Treg Cell Homeostasis Research Articles

Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is linked to various signs of advanced aging, such as premature immunosenescence which occurs due to decline in regenerative ability of T cells. RA T cells develop a unique aggressive inflammatory senescent phenotype with an imbalance of Th17/T regulatory (Treg) cell homeostasis and presence of CD28- T cells. The phenotypic analysis and characterization of T cell subsets become necessary to ascertain if any functional deficiencies exist within with the help of transcription factor (TF) analysis. These subset-specific TFs dictate the functional characteristics of T-cell populations, leading to the production of distinct effector cytokines and functions. Examining the expression, activity, regulation, and genetic sequence of TFs not only aids researchers in determining their importance in disease processes but also aids in immunological monitoring of patients enrolled in clinical trials, particularly in evaluating various T-cell subsets [Th17 (CD3+CD4+IL17+RORγt+) cells and T regulatory (Treg) (CD3+CD4+CD25+CD127-FOXP3+) cells], markers of T-cell aging [aged Th17 cells (CD3+CD4+IL17+RORγt+CD28-), and aged Treg cells (CD3+CD4+CD25+CD127-FOXP3+CD28-)]. In this context, we propose and outline the protocols for assessing the expression of TFs in aged Th17 and Treg cells, highlighting the crucial aspects of this cytometric approach.

Vps34 sustains Treg cell survival and function via regulating intracellular redox homeostasis.

The survival and suppressive function of regulatory T (Treg) cells rely on various intracellular metabolic and physiological processes. Our study demonstrates that Vps34 plays a critical role in maintaining Treg cell homeostasis and function by regulating cellular metabolic activities. Disruption of Vps34 in Treg cells leads to spontaneous fatal systemic autoimmune disorder and multi-tissue inflammatory damage, accompanied by a reduction in the number of Treg cells, particularly eTreg cells with highly immunosuppressive activity. Mechanistically, the poor survival of Vps34-deficient Treg cells is attributed to impaired endocytosis, intracellular vesicular trafficking and autophagosome formation, which further results in enhanced mitochondrial respiration and excessive ROS production. Removal of excessive ROS can effectively rescue the death of Vps34-deficient Treg cells. Functionally, acute deletion of Vps34 within established Treg cells enhances anti-tumor immunity in a malignant melanoma model by boosting T-cell-mediated anti-tumor activity. Overall, our results underscore the pivotal role played by Vps34 in orchestrating Treg cell homeostasis and function towards establishing immune homeostasis and tolerance.

Targeting p97-Npl4 interaction inhibits tumor Treg cell development to enhance tumor immunity.

Targeting tumor-infiltrating regulatory T (TI-Treg) cells is a potential strategy for cancer therapy. The ATPase p97 in complex with cofactors (such as Npl4) has been investigated as an antitumor drug target; however, it is unclear whether p97 has a function in immune cells or immunotherapy. Here we show that thonzonium bromide is an inhibitor of the interaction of p97 and Npl4 and that this p97-Npl4 complex has a critical function in TI-Treg cells. Thonzonium bromide boosts antitumor immunity without affecting peripheral Treg cell homeostasis. The p97-Npl4 complex bridges Stat3 with E3 ligases PDLIM2 and PDLIM5, thereby promoting Stat3 degradation and enabling TI-Treg cell development. Collectively, this work shows an important role for the p97-Npl4 complex in controlling Treg-TH17 cell balance in tumors and identifies possible targets for immunotherapy.

HSP90β shapes the fate of Th17 cells with the help of glycolysis-controlled methylation modification.

Ulcerative colitis (UC) is a refractory inflammatory disease associated with immune dysregulation. Elevated levels of heat shock protein (HSP) 90 in the β but not α subtype were positively associated with disease status in UC patients. This study validated the possibility that pharmacological inhibition or reduction of HSP90β would alleviate colitis, induced by dextran sulfate sodium, in mice and elucidated its mechanisms. Histopathological and biochemical analysis assessed disease severity, and bioinformatics and correlation analysis explained the association between the many immune cells and HSP90β. Flow cytometry was used to analyse the homeostasis and transdifferentiation of Th17 and Treg cells. In vitro inhibition and adoptive transfer assays were used to investigate functions of the phenotypically transformed Th17 cells. Metabolomic analysis, DNA methylation detection and chromatin immunoprecipitation were used to explore these mechanisms. The selective pharmacological inhibitor (HSP90βi) and shHSP90β significantly mitigated UC in mice and promoted transformation of Th17 to Treg cell phenotype, via Foxp3 transcription. The phenotypically-transformed Th17 cells by HSP90βi or shHSP90β were able to inhibit lymphocyte proliferation and colitis in mice. HSP90βi and shHSP90β selectively weakened glycolysis by stopping the direct association of HSP90β and GLUT1, the key glucose transporter, to accelerate ubiquitination degradation of GLUT1, and enhance the methylation of Foxp3 CNS2 region. Then, the mediator path was identified as the "lactate-STAT5-TET2" cascade. HSP90β shapes the fate of Th17 cells via glycolysis-controlled methylation modification to affect UC progression, which provides a new therapeutic target for UC.

