CD39 is involved in mediating suppression by Mycobacterium bovis BCG‐activated human CD8+CD39+ regulatory T cells

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4⁺ Treg cells, relatively little is known about the phenotype and function of CD8⁺ Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E‐NTPDase1) on Mycobacterium‐activated human CD8⁺ T cells. These CD8⁺CD39⁺ T cells significantly co‐expressed the Treg markers CD25, Foxp3, lymphocyte activation gene‐3 (LAG‐3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen‐specific CD4⁺ T helper‐1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8⁺ T cells and indicate that CD39 is functionally involved in suppression by CD8⁺ Treg cells.     

Circulating CD137+CD8+ T cells accumulate along with increased functional regulatory T cells and thoracic tumour burden in lung cancer patients

CD137 is a promising target for immunostimulation strategies against cancer. Previous studies showed that CD137⁺CD8⁺ T cells are enriched in antitumour effector T cells in both preclinical tumour models and cancer patients, but to date, such T cells in the blood of lung cancer patients have not been sufficiently investigated. In this study, circulating antigen‐activated CD8⁺ T cell subsets, identified as CD137⁺CD8⁺ or PD‐1⁺ (programmed cell death protein 1) CD8⁺, and regulatory T cells (Treg), identified as CD4⁺CD25⁺CD127^low/?, in 40 untreated lung cancer patients and in 49 age‐ and sex‐matched healthy controls (HCs) were assessed by flow cytometry. Results were evaluated for associations with lung cancer patient clinical characteristics. Correlations between antigen‐activated CD8⁺ T cells and effector Treg (CTLA‐4⁺ [cytotoxic T‐lymphocyte antigen 4] CD4⁺CD25⁺CD127^low/?) were also investigated. Higher percentages of PD‐1⁺, CD137⁺ and PD‐1⁺CD137⁺ amongst CD8⁺ T cells were observed in lung cancer patients compared with HCs. The percentages of CD137⁺CD8⁺ and PD‐1⁺CD137⁺CD8⁺ T cell subsets amongst CD8⁺ T cells were positively correlated with thoracic tumour burden and were strongly positively correlated with the percentage of effector Treg subset. Smoking patients harboured higher percentages of the PD‐1⁺CD8⁺ T cell subset compared with non‐smoking patients. This study demonstrated that circulating antigen‐activated CD8⁺ T cells accumulated in lung cancer patients along with increased effector Treg and thoracic tumour burden. These findings aid a better understanding of immune‐host interactions in lung cancer patients using peripheral blood, and further support immunotherapeutic intervention strategies using combination therapy for differential control of Treg and activation of tumour‐specific effector T cells.     

Co‐expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3⁺ cells present in CD25^high, CD25^low and even CD25^– subsets of CD4⁺ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4⁺CD25 ⁺ Treg included all of the FOXP3⁺ cells present in the CD4⁺CD25^high subset as well as a substantial proportion of the FOXP3⁺ cells present in the CD4⁺CD25^low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4⁺CD25⁺TNFR2⁺ subset than in the CD4⁺CD25^high subset. In addition, similar levels of FOXP3⁺ cells were identified in both the CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁺CD127^low/? subsets. Furthermore, the CD4⁺CD25⁺TNFR2⁺ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4 ⁺ CD25 ⁺ TNFR2 ⁺ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4 ⁺ CD25 ⁺ TNFR2 ^– and CD4 ⁺ CD25 ^– TNFR2 ⁺ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.     

Induction of organ-selective CD4+ regulatory T cell homing

Compelling evidence suggests that Foxp3⁺CD25⁺CD4⁺ Treg play a fundamental role in immunoregulation. We have previously demonstrated that Treg have to enter peripheral tissues to suppress ongoing inflammation. However, relatively little is known about how Treg acquire the expression of homing receptors required for tissue‐ or inflammation‐specific migration. Migratory properties of conventional naïve T cells are shaped by the tissue microenvironment and organ‐specific dendritic cells during priming. Here, we show that this basic concept also holds true for CD25⁺CD4⁺ Treg: Priming of Treg within peripheral LN led to the expression of selectin ligands, which facilitate migration into inflamed skin, whereas activation within mesenteric LN led to induction of the integrin α₄β₇, which is required for migration into mucosal tissues. Furthermore, we could establish in vitro culture systems containing either dendritic cells from mesenteric and peripheral LN, or retinoic acid and IL‐12 as polarizing compounds to induce mucosa‐ and skin‐seeking Treg, respectively. Together, our results demonstrate that Treg, similarly to conventional T cells, can be configured with organ‐selective homing properties allowing efficient targeting into distinct tissues.     

