Allogeneic induced human FOXP3+IFN‐γ+ T cells exhibit selective suppressive capacity

Human induced CD4⁺CD25⁺ T cells have been shown to express FOXP3, similar to naturally occurring Treg cells (nTreg). However, the suppressive capacity of these cells is still under debate. The current study was designed to investigate functional characteristics of CD25⁺FOXP3⁺ derived from CD25^? T cells. Stimulation of CD25^? PBMC with allogeneic PBMC resulted in production of CD4⁺CD25^high T cells. This process was more rapid and prominent when highly mature DC were used for stimulation. The resultant CD4⁺CD25^high population concurrently exhibited regulatory markers FOXP3, CTLA‐4, GITR, and inflammatory cytokines IL‐2 and IFN‐γ. These human‐induced FOXP3⁺IFN‐γ⁺ T cells were shown, for the first time, to markedly inhibit alloreactive T‐cell expansion, similar to nTreg. However, in contrast to nTreg, the induced CD4⁺CD25⁺FOXP3⁺ cells did not suppress proliferation against a third party donor stimulus or CMV. This suggested that the cell population possessed a more selective suppressive capacity targeted against the original stimulus only. The induced human CD4⁺CD25⁺FOXP3⁺ subset derived from CD25^? T cells, while expressing inflammatory cytokines, exhibits a suppressive cell contact‐dependent effect, restricted against T cells responding to the original stimulus. Such unique properties suggest that these cells are potentially ideal for the use as post‐transplant GVH disease prophylaxis.     

CD8α+ dendritic cells prime TCR‐peptide‐reactive regulatory CD4+FOXP3− T cells

CD4⁺ T cells with immune regulatory function can be either FOXP3⁺ or FOXP3^?. We have previously shown that priming of naturally occurring TCR‐peptide‐reactive CD4⁺FOXP3^? Treg specifically controls Vβ8.2⁺CD4⁺ T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVβ8.2‐chain, is not known. In this study we show that APC derived from splenocytes of naïve mice are able to stimulate cloned CD4⁺ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4⁺ Treg was dependent upon processing and presentation of TCR peptides from ingested Vβ8.2TCR⁺CD4⁺ T cells. Additionally, DC pulsed with TCR peptide or apoptotic Vβ8.2⁺ T cells were able to prime Treg in vivo and mediate protection from disease in a CD8‐dependent fashion. These data highlight a novel mechanism for the priming of CD4⁺ Treg by CD8α⁺ DC and suggest a pathway that can be exploited to prime antigen‐specific regulation of T‐cell‐mediated inflammatory disease.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

Functional islet‐specific Treg can be generated from CD4+CD25− T cells of healthy and type 1 diabetic subjects

CD4⁺CD25⁺FOXP3⁺ Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self‐antigen‐specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen‐specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen‐specific adaptive Treg were induced in vitro by activation of CD4⁺FOXP3^? T cells with glutamic acid decarboxylase and islet‐specific glucose‐6‐phosphate catalytic subunit‐related protein peptides in the context of T1D‐associated HLA‐DRβ alleles. Antigen‐specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza‐specific Treg from the same study population. The function of tetramer⁺ cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet‐specific Treg may potentially be used as a targeted immunotherapy in T1D.     

Phenotypic analysis of human peripheral blood regulatory T cells (CD4+FOXP3+CD127lo/–) ex vivo and after in vitro restimulation with malaria antigens

Regulatory T cells (Treg) play crucial roles in regulating autoimmune responses and immunity to tumors and infectious diseases. However, numerous subpopulations of Treg are now being described and the utility of various Treg markers is being reassessed. Here we report the results of a detailed phenotypic comparison of two supposedly regulatory human T‐cell populations, namely CD4⁺FOXP3⁺ T cells and CD4⁺CD25^hi T cells. We find that CD4⁺FOXP3⁺ cells are extremely heterogeneous with respect to CD25 expression and that FOXP3⁺ and CD25^hi CD4⁺ T cells differ in their expression of chemokine receptors (CCR), CD95 and Bcl‐2, suggestive of distinct migration characteristics and susceptibility to apoptosis. Further, we propose that CD25 expression should be regarded as an activation marker rather than as a defining marker of Treg. Lastly, CD4⁺FOXP3⁺ T cells activated in vitro with malaria antigen expressed the highest levels of CCR4 and CD95, and the lowest levels of CCR7, indicating that they are most likely generated from effector memory cells during an immune response and rapidly succumb to apoptosis at the end of the response.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

The adoptive transfer of CD4⁺CD25⁺ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127⁺ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3⁺ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4⁺CD25⁺CD127^low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA^?FOXP3⁺ memory‐type Treg as the main source of converting cells, whereas CD45RA⁺FOXP3⁺ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.     

