Polyclonally Derived Alloantigen-Specific T Regulatory Cells Exhibit Target-Specific Suppression and Capture MHC Class II from Dendritic Cells.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs in vitro and explored the fine specificity of their suppressive function and their mechanism of action in vitro and in vivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function in vivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

Regulatory T cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells.

Regulatory T cells (Treg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific Treg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (Tnaive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only Tnaive cells specific for cognate antigen in vivo. Antigen-specific Treg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of Tnaive cells to cognate antigen yet allowing bystander Tnaive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of Treg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for Treg cell-mediated suppression and may be a mechanism by which Treg cells maintain immune homeostasis.

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment.

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.

TCR Signaling and CD28/CTLA-4 Signaling Cooperatively Modulate T Regulatory Cell Homeostasis.

Foxp3+ T regulatory cells (Tregs), conventional CD4+Foxp3- T cells, and CD8+ T cells represent heterogeneous populations composed of naive phenotype (NP, CD44low) and memory phenotype (MP, CD44high) subpopulations. NP and MP subsets differ in their activation state, contribution to immune function, and capacity to proliferate in vivo. To further understand the factors that contribute to the differential homeostasis of NP/MP subsets, we examined the differential effects of CD28 and CTLA-4 interaction with CD80/CD86, as well as MHC class II-TCR interaction within mouse Treg pools and CD4+ and CD8+ T cell pools. Blockade of CD80/CD86 with CTLA-4-Ig markedly reduced the cycling and absolute numbers of MP Tregs and MP CD4+ T cells, with minimal effect on the NP T cell subpopulations. Blockade of MHC class II-TCR interaction led to selective expansion of MP Tregs and MP CD4+ and CD8+ T cells that was reversed upon cotreatment with CTLA-4-Ig. Treatment with anti-CTLA-4 mAb altered MP Treg and MP CD4+ and CD8+ T cell homeostasis in a manner similar to that observed with anti-MHC class II. We postulate a complex pathway in which CD28 is the primary driver of Treg proliferation and CTLA-4 functions as the main brake but is likely dependent on TCR signals and CD80/CD86. These findings have important implications for the use of biologic agents targeting such pathways to modulate autoimmune and neoplastic disease.

Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies.

Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4+ T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naïve CD4+ T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

The GARP/Latent TGF-ß1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance.

Treg cells can secrete latent TGF-ß1 (LTGF-ß1), but can also utilize an alternative pathway for transport and expression of LTGF-ß1 on the cell surface in which LTGF-ß1 is coupled to a distinct LTGF-ß binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF-ß1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF-ß1 in the induction of oral tolerance. When Foxp3(-) OT-II T cells were transferred to wild-type recipient mice followed by OVA feeding, the conversion of Foxp3(-) to Foxp3(+) OT-II cells was dependent on recipient Treg cells. Neutralization of IL-2 in the recipient mice also abrogated this conversion. The GARP/LTGF-ß1 complex on recipient Treg cells, but not dendritic cell-derived TGF-ß1, was required for efficient induction of Foxp3(+) T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvß8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF-ß1 complex.

Transcriptome profiling of human FoxP3+ regulatory T cells.

The major goal of this study was to perform an in depth characterization of the "gene signature" of human FoxP3(+) T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs.

Occupational exposure to carcinogens in Australian road transport workers.

BACKGROUND: Road transport workers (RTWs) are at high risk of exposure to several occupational carcinogens. However, there are gaps in knowledge regarding the extent and the circumstances of exposure. As a sub-study of the Australian Work Exposures Study, this study investigated the prevalence of occupational exposure in Australian RTWs. METHODS: A random sample of Australian working population was invited to a telephone interview regarding their current jobs. An automated expert-assessment procedure was applied to self-reported job-related tasks using a web-based application. 162 RTWs were included in this study. RESULTS: RTWs were exposed to diesel exhaust (97%), solar ultraviolet radiation (78%), environmental tobacco smoke (55%), benzene (29%), silica (15%), and asbestos (10%) at work. Besides driving on roads, vehicle maintenance-related tasks were the major source of carcinogen exposures among RTWs. DISCUSSION: Most RTWs are exposed to at least one carcinogen at work. We have identified tasks where the use of control measures could potentially reduce exposures.

Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.

Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.

Autoantibodies in scurfy mice and IPEX patients recognize keratin 14.

Scurfy mice have a deletion in the Foxp3 gene, resulting in a failure to generate Foxp3(+) regulatory T cells, and they subsequently develop severe CD4(+) T-cell-mediated autoimmune inflammation. Multiple organs are involved, but the skin is one of the main organs affected. During the course of disease, Scurfy mice develop autoantibodies; however, the targeted antigens are unknown. In this study, we show that Scurfy mice develop autoantibodies directed against skin antigens. Using western blot analysis, we found that Scurfy serum reacted with proteins in total skin lysate, as well as in a keratinocyte lysate. Most of the Scurfy sera tested identified a major band at 50 kDa. Transfer of Scurfy CD4(+) T cells into nu/nu mice yielded autoantibodies with similar reactivity. Further analysis using 2D western blots, followed by peptide mass fingerprinting, identified several keratins as targets. To confirm this observation, we chose one of the identified targets, keratin 14, and prepared recombinant proteins encompassing the N-terminal, middle, and C-terminal portions of the keratin 14 protein. Scurfy serum predominantly recognized the C-terminal fragment. Sera from patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, the human disease resulting from FOXP3 mutations, also recognized skin antigens, including keratin 14. Thus, the results of our study indicate that autoantibodies in Scurfy mice and patients with IPEX target keratins.

Pre-differentiated Th1 and Th17 effector T cells in autoimmune gastritis: Ag-specific regulatory T cells are more potent suppressors than polyclonal regulatory T cells.

Naïve antigen-specific CD4(+) T cells (TxA23) induce autoimmune gastritis when transferred into BALB/c nu/nu mice. Transfer of in vitro pre-differentiated Th1 or Th17 TxA23 effector T cells into BALB/c nu/nu recipients induces distinct histological patterns of disease. We have previously shown that co-transfer of polyclonal naturally occurring Treg (nTreg) suppressed development of Th1-, but not Th17-mediated disease. Therefore, we analysed the suppressive capacity of different types of Treg to suppress Th1- and Th17-mediated autoimmune gastritis. We compared nTreg with polyclonal TGFbeta-induced WT Treg (iTreg) or TGFbeta-induced antigen-specific TxA23 iTreg in co-transfer experiments with Th1 or Th17 TxA23 effector T cells. 6 weeks after transfer in vitro pre-differentiated TxA23 Th1 and Th17 effector cells induced destructive gastritis. Th1-mediated disease was prevented by co-transfer of nTreg and also antigen-specific iTreg, whereas WT iTreg did not show an effect. However, Th17-mediated disease was only suppressed by antigen-specific iTreg. Pre-activation of nTreg in vitro prior to transfer did not increase their suppressive activity in Th17-mediated gastritis. Thus, antigen-specific iTreg are potent suppressors of autoimmune gastritis induced by both, fully differentiated Th1 and Th17 effector cells.

Analysis of adhesion molecules, target cells, and role of IL-2 in human FOXP3+ regulatory T cell suppressor function.

