The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

Rabbit anti-thymocyte globulins (rATG) induce CD4⁺CD25⁺forkhead box P3 (FoxP3⁺) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4⁺CD25⁺FoxP3⁺CD127^−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25^neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25⁺ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4⁺CD25⁺FoxP3⁺CD127^−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3⁺T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3⁺ T cells. The rATG tacrolimus-induced CD25⁺ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4⁺CD25⁺FoxP3⁺CD127^−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25⁺ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25⁺ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4⁺CD25⁺ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.     

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.     

CD4+ CD25+ GARP+ regulatory T cells display a compromised suppressive function in patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a lethal inflammatory heart disease and closely connected with dysfunction of the immune system. Glycoprotein A repetitions predominant (GARP) expressed on activated CD4⁺ T cells with suppressive activity has been established. This study aimed to investigate the frequency and function of circulating CD4⁺   CD25⁺ GARP⁺ regulatory T (Treg) cells in DCM. Forty‐five DCM patients and 46 controls were enrolled in this study. There was a significant increase in peripheral T helper type 1 (Th1) and Th17 number and their related cytokines [interferon‐γ (IFN‐γ), interleukin (IL‐17)], and an obvious decrease in Treg number, transforming growth factor‐β₁ (TGF‐β₁) levels and the expression of forkhead box P3 (FOXP3) and GARP in patients with DCM compared with controls. In addition, the suppressive function of CD4⁺ CD25⁺ GARP⁺ Treg cells was impaired in DCM patients upon T‐cell receptor stimulation detected using CFSE dye. Lower level of TGF‐β₁ and higher levels of IFN‐γ and IL‐17 detected using ELISA were found in supernatants of the cultured CD4⁺ CD25⁺ GARP⁺ Treg cells in DCM patients compared with controls. Together, our results indicate that CD4⁺ CD25⁺ GARP⁺ Treg cells are defective in DCM patients and GARP seems to be a better molecular definition of the regulatory phenotype. Therefore, it might be an attractive stategy to pay more attention to GARP in DCM patients.     

End‐stage renal disease,dialysis, kidney transplantation and their impact on CD4+ T‐cell differentiation

Premature aging of both CD4⁺ regulatory T (Treg) and CD4⁺ responder‐T (Tresp) cells in patients with end‐stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T‐cell populations are released from the thymus as inducible T‐cell co‐stimulator‐positive (ICOS⁺) and ICOS^? recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS⁺ and ICOS^? RTE Treg/Tresp cells into ICOS⁺ CD31^? or ICOS^? CD31^? memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS⁺ and ICOS^? Treg cells in co‐culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS⁺ RTE Treg/Tresp cells and ICOS^? RTE Treg cells through CD31⁺ memory Treg/Tresp cells into CD31^? memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS^? RTE Tresp cells showed an increased differentiation via ICOS^? mature naive (MN) Tresp cells into CD31^? memory Tresp cells. Thereby, the ratio of ICOS⁺ Treg/ICOS⁺ Tresp cells was not changed, whereas that of ICOS^? Treg/ICOS^? Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS⁺ and ICOS^? RTE Tresp cells proceeded, whereas that of ICOS⁺ RTE Treg cells ceased and that of ICOS^? RTE Treg cells switched to an increased differentiation via ICOS^? MN Treg cells. Consequently, the ratios of ICOS⁺ Treg/ICOS⁺ Tresp cells and of ICOS^? Treg/ICOS^? Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance‐inducing differentiation of ICOS⁺ and ICOS^? Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long‐term renal replacement therapy.     

Avian CD4CD25 regulatory T cells: Properties and therapeutic applications

Regulatory T cells (Tregs) are a subset of T cells that specialize in immune suppression. CD4⁺CD25⁺FoxP3⁺ T cells have been characterized as Tregs and extensively studied in mammals. In the absence of a putative FoxP3 ortholog in avians, CD4⁺CD25⁺ cells is characterized as Tregs in avians. Avian CD4⁺CD25⁺ cells produce high amounts of IL-10, TGF-β, CTLA-4, and LAG-3 mRNA; lack IL-2 mRNA; and suppress T cell proliferation in vitro through both contact-dependent and -independent pathways. Depleting avian CD4⁺CD25⁺ cells increases the proliferation of, IL-2 amount, and IFNγ mRNA amount of CD4⁺CD25⁻ cells. Avian CD4⁺CD25⁺ cells lose their suppressive properties immediately after inflammation and acquire supersuppressive properties once inflammation subsides. Although Treg activity could be beneficial to the host, Tregs simultaneously inhibit host immunity and cause persistent infections of certain pathogens. Therapy targeted toward alleviating Treg mediated immune suppression can improve host immunity against those persistent pathogens and benefit poultry production.     