CD4+ CD25+ Treg regulate the contribution of CD8+ T-cell subsets in repopulation of the lymphopenic environment

Peripheral T‐cell expansion is of major relevance for immune function after lymphopenia. In order to promote regeneration, the process should result in a peripheral T‐cell pool with a similar subpopulation structure as before lymphopenia. We investigated the repopulation of the CD8⁺ central‐memory T cells (T_CM) and effector‐memory T cells (T_EM) pools after adoptive transfer of sorted CD8⁺ T cells from naïve, T_CM and T_EM subsets into T‐cell‐deficient hosts. We show that the initial kinetics of expansion are distinct for each subset and that the contribution to the repopulation of the CD8⁺ T‐cell pool by the progeny of each subset is not a mere function of its initial expansion. We demonstrate that CD4⁺CD25⁺ Treg play a major role in the repopulation of the CD8⁺ T‐cell pool and that CD8⁺ T‐cell subsets impact on each other. In the absence of CD4⁺CD25⁺ Treg, a small fraction of naïve CD8⁺ T cells strongly proliferates, correlating with further expansion and differentiation of co‐expanding CD8⁺ T cells. CD4⁺CD25⁺ Treg suppress these responses and lead to controlled repopulation, contributing decisively to the maintenance of recovered T_CM and T_EM fractions, and leading to repopulation of each pool with progeny of its own kind.     

CTLA‐4 is required by CD4+CD25+ Treg to control CD4+ T‐cell lymphopenia‐induced proliferation

CTLA‐4 is constitutively expressed by CD4⁺CD25⁺Foxp3⁺ Treg but its precise role in Treg function is not clear. Although blockade of CTLA‐4 interferes with Treg function, studies using CTLA‐4‐deficient Treg have failed to reveal an essential requirement for CTLA‐4 in Treg suppression in vivo. Conditional deletion of CTLA‐4 in Foxp3⁺ T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA‐4 deletion have not been determined. We demonstrate that Treg expression of CTLA‐4 is essential for Treg control of lymphopenia‐induced CD4 T‐cell expansion. Despite IL‐10 expression, CTLA‐4‐deficient Treg were unable to control the expansion of CD4⁺ target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA‐4‐deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA‐4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non‐redundant role for CTLA‐4 in Treg function in vivo.     

CD4+CD25+ Treg induction by an HSP60‐derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent

Chronic schistosome infection results in the suppression of host immune responses, allowing long‐term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4⁺CD25⁺ T cells by Schistosoma japonicum HSP60 (SjHSP60)‐derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4⁺CD25⁺ T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4⁺CD25⁺ T‐cell populations. Release of IL‐10 and TGF‐β by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1‐induced CD4⁺CD25⁺ T cells inhibited delayed‐type hypersensitivity in BALB/c mice. Additionally, SJMHE1‐treated APC were tolerogenic and induced CD4⁺ cells to differentiate into suppressive CD4⁺CD25⁺ Treg. Furthermore, our data support a role for TLR2 in SJMHE1‐mediated CD4⁺CD25⁺ Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum–host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.     

CD122+CD8+ Treg suppress vaccine‐induced antitumor immune responses in lymphodepleted mice

Lymphodeleption prior to adoptive transfer of tumor‐specific T cells greatly improves the clinical efficacy of adoptive T‐cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates “space” for enhanced expansion and survival of tumor‐specific T cells. Within the lymphodepleted host, Ag‐specific T cells still need to compete with other lymphocytes that undergo lymphopenia‐driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia‐driven proliferation. We found that the major population that underwent lymphopenia‐driven proliferation was the CD122⁺ memory‐like T‐cell population (CD122⁺CD8⁺ Treg), and these cells competed with Ag‐driven proliferation of melanoma‐specific T cells. Removal of CD122⁺CD8⁺ Treg resulted in a greater expansion of tumor‐specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia‐driven proliferation of CD122⁺CD8⁺ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor‐specific T cells.     

Mapping urinary chemokines in human lupus nephritis: Potentially redundant pathways recruit CD4+ and CD8+ T cells and macrophages

Renal infiltration of inflammatory cells contributes to the pathogenesis of lupus nephritis (LN). Current knowledge on the recruitment mechanisms relies mainly on findings in rodent models. Here, we assess various chemokine pathways in human LN by comparing urinary chemokine concentrations (in 25 patients with acute LN and in 78 lupus patients without active LN) with the expression of corresponding chemokine receptors on urinary leukocytes (in ten acute LN patients). Nine urinary chemokines were significantly elevated in LN patients and correlated with renal disease activity and urinary cell counts; however, their concentrations displayed considerable interindividual heterogeneity. Analysis of the corresponding receptors revealed abundance of urinary CD8⁺ T cells for CCR5 and CXCR3, while CD4⁺ T cells were additionally enriched for CCR1, CCR6 and CXCR6. Urinary Treg showed similar CCR expression, and urinary CD14⁺ macrophages were enriched for CCR5 expressing cells. In conclusion, cell specific recruitment patterns seem to involve CCR5 and CXCR3 in all cells studied, while CD4⁺ T‐cell subset recruitment is probably much more varied. However, urinary chemokine abundance in active LN is individually variable in our cohort and does not offer a singular chemokine usable as universal biomarker or potential future treatment target.     