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of self-tolerance and immune homeostasis. The mechanisms of action and cellular targets for Treg-mediated suppression remain controversial. The critical adhesion molecules utilized by Tregs for the interaction with their target cells have not been well characterized. We show that human CD4(+)FOXP3(+)CD25(high) cells (hTregs) suppress the activation of mouse responders as efficiently as mouse Tregs. LFA-1 (CD11a/CD18) on the hTregs is critical for their suppressor function, since suppression can be reversed with blocking anti-hCD11a or anti-hCD18 mAb. Tregs from patients with LFA-1 deficiency fail to suppress human and mouse responders. Mouse CD4(+) T cells deficient in ICAM-1 can be suppressed by hTregs, indicating that the hTregs target mouse dendritic cells (DCs) through the binding of human LFA-1 to mouse ICAM-1. Coculture of mouse DCs with hTregs, but not hTregs from LFA-1-deficient patients, prevented the up-regulation of CD80/CD86 on the DCs and their capacity to activate responder T cells. Lastly, IL-2 is not required for hTreg suppressor function under optimal stimulatory condition and IL-2 consumption plays no role in hTreg-mediated suppression. Taken together, one of the mechanisms of Treg-mediated suppression functions across species and mediates an LFA-1/ICAM-1-dependent interaction between Tregs and DCs.

Cutting edge: antigen-specific TGF beta-induced regulatory T cells suppress Th17-mediated autoimmune disease.

CD4(+) T cells from the TCR transgenic TxA23 mouse recognize a peptide from the H/K-ATPase alpha-chain. When TxA23 CD4(+) thymocytes are differentiated into Th1, Th2, and Th17 lines, all three subpopulations induced autoimmune gastritis (AIG) upon transfer into nu/nu recipients. The induction of AIG by naive T cells or by Th1 or Th2 cell lines could be prevented by the cotransfer of polyclonal Foxp3(+) T regulatory cells (nTreg), whereas Th17-induced AIG was resistant to suppression. We compared the capacity of different types of Treg to suppress Th17-mediated AIG. Cotransfer of either nTreg or polyclonal TGFbeta-induced Treg (iTreg) did not prevent AIG, while cotransfer of TGFbeta-induced Ag-specific TxA23 iTreg completely prevented the development of disease. Ag-specific iTreg were able to suppress Th17-mediated disease when injected 6 days after the Th17 effectors. The implications of these results for the use of Treg for the cellular biotherapy of autoimmune disease are discussed.

TGF-beta-induced Foxp3+ regulatory T cells rescue scurfy mice.

Scurfy mice have a deletion in the forkhead domain of the forkhead transcription factor p3 (Foxp3), fail to develop thymic-derived, naturally occurring Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3(-) via the TCR in the presence of TGF-beta and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGF-beta-induced Treg (iTreg) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTreg. Scurfy mice treated with iTreg do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTreg retained their expression of Foxp3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGF-beta-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.

CD4+CD25+ T cells prevent the development of organ-specific autoimmune disease by inhibiting the differentiation of autoreactive effector T cells.

Thymic-derived, naturally occurring, CD4+CD25+ regulatory T cells (nTreg) are potent suppressors of immune responses. A detailed understanding of which components of the development and activation of pathogenic effector T cells are inhibited by nTreg during the course of T cell-mediated, organ-specific autoimmunity is as yet unknown. We have analyzed the effects of polyclonal nTreg on the development of autoimmune gastritis. The nTreg inhibited the development of disease, but failed to inhibit the migration of effector cells into the gastric lymph node or stomach. Notably, nTreg did not inhibit the expansion of autoreactive T cells in the gastric lymph node. The primary effect of nTreg appeared to be inhibition of differentiation of autoantigen-specific T cells to Th1 effector cells, as reflected by a decrease in Ag-stimulated IFN-gamma production and a reduction in T-bet expression.

Cutting Edge: IL-10-producing CD4+ T cells mediate tumor rejection.

IL-10 has potent immunosuppressive properties, and IL-10-producing CD4+ Tr1 cells have been characterized as regulators of Th1-mediated immunity. In this study, using a s.c. model of glioma cell growth in mice, we demonstrate that CD4+, but not CD8+, T cells play a critical role in tumor rejection following vaccination with irradiated glioma cells. Surprisingly, glioma-specific CD4+ T cells produce IL-10 but neither IL-4 nor IFN-gamma, and glioma rejection is compromised in IL-10(-/-) hosts. Hence, our findings demonstrate that IL-10-producing CD4+ T cells can manifest antitumor functions and suggest that IL-10 may have proinflammatory effects in disease states.