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.     

Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4⁺ or Tc : CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA⁻CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA⁻CCR7⁻ and late differentiated; CD27⁻CD28⁻CD45RA⁻CCR7⁻), terminally differentiated effector cells (TEMRA; CD27⁻CD28⁻CD45RA⁺CCR7⁻) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127⁻) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P < 0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127⁻) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129⁺ (P < 0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.     

Chemokines,chemokine receptors and CD4+CD25+ regulatory T cells

The fields of regulatory T (Treg) cells and chemokines/chemokine receptors have progressed rapidly in the last few years. Treg cells, especially CD4⁺CD25⁺ Treg cells, play a critical role in maintaining self-tolerance and immune homeostasis. Chemokines and chemokine receptors are crucial for lymphoid development, homing and immunological regulation. This review will discuss the biological effects of chemokines and chemokine receptors on regulating the migration and development of CD4⁺CD25⁺ Treg cells, and the potential clinical implications of these findings when considering chemokine receptors as therapeutic targets.     

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.     

Imbalance of different types of CD4+forkhead box protein 3 (FoxP3)+ T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to examine the numbers of CD4⁺CD25⁻forkhead box protein 3 (FoxP3)⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4⁺CD25⁻FoxP3⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4⁺FoxP3⁺ T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells, but increased numbers of CD4⁺CD25⁻FoxP3⁺ T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3⁺ T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4⁺CD25⁺FoxP3⁺ T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4⁺CD25⁻FoxP3⁺ T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4⁺CD25⁺FoxP3⁺ T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3⁺CD4⁺ T cells may contribute to the development of SLE in Chinese patients.     

Increased frequency and suppressive activity of CD127low/− regulatory T cells in the peripheral circulation of patients with head and neck squamous cell carcinoma are associated with advanced stage and nodal involvement

The presence of regulatory T (Treg) cells is thought to be an important mechanism by which head and neck squamous cell carcinoma (HNSCC) successfully evades the immune system. Using multicolour flow cytometry, the frequency and functional capacity of two CD4⁺ CD127^low/− Treg cell populations, separated on the basis of different levels of CD25 expression (CD25^inter and CD25^high), from the peripheral circulation of newly presenting HNSCC patients were assessed with regard to clinicopathological features and healthy controls. The frequency of circulating Treg cells was similar between HNSCC patients and healthy controls, and for patients with HNSCC developing from different subsites (laryngeal compared with oropharyngeal). However, patients with advanced stage tumours and those with nodal involvement had significantly elevated levels of CD4⁺ CD25^high CD127^low/− Treg cells compared with patients who had early stage tumours (P = 0·03) and those without nodal involvement (P = 0·03), respectively. CD4⁺ CD25^high CD127^low/− Treg cells from the entire HNSCC patient cohort and from patients whose tumours had metastasized to the lymph nodes were also shown to suppress the proliferation of effector T cells significantly more, compared with those from healthy controls (P = 0·04) or patients with no nodal involvement (P = 0·04). Additionally, CD4⁺ CD25^inter CD127^low/− Treg cells consistently induced greater suppressive activity than CD4⁺ CD25^high CD127^low/− Treg cells on the proliferation of the effector T-cell populations (CD4⁺ CD25⁻ CD127^−/+ and CD4⁺ CD25⁺ CD127⁺). Peripheral Treg cells, identified by the CD127^low/− phenotype, have been shown to be influenced by a patient''s tumour stage and/or nodal status in HNSCC; suggesting a role in tumour progression that could be manipulated by future immunotherapy.     

Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens

Regulatory T cells (Tregs) are defined as CD4⁺CD25⁺ cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4⁺CD25⁻ cells in Treg-depleted birds. The CD4⁺CD25⁺ cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4⁺CD25⁺ cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4⁺CD25⁺ cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4⁺CD25⁻ cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4⁺CD25⁺ cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4⁺CD25⁻ cells at 12 d post injection.     