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.     

Mouse and human CD8+ CD28low regulatory T lymphocytes differentiate in the thymus

Regulatory T (Treg) lymphocytes play a central role in the control of immune responses and so maintain immune tolerance and homeostasis. In mice, expression of the CD8 co‐receptor and low levels of the co‐stimulatory molecule CD28 characterizes a Treg cell population that exerts potent suppressive function in vitro and efficiently controls experimental immunopathology in vivo. It has remained unclear if CD8⁺ CD28^low Treg cells develop in the thymus or represent a population of chronically activated conventional T cells differentiating into Treg cells in the periphery, as suggested by their CD28^low phenotype. We demonstrate that functional CD8⁺ CD28^low Treg cells are present in the thymus and that these cells develop locally and are not recirculating from the periphery. Differentiation of CD8⁺ CD28^low Treg cells requires MHC class I expression on radioresistant but not on haematopoietic thymic stromal cells. In contrast to other Treg cells, CD8⁺ CD28^low Treg cells develop simultaneously with CD8⁺ CD28^high conventional T cells. We also identified a novel homologous naive CD8⁺ CD28^low T‐cell population with immunosuppressive properties in human blood and thymus. Combined, our data demonstrate that CD8⁺ CD28^low cells can develop in the thymus of mice and suggest that the same is true in humans.     

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

Human Ag‐specific CD4⁺ T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag‐specific cells consistently identifies a substantial population of CD4⁺CD25⁺CD134⁺CD39⁺ T cells that have a Treg‐cell‐like phenotype and mostly originate from bulk memory CD4⁺CD45RO⁺CD127^lowCD25^highCD39⁺ Treg cells. Viable, Ag‐specific CD25⁺CD134⁺CD39⁺ T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA‐4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV‐Gag responses in HIV⁺ patients before and after anti‐retroviral therapy (ART). Interestingly, we found that the percentage of CD39^? cells within baseline CD4⁺ T‐cell responses to HIV‐Gag was negatively correlated with HIV viral load pre‐ART and positively correlated with CD4⁺ T‐cell recovery over 96 weeks of ART. Collectively, our data show that Ag‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg‐cell‐like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics.     

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.     

CD4+CD25+ regulatory T cells utilize FasL as a mechanism to restrict DC priming functions in cutaneous immune responses

Recent studies have suggested Fas‐mediated elimination of antigen‐presenting cells as an important mechanism down‐regulating the induction of autoimmune responses. It remains unknown whether this mechanism restricts the magnitude of immune responses to non‐self antigens. We used a mouse model of a cutaneous CD8⁺ T‐cell‐mediated immune response (contact hypersensitivity, CHS) to test if CD4⁺CD25⁺ T cells expressing FasL regulate hapten‐specific effector CD8⁺ T cell expansion through the elimination of Fas‐expressing hapten‐presenting DC. In WT mice, attenuation of CD4⁺CD25⁺ T regulatory cell activity by anti‐CD25 mAb increased hapten‐presenting DC numbers in skin‐draining LN, which led to increased effector CD8⁺ T‐cell priming for CHS responses. In contrast, CD4⁺CD25⁺ T cells did not regulate hapten‐specific CD8⁺ T‐cell priming and CHS responses initiated by Fas‐defective (lpr) DC. Thus, restricting DC priming functions through Fas–FasL interactions is a potent mechanism employed by CD4⁺CD25⁺ regulatory cells to restrict CD8⁺ T‐cell‐mediated allergic immune responses in the skin.     

Epithelial expression of human papillomavirus type 16 E7 protein results in peripheral CD8 T‐cell suppression mediated by CD4+CD25+ T cells

The role of thymic versus peripheral epithelium in the regulation of the antigen‐specific CD8 T‐cell repertoire is still largely unresolved. We generated TCR‐β chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H‐2D^b MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T‐cell development in the thymus was observed in these double‐transgenic mice. In contrast, peripheral CD8 T‐cell responses in the single‐transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T‐cell responses and antibody production are unchanged in these mice. Peripheral non‐responsiveness among CD8 T cells was mediated largely by CD4⁺CD25⁺ T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down‐regulate CD8 T‐cell responses in the periphery. This may have important consequences for the treatment of cervical pre‐cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.     

