Regulatory T cell expression of herpesvirus entry mediator suppresses the function of B and T lymphocyte attenuator-positive effector T cells

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA(-/-) CD4(+)CD25(+) Tregs had normal suppressive activity, whereas BTLA(-/-) Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM(-/-) Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM(-/-) Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

Normalisation to blood activity is required for the accurate quantification of Na/I symporter ectopic expression by SPECT/CT in individual subjects

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used (99m)Tc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to 'noisy', and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.     

Differential suppression of tumor-specific CD8+ T cells by regulatory T cells

In the CT26 BALB/c murine model of colorectal carcinoma, depletion of regulatory T cells (Tregs) prior to tumor inoculation results in protective immunity to both CT26 and other BALB/c-derived tumors of diverse histological origin. In this paper, we show that cross-protection can be conferred by adoptively transferred CD8(+) CTLs. Other schedules for inducing immunity to CT26 have been described, but they do not lead to cross-protection. We show that Treg ablation facilitates the development of new CTL specificities that are normally cryptic, and have mapped the root epitope of one of these responses. This work has allowed us to demonstrate how the specificity of CTL responses to tumor Ags can be controlled via differential suppression of CTL specificities by Tregs, and how this can result in very different physiological outcomes.     

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.     

T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.     

Cutting edge: CD47 controls the in vivo proliferation and homeostasis of peripheral CD4+ CD25+ Foxp3+ regulatory T cells that express CD103

Peripheral CD103(+)Foxp3(+) regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived, expanded/differentiated natural Tregs. We here show that CD47 expression, a marker of self on hematopoietic cells, selectively regulated CD103(+)Foxp3(+) Treg homeostasis at the steady state. First, the proportion of effector/memory-like (CD44(high)CD62L(low)) CD103(+)Foxp3(+) Tregs rapidly augmented with age in CD47-deficient mice (CD47(-/-)) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44(low)CD62L(high)) CD103(-)Foxp3(+) Tregs remained stable. Second, the increased proliferation rate (BrdU incorporation) observed within the CD47(-/-)Foxp3(+) Treg subpopulation was restricted to those Tregs expressing CD103. Third, CD47(-/-) Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103(+) Tregs that may overwhelmingly inhibit Ag-specific T cell responses.     

CTLA4 expression is an indicator and regulator of steady-state CD4+ FoxP3+ T cell homeostasis

The presence of FoxP3(+) regulatory T cells (Tregs) is necessary for control of deleterious immune responses in the steady state; however, mechanisms for maintaining the frequency and quality of endogenous Tregs are not well defined. In this study, we used in vivo modulators of the CD28 and CTLA4 pathways administered to intact mice to reveal mechanisms controlling the homeostasis and phenotype of endogenous Tregs. We demonstrate that expression of the negative costimulatory regulator CTLA4 on FoxP3(+) Tregs in vivo is a direct consequence of their rapid, perpetual homeostasis. Up-regulation of CTLA4 expression occurs only on FoxP3(+) Tregs undergoing extensive proliferation and can be abrogated by inhibiting the CD28 pathway, coinciding with a reduction in FoxP3(+) Treg proliferation and frequency. We further demonstrate that CTLA4 negatively regulates steady-state Treg homeostasis, given that inhibiting CTLA4 signaling with an anti-CTLA4 blocking Ab greatly enhances Treg proliferation and overall Treg frequency. Our findings provide new insight into the origin and role of CTLA4 expression on natural FoxP3(+) Tregs and reveal opposing effects of costimulation modulators on the steady-state level and quality of Tregs, with implications regarding their effects on endogenous Tregs in patients receiving immunotherapy.     

A self-reactive TCR drives the development of Foxp3+ regulatory T cells that prevent autoimmune disease

Although Foxp3(+) regulatory T cells (Tregs) are thought to express autoreactive TCRs, it is not clear how individual TCRs influence Treg development, phenotype, and function in vivo. We have generated TCR transgenic mice (termed SFZ70 mice) using Tcra and Tcrb genes cloned from an autoreactive CD4(+) T cell isolated from a Treg-deficient scurfy mouse. The SFZ70 TCR recognizes a cutaneous autoantigen and drives development of both conventional CD4(+) Foxp3(-) T cells (T(conv)) and Foxp3(+) Tregs. SFZ70 Tregs display an activated phenotype evidenced by robust proliferation and expression of skin-homing molecules such as CD103 and P-selectin ligand. Analysis of Foxp3-deficient SFZ70 mice demonstrates that Tregs inhibit T(conv) cell expression of tissue-homing receptors and their production of proinflammatory cytokines. In addition, Treg suppression of SFZ70 T(conv) cells can be overcome by nonspecific activation of APCs. These results provide new insights into the differentiation and function of tissue-specific Tregs in vivo and provide a tractable system for analyzing the molecular requirements of Treg-mediated tolerance toward a cutaneous autoantigen.     

Murine lupus susceptibility locus Sle1a controls regulatory T cell number and function through multiple mechanisms

The Sle1 locus is a key determinant of lupus susceptibility in the NZM2410 mouse model. Within Sle1, we have previously shown that Sle1a expression enhances activation levels and effector functions of CD4(+) T cells and reduces the size of the CD4(+)CD25(+)Foxp3(+) regulatory T cell subset, leading to the production of autoreactive T cells that provide help to chromatin-specific B cells. In this study, we show that Sle1a CD4(+) T cells express high levels of ICOS, which is consistent with their increased ability to help autoreactive B cells. Furthermore, Sle1a CD4(+)CD25(+) T cells express low levels of Foxp3. Mixed bone marrow chimeras demonstrated that these phenotypes require Sle1a to be expressed in the affected CD4(+) T cells. Expression of other markers generally associated with regulatory T cells (Tregs) was similar regardless of Sle1a expression in Foxp3(+) cells. This result, along with in vitro and in vivo suppression studies, suggests that Sle1a controls the number of Tregs rather than their function on a per cell basis. Both in vitro and in vivo suppression assays also showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells.     

