH17 differentiation and experimental autoimmune encephalomyelitis by controlling the homeostasis of T-bet/Hlx axis.

Unlike genetic alterations, epigenetic modifications are reversible and amenable to pharmacological interventions, which make them appealing targets for clinical therapy. However, little is known about epigenetic regulation in experimental autoimmune encephalomyelitis (EAE). Here we demonstrated that methyl-CpG-binding domain protein 2 (MBD2), an epigenetic regulator, controls autoimmunity and EAE through T-bet/Hlx. Tbx21 and Hlx underwent a DNA methylation turnover upon polarizations and a unique methylation pattern was essential for TH17 development. Loss of Mbd2 resulted in a defect for reading the information encoded by this methylation turnover, which disrupted the homeostasis of T-bet/Hlx axis and suppressed TH17 differentiation. DNA demethylation induced similar effect on helper T cell differentiation. Therefore, Mbd2(-/-) mice were completely protected from EAE. Pathogenic splenocytes isolated from wild-type mice challenged with MOG35-55 could adoptively transfer disease to Mbd2(-/-) mice. In addition, Mbd2(-/-) mice reconstituted with unstimulated wild-type splenocytes developed EAE as wild-type mice did. These data would provide novel insights into epigenetic regulation of EAE.

Connexin 43 Signaling Enhances the Generation of Foxp3+ Regulatory T Cells

Despite their importance for the functioning of the immune system, thymic development and peripheral maintenance of Foxp3+ regulatory T (TR) cells are poorly understood. We have found that connexin 43 (Cx43), expressed by thymic TR cells progenitors, supports TR development. Mice with deletion of the Cx43 gene induced in T cells produce only few TR cells and had increased proportion of activated T cells in the lymph nodes, suggesting impaired peripheral tolerance. Reduction of the TR cell numbers was accompanied by increased presence of CD4+CD25+GITR+Foxp3− T cells, which did not produce inflammatory cytokines and lost suppressor function. These results strongly argue that we have discovered a novel signaling pathway, controlled by Cx43, that enhances the generation of TR cells. We propose that a possible mechanism of Cx43 activity is by regulating Foxp3 expression in TR lineage cells.

Intratumoral Convergence of the TCR Repertoires of Effector and Foxp3+ CD4+ T cells

The presence of Foxp3+ regulatory CD4+ T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral Treg cells represent Treg cells pre-existing in healthy mice, or arise from tumor-specific effector CD4+ T cells and thus representing adaptive Treg cells. The generation of Treg population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of Treg cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied Treg cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4+ T cells and which preserve the heterogeneity of the Treg population. The majority of Treg cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4+ T cells. A small Treg subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and Treg cells. However, the population of Treg cells in tumors was dominated by cells expressing TCRs shared with effector CD4+ T cells. In contrast, Treg cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all Treg cells in tumor lesions. Our results suggest that the Treg repertoire in tumors is generated by conversion of effector CD4+ T cells or expansion of a minor subset of Treg cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4+ T cells and/or selectively inhibiting the expansion of a minor Treg subset.

IL-7 signaling imparts polyfunctionality and stemness potential to CD4+ T cells

ABSTRACT The functional status of CD4+ T cells is a critical determinant of antitumor immunity. Polyfunctional CD4+ T cells possess the ability to concomitantly produce multiple Th1-type cytokines, exhibiting a functional attribute desirable for cancer immunotherapy. However, the mechanisms by which these cells are induced are neither defined nor it is clear if these cells can be used therapeutically to treat cancer. Here, we report that CD4+ T cells exposed to exogenous IL-7 during antigenic stimulation can acquire a polyfunctional phenotype, characterized by their ability to simultaneously express IFNγ, IL-2, TNFα and granzyme B. This IL-7-driven polyfunctional phenotype was associated with increased histone acetylation in the promoters of the effector genes, indicative of increased chromatin accessibility. Moreover, forced expression of a constitutively active (CA) form of STAT5 recapitulated IL-7 in inducing CD4+ T-cell polyfunctionality. Conversely, the expression of a dominant negative (DN) form of STAT5 abolished the ability of IL-7 to induce polyfunctional CD4+ T cells. These in-vitro-generated polyfunctional CD4+ T cells can traffic to tumor and expand intratumorally in response to immunization. Importantly, adoptive transfer of polyfunctional CD4+ T cells following lymphodepletive chemotherapy was able to eradicate large established tumors. This beneficial outcome was associated with the occurrence of antigen epitope spreading, activation of the endogenous CD8+ T cells and persistence of donor CD4+ T cells exhibiting memory stem cell attributes. These findings indicate that IL-7 signaling can impart polyfunctionality and stemness potential to CD4+ T cells, revealing a previously unknown property of IL-7 that can be exploited in adoptive T-cell immunotherapy.