Enhanced suppressor function of TIM‐3+FoxP3+ regulatory T cells

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) is an Ig‐superfamily member expressed on IFN‐γ‐secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM‐3 is expressed by a subset of activated CD4⁺ T cells, and anti‐CD3/anti‐CD28 stimulation increases both the level of expression and the number of TIM‐3⁺ T cells. In mice, TIM‐3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4⁺ T‐cell differentiation. Here, we examined TIM‐3 expression on human Treg cells to determine its role in T‐cell suppression. In contrast to mice, TIM‐3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM‐3⁺ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM‐3^? Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT‐3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM‐3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1‐ and Th17‐cell responses.     

Surface expression of CD39 identifies an enriched Treg‐cell subset in the rheumatic joint,which does not suppress IL‐17A secretion

Treg cells are important for the maintenance of self‐tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP‐hydrolyzing enzymes CD39 and CD73 and the resulting anti‐inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3⁺ Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3⁺ Treg cells displayed high expression levels of rate‐limiting CD39, whereas CD73 was diminished. FOXP3⁺CD39⁺ Treg cells were also abundant in synovial tissue. Furthermore, FOXP3⁺CD39⁺ Treg cells did not secrete the proinflammatory cytokines IFN‐γ and TNF after in vitro stimulation in contrast to FOXP3⁺CD39^? T cells. FOXP3⁺CD39⁺ Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg‐specific demethylated region and coculture assays confirmed that CD25⁺CD39⁺ T cells have suppressive capacity, while their CD39^? counterparts do not. Overall, our data show that FOXP3⁺CD39⁺ Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T‐cell functions including production of IFN‐γ, TNF, and IL‐17F but do not limit IL‐17A secretion.     

Ribavirin modulates the conversion of human CD4+ CD25− T cell to CD4+ CD25+ FOXP3+ T cell via suppressing interleukin‐10‐producing regulatory T cell

Because regulatory T (Treg) cells play an important role in modulating the immune system response against both endogenous and exogenous antigens, their control is critical to establish immunotherapy against autoimmune disorders, chronic viral infections and tumours. Ribavirin (RBV), an antiviral reagent used with interferon, is known to polarize the T helper (Th) 1/2 cell balance toward Th1 cells. Although the immunoregulatory mechanisms of RBV are not fully understood, it has been expected that RBV would affect T reg cells to modulate the Th1/2 cell balance. To confirm this hypothesis, we investigated whether RBV modulates the inhibitory activity of human peripheral CD4⁺ CD25⁺ CD127⁻ T cells in vitro. CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV lose their ability to inhibit the proliferation of CD4⁺ CD25⁻ T cells. Expression of Forkhead box P3 (FOXP3) in CD4⁺ CD25⁻ T cells was down-modulated when they were incubated with CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV without down-modulating CD45RO on their surface. In addition, transwell assays and cytokine-neutralizing assays revealed that this effect depended mainly on the inhibition of interleukin-10 (IL-10) produced from CD4⁺ CD25⁺ CD127⁻ T cells. These results indicated that RBV might inhibit the conversion of CD4⁺ CD25⁻ FOXP3⁻ naive T cells into CD4⁺ CD25⁺ FOXP3⁺ adaptive Treg cells by down-modulating the IL-10-producing Treg 1 cells to prevent these effector T cells from entering anergy and to maintain Th1 cell activity. Taken together, our findings suggest that RBV would be useful for both elimination of long-term viral infections such as hepatitis C virus infection and for up-regulation of tumour-specific cellular immune responses to prevent carcinogenesis, especially hepatocellular carcinoma.     