Metabolic Plasticity of Regulatory T Cells in Health and Autoimmunity.

Immunometabolism has been demonstrated to control immune tolerance and the pathogenic events leading to autoimmunity. Compelling experimental evidence also suggests that intracellular metabolic programs influence differentiation, phenotype, proliferation, and effector functions of anti-inflammatory CD4+CD25+Foxp3+ regulatory T (Treg) cells. Indeed, alterations in intracellular metabolism associate with quantitative and qualitative impairments of Treg cells in several pathological conditions. In this review, we summarize the most recent advances linking how metabolic pathways control Treg cell homeostasis and their alterations occurring in autoimmunity. Also, we analyze how metabolic manipulations could be employed to restore Treg cell frequency and function with the aim to create novel therapeutic opportunities to halt immune-mediated disorders.

Lupus susceptibility gene Pbx1 controls the development, stability, and function of regulatory T cells via Rtkn2 expression.

The maintenance of regulatory T (Treg) cells critically prevents autoimmunity. Pre-B cell leukemia transcription factor 1 (Pbx1) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4+ T cells. Pbx1-d overexpression impaired Treg cell homeostasis and promoted inflammatory CD4+ T cells. Here, we showed a high expression of Pbx1 in human and murine Treg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of Treg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in Treg cells. Intriguingly, Rtkn2, a Rho-GTPase previously associated with Treg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of Treg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.

RIPK1 plays a crucial role in maintaining regulatory T-Cell homeostasis by inhibiting both RIPK3- and FADD-mediated cell death.

Regulatory T (Treg) cells play an essential role in maintaining immune balance across various physiological and pathological conditions. However, the mechanisms underlying Treg homeostasis remain incompletely understood. Here, we report that RIPK1 is crucial for Treg cell survival and homeostasis. We generated mice with Treg cell-specific ablation of Ripk1 and found that these mice developed fatal systemic autoimmunity due to a dramatic reduction in the Treg cell compartment caused by excessive cell death. Unlike conventional T cells, Treg cells with Ripk1 deficiency were only partially rescued from cell death by blocking FADD-dependent apoptosis. However, simultaneous removal of both Fadd and Ripk3 completely restored the homeostasis of Ripk1-deficient Treg cells by blocking two cell death pathways. Thus, our study highlights the critical role of RIPK1 in regulating Treg cell homeostasis by controlling both apoptosis and necroptosis, thereby providing novel insights into the mechanisms of Treg cell homeostasis.

USP47 inhibits m6A-dependent c-Myc translation to maintain regulatory T cell metabolic and functional homeostasis.

The functional integrity of Tregs is interwoven with cellular metabolism; however, the mechanisms governing Treg metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited c-Myc translation mediated by the RNA N6-methyladenosine (m6A) reader YTHDF1 to maintain Treg metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg signature in samples from patients with colorectal cancer and gastric cancer. USP47 ablation compromised Treg homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Tregs triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting of USP47.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector Tcells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Molecular mechanisms of PBX1 regulation of CD4 +T cell inflammatory phenotypes

Abstract The pre-B cell leukemia homeobox1 (Pbx1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, Pbx1-d that lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4 +T cells from lupus-prone mice and SLE patients than healthy controls. We have shown that overexpression of Pbx1-d in CD4 +T cells impaired Treg cell homeostasis and expanded inflammatory CD4 +T cells. Here, we investigated the molecular mechanisms by which PBX1 regulates T cells. We showed that PBX1 is preferentially expressed in Treg cells over other CD4+ T cell subsets, and that it is negatively regulated by TCR activation. Furthermore, PBX1-D protein was less stable than the normal isoform, PBX1-B, in a pulse chase experiment. PBX1-B but not PBX1-D has two predictive ubiquitin binding sites, K292 and K293, located in the DNA binding domain. Mutation of these two sites reduced PBX1-B stability to a level similar to PBX1-D, suggesting that a loss of function in the lupus associated allele through the ubiquitin-proteasome dependent pathway. In addition, contrary to PBX1-B, PBX1-D is retained in the cytoplasm without the help of the MEIS or PREP1 co-factors, indicating different requirements for nuclear translocation. Overall, these findings suggest that multiple transcriptional and post-transcriptional mechanisms are responsible for PBX1-D loss of function and induction of CD4 +T cell inflammatory phenotypes in SLE. This work was supported by National Institutes of Health Grants AI045050

BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

A Stk4-Foxp3-NF-κB p65 transcriptional complex promotes Treg cell activation and homeostasis.

The molecular programs involved in regulatory T (Treg) cell activation and homeostasis remain incompletely understood. Here, we show that T cell receptor (TCR) signaling in Treg cells induces the nuclear translocation of serine/threonine kinase 4 (Stk4), leading to the formation of an Stk4-NF-κB p65-Foxp3 complex that regulates Foxp3- and p65-dependent transcriptional programs. This complex was stabilized by Stk4-dependent phosphorylation of Foxp3 on serine-418. Stk4 deficiency in Treg cells, either alone or in combination with its homolog Stk3, precipitated a fatal autoimmune lymphoproliferative disease in mice characterized by decreased Treg cell p65 expression and nuclear translocation, impaired NF-κB p65-Foxp3 complex formation, and defective Treg cell activation. In an adoptive immunotherapy model, overexpression of p65 or the phosphomimetic Foxp3S418E in Stk3/4-deficient Treg cells ameliorated their immune regulatory defects. Our studies identify Stk4 as an essential TCR-responsive regulator of p65-Foxp3-dependent transcription that promotes Treg cell-mediated immune tolerance.

Migration and homeostasis of regulatory T cells in rheumatoid arthritis.

Regulatory T (Treg) cells are garnering increased attention in research related to autoimmune diseases, including rheumatoid arthritis (RA). They play an essential role in the maintenance of immune homeostasis by restricting effector T cell activity. Reduced functions and frequencies of Treg cells contribute to the pathogenesis of RA, a common autoimmune disease which leads to systemic inflammation and erosive joint destruction. Treg cells from patients with RA are characterized by impaired functions and by an altered phenotype. They show increased plasticity towards Th17 cells and a reduced suppressive capacity. Besides the suppressive function of Treg cells, their effectiveness is determined by their ability to migrate into inflamed tissues. In the past years, new mechanisms involved in Treg cell migration have been identified. One example of such a mechanism is the phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Efficient migration of Treg cells requires the presence of VASP. IL-6, a cytokine which is abundantly present in the peripheral blood and in the synovial tissue of RA patients, induces posttranslational modifications of VASP. Recently, it has been shown in mice with collagen-induced arthritis (CIA) that this IL-6 mediated posttranslational modification leads to reduced Treg cell trafficking. Another protein which facilitates Treg cell migration is G-protein-signaling modulator 2 (GPSM2). It modulates G-protein coupled receptor functioning, thereby altering the cellular activity initiated by cell surface receptors in response to extracellular signals. The almost complete lack of GPSM2 in Treg cells from RA patients contributes to their reduced ability to migrate towards inflammatory sites. In this review article, we highlight the newly identified mechanisms of Treg cell migration and review the current knowledge about impaired Treg cell homeostasis in RA.

Regulatory T cells and immunoglobulin E: A new therapeutic link for autoimmunity?

Autoimmune diseases have a prevalence of approximately 7 to 9% and are classified as either organ-specific diseases, including type I diabetes, multiple sclerosis, inflammatory bowel disease and myasthenia gravis, or systemic diseases, including systemic lupus erythematosus, rheumatoid arthritis and Sjögren's syndrome. While many advancements have been made in understanding of the mechanisms of autoimmune disease, including the nature of self-tolerance and its breakdown, there remain unmet needs in terms of effective and highly targeted treatments. T regulatory cells (Tregs) are key mediators of peripheral tolerance and are implicated in many autoimmune diseases, either as a result of reduced numbers or altered function. Tregs may be broadly divided into those generated in the thymus (tTregs) and those generated in the periphery (pTregs). Tregs target many different immune cell subsets and tissues to suppress excessive inflammation and to support tissue repair and homeostasis: there is a fine balance between Treg cell stability and the plasticity that is required to adjust Tregs' regulatory purposes to particular immune responses. The central role of immunoglobulin E (IgE) in allergic disease is well recognized, and it is becoming increasingly apparent that this immunoglobulin also has a wider role encompassing other diseases including autoimmune disease. Anti-IgE treatment restores the capacity of plasmacytoid dendritic cells (pDCs) impaired by IgE- high-affinity IgE receptor (FcεR1) cross-linking to induce Tregs in vitro in atopic patients. The finding that anti-IgE therapy restores Treg cell homeostasis, and that this mechanism is associated with clinical improvement in asthma and chronic spontaneous urticaria suggests that anti-IgE therapy may also have a potential role in the treatment of autoimmune diseases in which Tregs are involved.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