Cx3cr1 deficiency in mice attenuates hepatic granuloma formation during acute schistosomiasis by enhancing the M2-type polarization of macrophages.

ABSTRACT Acute schistosomiasis is characterized by pro-inflammatory responses against tissue- or organ-trapped parasite eggs along with granuloma formation. Here, we describe studies in Cx3cr1−/− mice and demonstrate the role of Cx3cr1 in the pathoetiology of granuloma formation during acute schistosomiasis. Mice deficient in Cx3cr1 were protected from granuloma formation and hepatic injury induced by Schistosoma japonicum eggs, as manifested by reduced body weight loss and attenuated hepatomegaly along with preserved liver function. Notably, S. japonicum infection induced high levels of hepatic Cx3cr1 expression, which was predominantly expressed by infiltrating macrophages. Loss of Cx3cr1 rendered macrophages preferentially towards M2 polarization, which then led to a characteristic switch of the host immune defense from a conventional Th1 to a typical Th2 response during acute schistosomiasis. This immune switch caused by Cx3cr1 deficiency was probably associated with enhanced STAT6/PPAR-γ signaling and increased expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that promotes M2 polarization of macrophages. Taken together, our data provide evidence suggesting that CX3CR1 could be a viable therapeutic target for treatment of acute schistosomiasis. Highlighted Article: A reduction in CX3CR1 signaling provides protection for mice against pro-inflammatory responses and hepatic granuloma formation during acute schistosomiasis.

Modulation of bone morphogenic protein signaling in T-cells for cancer immunotherapy

Abstract Immunotherapy is becoming an increasingly attractive therapeutic alternative for conventional cancer therapy. In recent years Foxp3+ regulatory T-cells (TR) were identified as the major obstacle to effective cancer immunotherapy. The abundance of these cells in peripheral blood is increased in patients with multiple types of cancer and their prevalence among tumor-infiltrating lymphocytes correlated with poor clinical prognosis. In contrast, removal or inactivation of TR cells led to enhanced anti-tumor immune response and better efficacy of cancer vaccines. This study reports that Bone Morphogenic Protein Receptor 1α (BMPR1α, Alk-3) is expressed by activated effector CD4+ and TR cells and modulates functions of both cell types. Bone Morphogenic Proteins (BMPs) belong to the transforming growth factor (TGF)-β family of cytokines that also include TGFβ and activins. BMPs play crucial roles in embryonic development, tissue differentiation and homeostasis, and development of cancer. It was demonstrated that BMPs and activins synergize with TGFβ to regulate thymic T-cell development, maintain TR cells, and control peripheral tolerance. Inactivation of BMPR1α in T-cells results in impaired thymic and peripheral generation of TR cells. BMPR1α-deficient activated T-cells produced a higher level of interferon (IFN)-γ than BMPR1α-sufficient T-cells. Moreover, transplanted B16 melanoma tumors grew smaller in mice lacking expression of BMPR1α in T-cells and tumors had few infiltrating TR cells and a higher proportion of CD8+ T-cells than wild-type mice.

Altered Connexin 43 Expression Underlies Age-Dependent Decrease of Regulatory T Cell Suppressor Function in Nonobese Diabetic Mice

Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell–mediated destruction of insulin-producing β cells are still not well understood. In this study, we show that regulatory T cells (Tregs) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate Tregs in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-β, and IL-2, which enhance connexin 43 and Foxp3 expression in Tregs and restore the ability of conventional CD4+ T cells to upregulate Foxp3 and generate peripherally derived Tregs. Moreover, we demonstrate that suppression mediated by Tregs from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction–mediated intercellular communication as an important component of the Treg suppression mechanism compromised in NOD mice and suggests how Treg mediated immune regulation can be improved.

Bone Morphogenetic Protein Signaling Regulates Development and Activation of CD4(+) T Cells.

Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β (transforming growth factor β) superfamily. BMPs were found to regulate multiple cell processes such as proliferation, survival, differentiation, and apoptosis. They were originally described to play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites but were found to play a significant role in embryogenesis and development of multiple tissues and organs. Activities of BMPs are regulated by a number of secreted proteins, which modulate their availability to bind cellular receptors. The functions of individual BMPs are highly redundant due to binding the same receptors and inducing overlapping signal transduction pathways. Recently, BMPs were found to regulate cells of the innate and adaptive immune system. BMPs are involved in thymic development of T cells at the early, double negative, as well as later, double positive, stages of thymopoesis. They specifically modulate thymic development of regulatory T cells (T(reg)). In the periphery, BMPs affect T cell activation, promoting generation of T(reg) cells. We found that mice deficient for one of the receptors activated by BMPs demonstrated slower growth of transplantable melanoma tumors.