Human adipose‐tissue derived mesenchymal stem cells induce functional de‐novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4⁺CD25⁺CD127^–forkhead box P3 (FoxP3)⁺ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT_reg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25⁺CD127^–FoxP3⁺ cells within the CD4⁺ T cell population from allostimulated CD25^–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iT_reg) inhibited effector cell proliferation as effectively as nT_reg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nT_reg origin. In conclusion, ASC induce T_reg from effector T cells. These iT_reg have immunosuppressive capacities comparable to those of nT_reg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.     

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg

CD25⁺ FOXP3⁺CD4⁺ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high‐level expression of FOXP3 (FOXP3^high) is sufficient to confer suppressive activity to normal non‐Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3^high but not FOXP3^low Treg. Such VEGFR2⁺ Treg exist in several tissues including PBMC and malignant effusion‐derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.     

Staphylococcus aureus convert neonatal conventional CD4+ T cells into FOXP3+ CD25+ CD127low T cells via the PD‐1/PD‐L1 axis

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4⁺ T cells for the induction of CD25⁺ CD127^low Treg cells in vitro. The proportion of circulating CD25⁺ CD127^low Treg cells and their expression of FOXP3, Helios and CTLA‐4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet‐stained non‐Treg cells from cord or peripheral blood from adults were co‐cultured with autologous CD25⁺ CD127^low Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25⁺ CD127^low Treg cells than adults, but these cells were Helios⁺ and CTLA‐4⁺ to a higher extent than in adults. FOXP3⁺ CD25⁺ CD127^low T cells were induced mainly in neonatal CellTrace‐stained non‐Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25⁺ CD127^low T cells only if sorted naive CD45RA⁺ non‐Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25⁺ CD127^low T cells from S. aureus‐stimulated cultures were still suppressive. Finally, blocking PD‐L1 during stimulation reduced the induction of FOXP3⁺ CD25⁺ CD127^low T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios⁺ Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4⁺ T cells into FOXP3⁺ CD25⁺ CD127^low Treg cells via the PD‐1/PD‐L1 axis.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

Sympathetic neural signaling via the β2‐adrenergic receptor suppresses T‐cell receptor‐mediated human and mouse CD8+ T‐cell effector function

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2‐adrenergic receptor (ADRB2) on primary human CD8⁺ effector memory T cells. Treatment of both human and murine CD8⁺ T cells with NE decreased IFN‐γ and TNF‐α secretion and suppressed their cytolytic capacity in response to T‐cell receptor (TCR) activation. The effects of NE were specifically reversed by β 2‐specific antagonists. Adrb2^?/? CD8⁺ T cells were completely resistant to the effects of NE. Further, the ADRB2‐specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8⁺ T cells. While both TCR activation and stimulation with IL‐12 + IL‐18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN‐γ secretion in response to IL‐12 + IL18. Finally, the long‐acting ADRB2‐specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8⁺ T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8⁺ T‐cell function and underscores the novel role this pathway plays in adaptive T‐cell responses to infection.     

β‐cell‐specific IL‐35 therapy suppresses ongoing autoimmune diabetes in NOD mice

IL‐35 is a recently identified cytokine exhibiting potent immunosuppressive properties. The therapeutic potential and effects of IL‐35 on pathogenic T effector cells (Teff) and Foxp3⁺ Treg, however, are ill defined. We tested the capacity of IL‐35 to suppress ongoing autoimmunity in NOD mice. For this purpose, an adeno‐associated virus vector in which IL‐35 transgene expression is selectively targeted to β cells via an insulin promoter (AAV8mIP‐IL35) was used. AAV8mIP‐IL35 vaccination of NOD mice at a late preclinical stage of type 1 diabetes (T1D) suppressed β‐cell autoimmunity and prevented diabetes onset. Numbers of islet‐resident conventional CD4⁺ and CD8⁺ T cells, and DCs were reduced within 4 weeks of AAV8mIP‐IL35 treatment. The diminished islet T‐cell pool correlated with suppressed proliferation, and a decreased frequency of IFN‐γ‐expressing Teff. Ectopic IL‐35 also reduced islet Foxp3⁺ Treg numbers and proliferation, and protection was independent of induction/expansion of adaptive islet immunoregulatory T cells. These findings demonstrate that IL‐35‐mediated suppression is sufficiently robust to block established β‐cell autoimmunity, and support the use of IL‐35 to treat T1D and other T‐cell‐mediated autoimmune diseases.