Activation of CD4+ CD25+ regulatory T cell suppressor function by analogs of the selecting peptide

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells can undergo both thymic selection and peripheral expansion in response to self peptides that are agonists for their T cell receptors (TCR). However, the specificity by which these TCR must recognize peptide:MHC complexes to activate Treg cell function is not known. We show that CD4⁺CD25⁺Foxp3⁺ Treg cells can mediate suppression in response to peptides that are only weakly cross‐reactive with the self peptide that induced their formation in vivo. Moreover, suppression could be efficiently activated by peptide analogs that were inefficient at inducing CD69 up‐regulation, and that also induced little or no proliferation of naïve CD4⁺CD25^–Foxp3^– T cells expressing the same TCR. These findings provide evidence that self peptide‐specific CD4⁺CD25⁺Foxp3⁺ Treg cells can exert regulatory function in response to self‐ and/or pathogen‐derived peptides with which they are only weakly cross‐reactive.     

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (T_reg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of T_reg (CD4⁺CD25^highforkhead box protein 3⁺), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4⁺CD25⁺, CD4⁺CD25^highFoxP3⁺ and CD8⁺CD25⁺ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4⁺CD25^low/negIL‐10⁺ in PB and CD4⁺CD25^low/negIFN‐γ⁺ in PF; meanwhile, CD25^high mainly expressed IL‐10 in both compartments. A high proportion of CD4⁺CD107⁺ and CD8⁺CD107⁺ cells was observed in PF. T_reg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4⁺ and CD8⁺ degranulation responses and decreased CD4⁺IL‐10⁺ cells in PF. Our results demonstrated that in TB pleurisy T_reg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4⁺ and CD8⁺ cells to degranulate in response to M. tuberculosis.     

Recurrent superantigen exposure in vivo leads to highly suppressive CD4+CD25+ and CD4+CD25‐ T cells with anergic and suppressive genetic signatures

Staphylococcal enterotoxin B (SEB) activates T cells via non‐canonical signalling through the T cell receptor and is an established model for T cell unresponsiveness in vivo. In this study, we sought to characterize the suppressive qualities of SEB‐exposed CD4⁺ T cells and correlate this with genetic signatures of anergy and suppression. SEB‐exposed CD25⁺ and CD25^‐Vβ8⁺CD4⁺ T cells expressed forkhead box P3 (FoxP3) at levels comparable to naive CD25⁺ T regulatory cells and were enriched after exposure in vivo. Gene related to anergy in lymphocytes (GRAIL), an anergy‐related E3 ubiquitin ligase, was up‐regulated in the SEB‐exposed CD25⁺ and CD25^‐FoxP3⁺Vβ8⁺CD4⁺ T cells and FoxP3^‐CD25^‐Vβ8⁺CD4⁺ T cells, suggesting that GRAIL may be important for dominant and recessive tolerance. The SEB‐exposed FoxP3⁺GRAIL⁺ T cells were highly suppressive and non‐proliferative independent of CD25 expression level and via a glucocorticoid‐induced tumour necrosis factor R‐related protein‐independent mechanism, whereas naive T regulatory cells were non‐suppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3⁺ regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non‐canonical T cell receptor engagement leading to highly suppressive FoxP3⁺GRAIL⁺CD4⁺ T cells.     

Increased numbers of thymic and peripheral CD4+ CD25+Foxp3+ cells in the absence of CD5 signaling

It has been suggested that high affinity/avidity interactions are required for the thymic selection of Treg. Here, we investigated the role of CD5, a negative regulator of TCR signaling, in the selection and function of “naturally occurring” CD4⁺CD25⁺ Treg (nTreg). Analysis of CD5^?/? mice showed a significant increase in the percentage and absolute numbers of CD4⁺ CD25⁺Foxp3⁺ thymocytes and peripheral T lymphocytes, compared with BALB/c mice. Thymi from CD5^?/? mice showed reduced cellularity due to increased apoptosis, which preferentially affected naïve T cells. To characterize nTreg selection at the molecular level we investigated the phosphorylation of Erk, c‐Cbl, PI3K and Akt. CD5^?/? nTreg showed increased basal levels of p‐Erk compared with wild‐type nTreg. Interestingly, in response to CD3 plus CD28 costimulation, CD5^?/? naïve T cells but not CD5^?/? nTreg showed lower levels of p‐Akt. Finally, CD5^?/? nTreg were thymus‐derived and fully functional. We conclude that the enrichment of nTreg observed in the absence of CD5 signaling is due to de novo generation of nTreg and selective reduction of CD4⁺CD25^? naïve thymocytes. Furthermore, we provide new evidence supporting a potential role of CD5 in thymocyte survival, through a mechanism that may involve the phosphorylation of Akt.