Preferential Expansion of Foxp3+ T Regulatory Cells in CTLA-4-Deficient and CTLA-4-Haploinsufficient C57BL/6 Mice.

Foxp3+ cells and CTLA-4 have been ascribed major roles in downregulating immune responses. To address the relationship between CTLA-4 expression and Foxp3+ cells, we generated littermate CTLA-4-sufficient (Ctla4 +/+), CTLA-4-haploinsufficient (Ctla4 +/-), and CTLA-4-deficient (Ctla4 -/-) Foxp3-gfp knock-in C57BL/6 mice, permitting us to characterize the phenotype of Foxp3+ cells and to test their ex vivo T regulatory (Treg) suppressor activity. CD3+, CD4+, and CD8+ cells, but not CD19+ cells, were markedly expanded in Ctla4 -/- mice compared with Ctla4 +/+ or Ctla4 +/- mice. In Ctla4 -/- mice, the relative expansion of the Foxp3+ population was greater than that of the CD3+, CD4+, or CD8+ populations because of increased survival of Foxp3+ cells. Foxp3+ Treg cells from Ctla4 -/- mice and Foxp3+ Treg cells from Ctla4 +/+ mice exerted identical ex vivo suppressor function. This may be related to differential expression of GITR, CD73, and CD39 on Foxp3+ Treg cells from Ctla4 -/- mice versus that on corresponding cells from littermate Ctla4 +/+ or Ctla4 +/- mice, with GITR and CD39 being upregulated and CD73 being downregulated on Foxp3+ Treg cells from Ctla4 -/- mice. Moreover, CTLA-4 expression in Ctla4 +/+, Ctla4 +/-, and Ctla4 -/- mice correlated with their percentages of Foxp3+ cells, suggesting an important role for CTLA-4 expression in Treg cell homeostasis. This may have vital ramifications for the treatment of patients for whom augmentation of suppressor function would be beneficial (e.g., patients with autoimmune diseases) and for whom diminution of suppressor function would be beneficial (e.g., patients with cancer).

Differentiation and homeostasis of effector Treg cells are regulated by inositol polyphosphates modulating Ca2+ influx

Activated Foxp3+ regulatory T (Treg) cells differentiate into effector Treg (eTreg) cells to maintain peripheral immune homeostasis and tolerance. T cell receptor (TCR)-mediated induction and regulation of store-operated Ca2+ entry (SOCE) is essential for eTreg cell differentiation and function. However, SOCE regulation in Treg cells remains unclear. Here, we show that inositol polyphosphate multikinase (IPMK), which generates inositol tetrakisphosphate and inositol pentakisphosphate, is a pivotal regulator of Treg cell differentiation downstream of TCR signaling. IPMK is highly expressed in TCR-stimulated Treg cells and promotes a TCR-induced Treg cell program. IPMK-deficient Treg cells display aberrant T cell activation and impaired differentiation into RORγt+ Treg cells and tissue-resident Treg cells. Mechanistically, IPMK controls the generation of higher-order inositol phosphates, thereby promoting Ca2+ mobilization and Treg cell effector functions. Our findings identify IPMK as a critical regulator of TCR-mediated Ca2+ influx and highlight the importance of IPMK in Treg cell-mediated immune homeostasis.

Driving Role of Interleukin-2-Related Regulatory CD4+ T Cell Deficiency in the Development of Lung Fibrosis and Vascular Remodeling in a Mouse Model of Systemic Sclerosis.

Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by severe lung outcomes resulting in reduced life expectancy. Fra-2-transgenic mice offer the opportunity to decipher the relationships between the immune system and lung fibrosis. This study was undertaken to investigate whether the Fra-2-transgenic mouse lung phenotype may result from an imbalance between the effector and regulatory arms in the CD4+ T cell compartment. We first used multicolor flow cytometry to extensively characterize homeostasis and the phenotype of peripheral CD4+ T cells from Fra-2-transgenic mice and control mice. We then tested different treatments for their effectiveness in restoring CD4+ Treg cell homeostasis, including adoptive transfer of Treg cells and treatment with low-dose interleukin-2 (IL-2). Fra-2-transgenic mice demonstrated a marked decrease in the proportion and absolute number of peripheral Treg cells that preceded accumulation of activated, T helper cell type 2-polarized, CD4+ T cells. This defect in Treg cell homeostasis was derived from a combination of mechanisms including impaired generation of these cells in both the thymus and the periphery. The impaired ability of peripheral conventional CD4+ T cells to produce IL-2 may greatly contribute to Treg cell deficiency in Fra-2-transgenic mice. Notably, adoptive transfer of Treg cells, low-dose IL-2 therapy, or combination therapy changed the phenotype of Fra-2-transgenic mice, resulting in a significant reduction in pulmonary parenchymal fibrosis and vascular remodeling in the lungs. Immunotherapies for restoring Treg cell homeostasis could be relevant in SSc. An intervention based on low-dose IL-2 injections, as is already proposed in other autoimmune diseases, could be the most suitable treatment modality for restoring Treg cell homeostasis for future research.

The Ube2m-Rbx1 neddylation-Cullin-RING-Ligase proteins are essential for the maintenance of Regulatory T cell fitness

Neddylation-mediated activation of Cullin-RING E3 Ligases (CRLs) are necessary for the degradation of specific immune regulatory proteins. However, little is known about how these processes govern the function of regulatory T (Treg) cells. Here we show that mice with Treg cell-specific deletion of Rbx1, a dual E3 for both neddylation and ubiquitylation by CRLs, develop an early-onset fatal inflammatory disorder, characterized by disrupted Treg cell homeostasis and suppressive functions. Specifically, Rbx1 is essential for the maintenance of an effector Treg cell subpopulation, and regulates several inflammatory pathways. Similar but less severe phenotypes are observed in mice having Ube2m, a neddylation E2 conjugation enzyme, deleted in their Treg cells. Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, components of a similar but distinct neddylation-CRL complex, yields no obvious phenotype. Thus, our work demonstrates that the Ube2m-Rbx1 axis is specifically required for intrinsic regulatory processes in Treg cells; and that Rbx1 might also play Ube2m-independent roles in maintaining the fitness of Treg cells, suggesting a layer of complexity in neddylation-dependent activation of CRLs.

Homeodomain protein Pbx1 regulates regulatory T cell development, stability and suppressive function

Abstract Pre-B cell leukemia transcription factor 1 (Pbx1) regulates multiple processes in various cell types. The Pbx1 gene is located in the Sle1a1 lupus susceptibility locus in the NZM2410 lupus-prone mouse model. The Pbx1-d dominant negative isoform lacking the DNA binding domain is more frequent in the CD4+ T cells from NZM240 mice as well as lupus patients as compared to healthy controls. In addition, the transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with impaired Foxp3+ Treg cell homeostasis as well as increased inflammatory CD4+ T cells. To study the role of Pbx1 in Treg cells, we generated Foxp3-specific Pbx1 knock-out mice (Foxp3-Pbx1 KO), which were bred to CD4-Pbx1-d-Tg mice to generate Pbx1-d Tg-KO mice that have Treg cells solely expressing Pbx1-d. Foxp3-Pbx1 KO mice showed reduced numbers of Treg cells with impaired suppressive activity. The numbers of natural Treg (nTrg) cells are decreased in thymus but not in spleen of Foxp3-Pbx1 KO mice, while the number of induced Treg (iTreg) cells are increased in the thymus and decreased in the periphery, suggesting that Pbx1 regulates Treg cell development and stability. The results were enhanced in Pbx1-d Tg-KO mice compared with Foxp3-Pbx1 KO mice, leading to autoimmune phenotypes, suggesting that the expression of Pbx1 functional isoform is necessary for the maintenance and function of Treg cells. RNAseq analyses with Pbx1-KO Treg cells indicated that Pbx1 regulates cell cycle, apoptosis and migration. Overall, our results suggest that Pbx1 regulates Treg cell homeostasis, stability and their suppressive functions and that Pbx1-d promotes lupus development by decreasing Treg cell development and functions. Supported by a grant from the NIH (R01 AI045050)