Transient local depletion of Foxp3+ regulatory T cells during recovery from colitis via Fas/Fas ligand-induced death

Regulatory T cells (Tregs) play a fundamental role in regulating the immune system in health and disease. Considerable evidence exists demonstrating that transfer of Tregs can cure colitis and a variety of other inflammatory disorders. However, little is known about the effects of inflammation on resident Tregs. Mice (BALB/c or C57BL/6) treated with an intrarectal instillation of the haptenizing agent 2,4-dinitrobenzene sulfonic acid (DNBS) develop an acute inflammatory disease, the histopathology of which peaks at 3 days posttreatment and resolves spontaneously thereafter. In this study we demonstrate that DNBS (or oxazolone)-induced colitis causes a depletion of colonic Foxp3+ Tregs 8 days posttreatment, while the proportion of Foxp3+ cells in the ileum, mesenteric lymph nodes, and spleen remains unchanged. Replenishment of the colonic Treg population was associated with the reappearance of mucosal homing (alpha4beta7+) CD4+Foxp3+ Tregs. Assessing the mechanism of local Treg depletion, we found no evidence to implicate cytokine-induced phenotypic switching in the Foxp3+ population or increased SMAD7 expression despite the essential role that TGF-beta has in Foxp3+ Treg biology. Increased Fas ligand (FasL) expression was observed in the colon of colitic mice and in vitro stimulation with a Fas cross-linking Ab resulted in apoptosis of CD4+Foxp3+ but not CD4+Foxp3- cells. Furthermore, DNBS-induced colitis in Fas/FasL-deficient mice did not result in depletion of colonic Tregs. Finally, adoptively transferred synergic Fas-/- but not Fas+/+ Tregs were protected from depletion in the colon 8 days post-DNBS treatment, thus substantiating the hypothesis that inflammation-induced local depletion of Foxp3+ Tregs in the colon of mice occurs via Fas/FasL-mediated death.     

Requirement for diverse TCR specificities determines regulatory T cell activity in a mouse model of autoimmune arthritis

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are required to restrain the immune system from mounting an autoaggressive systemic inflammatory response, but why their activity can prevent (or allow) organ-specific autoimmunity remains poorly understood. We have examined how TCR specificity contributes to Treg activity using a mouse model of spontaneous autoimmune arthritis, in which CD4(+) T cells expressing a clonotypic TCR induce disease by an IL-17-dependent mechanism. Administration of polyclonal Tregs suppressed Th17 cell formation and prevented arthritis development; notably, Tregs expressing the clonotypic TCR did not. These clonotypic Tregs exerted Ag-specific suppression of effector CD4(+) T cells using the clonotypic TCR in vivo, but failed to mediate bystander suppression and did not prevent Th17 cells using nonclonotypic TCRs from accumulating in joint-draining lymph nodes of arthritic mice. These studies indicate that the availability of Tregs with diverse TCR specificities can be crucial to their activity in autoimmune arthritis.     

hNIS-IRES-eGFP dual reporter gene imaging

The human and rodent sodium iodide symporters (NIS) have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked human NIS (hNIS)-enhanced green fluorescent protein (eGFP) hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4- to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.     

The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells

The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1-deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.     

Expansion of CD4+CD25+and FOXP3+ regulatory T cells during the follicular phase of the menstrual cycle: implications for human reproduction

Regulatory T cells (Tregs) are thought to affect the severity of various infectious and autoimmune diseases. The incidence of autoimmune disease is higher in fertile women than in men. Thus, we investigated whether Treg numbers were modulated during the menstrual cycle by sex hormones. In fertile nonpregnant women, we detected an expansion of CD4(+)CD25(+)FOXP3(+) Tregs in the late follicular phase of the menstrual cycle. This increase was tightly correlated with serum levels of estradiol and was followed by a dramatic decrease in Treg numbers at the luteal phase. Women who have had recurrent spontaneous abortions (RSA) showed similarly low numbers of Tregs at both the follicular and luteal phases, comparable to numbers we observed in postmenopausal women. In addition to decreased numbers, Tregs from women with RSA were also functionally deficient, as higher numbers were required to exert a similar magnitude of suppression to CD4(+)CD25(+)FOXP3(+) cells from fertile women. Consequently, reproductive failure might result from the inability of Tregs in women with RSA to expand during the preimplantatory phase combined with their lower functional capacity. Additionally, the modulation of Treg numbers we observed in fertile women suggests that the stage of the menstrual cycle should be taken into account when Treg numbers are investigated clinically.     

Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ(+) subset by flow cytometry. Purified IFN-γ(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-γ(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.     

Endogenous regulatory T cells adhere in inflamed dermal vessels via ICAM-1: association with regulation of effector leukocyte adhesion

Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.     

Thymus exosomes-like particles induce regulatory T cells

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3(+) regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4(+)CD25(-) T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4(+)CD25(-) T cells in vitro and in vivo. We further show that neutralization of TGF-beta in ELPs partially reverses thymic ELP-mediated induction of CD4(+)Foxp3(+) T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3(+) Tregs. Also, TGF-beta of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.