Manipulation of regulatory T cells and antigen-specific cytotoxic T lymphocyte-based tumour immunotherapy

The most potent killing machinery in our immune system is the cytotoxic T lymphocyte (CTL). Since the possibility for self-destruction by these cells is high, many regulatory activities exist to prevent autoimmune destruction by these cells. A tumour (cancer) grows from the cells of the body and is tolerated by the body''s immune system. Yet, it has been possible to generate tumour-associated antigen (TAA) -specific CTL that are also self-antigen specific in vivo, to achieve a degree of therapeutic efficacy. Tumour-associated antigen-specific T-cell tolerance through pathways of self-tolerance generation represents a significant challenge to successful immunotherapy. CD4⁺ CD25⁺ FoxP3⁺ T cells, referred to as T regulatory (Treg) cells, are selected in the thymus as controllers of the anti-self repertoire. These cells are referred to as natural T regulatory (nTreg) cells. According to the new consensus (Nature Immunology 2013; 14:307–308) these cells are to be termed as (tTreg). There is another class of CD4⁺ Treg cells also involved in regulatory function in the periphery, also phenotypically CD4⁺ CD25^±, classified as induced Treg (iTreg) cells. These cells are to be termed as peripherally induced Treg (pTreg) cells. In vitro-induced Treg cells with suppressor function should be termed as iTreg. These different Treg cells differ in their requirements for activation and in their mode of action. The current challenges are to determine the degree of specificity of these Treg cells in recognizing the same TAA as the CTL population and to circumvent their regulatory constraints so as to achieve robust CTL responses against cancer.     

T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.     

Flt3 ligand expands CD4+FoxP3+ regulatory T cells in human subjects

CD4⁺CD25⁺FoxP3⁺ naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8⁺ T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg‐cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L‐induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L‐expanded pool of CD1c⁺ myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.     

Expansion of regulatory T cells from umbilical cord blood and adult peripheral blood CD4+CD25+ T cells

CD4⁺CD25⁺ regulatory T cells (Treg), if properly expanded from umbilical cord blood (UCB), may provide a promising immunotherapeutic tool. Our previous data demonstrated that UCB CD4⁺CD25⁺ T cells with 4-day stimulation have comparable phenotypes and suppressive function to that of adult peripheral blood (APB) CD4⁺CD25⁺ T cells. We further examined whether 2-week culture would achieve higher expansion levels of Tregs. UCB CD4⁺CD25⁺ T cells and their APB counterparts were stimulated with anti-CD3/anti-CD28 in the presence of IL-2 or IL-15 for 2 weeks. The cell proliferation and forkhead box P3 (FoxP3) expression were examined. The function of the expanded cells was then investigated by suppressive assay. IL-21 was applied to study whether it counteracts the function of UCB and APB CD4⁺CD25⁺ T cells. The results indicate that UCB CD4⁺CD25⁺ T cells expanded much better than their APB counterparts. IL-2 was superior to expand UCB and APB Tregs for 2 weeks than IL-15. FoxP3 expression which peaked on Day 10–14 was comparable. Most importantly, expanded UCB Tregs showed greater suppressive function in allogeneic mixed lymphocyte reaction. The addition of IL-21, however, counteracted the suppressive function of expanded UCB and APB Tregs. The results support using UCB as a source of Treg cells.     

The imbalance of T helper 17/regulatory T cells and memory B cells during the early post‐transplantation period in peripheral blood of living donor liver transplantation recipients under calcineurin inhibitor‐based immunosuppression

There is limited clinical research regarding the changes in peripheral lymphocyte subsets during the early post-operative period of liver transplantation. Serial changes of T cells and B cells in living donor liver transplantation (LDLT) recipients during the early post-transplantion period were prospectively investigated. From June 2010 to February 2011, 27 consecutive LDLT recipients were enrolled. Percentages of T helper type 1 (Th1; interferon-γ-producing), Th2 (interleukin-4-producing), Th17 (interleukin-17-producing), regulatory T (Treg; CD4⁺ CD25⁺ FoxP3⁺), memory B (CD19⁺ CD24^hi CD38⁻) and mature B (CD19⁺ CD24^int CD38^int) cells were measured using fluorescence-activated cell sorting. Patients were followed up for a median of 9·9 months (range 6·8−15·5 months) after transplantation. Serial monitoring of immunological profiles showed no significant suppression of Th1, Th2, Th17, mature B or memory B cells, whereas frequencies of Treg cells significantly decreased. Interleukin-17 production by central and effector memory cells was not suppressed during the early post-operative period. The continuous production of interleukin-17 by the memory T cells may contribute to the persistence of Th17 cells. This prospective study demonstrated that current immunosuppression maintained the effector T or memory B cells during the early post-transplantation period but significantly suppressed Treg cells. Serial immune monitoring may suggest clues for optimal or individualized immunosuppression during the early post-operative period in clinical practice.     

CD4(+) CD25(-) FoxP3(+) regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

The distribution and dynamic changes of CD4⁺ CD25⁺ FoxP3⁺ and CD4⁺ CD25⁻ FoxP3⁺ regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4⁺ CD25⁻ Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4⁺ CD25⁻ Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = −1, P < 0·0001). Hence, higher frequencies of functional CD4⁺ CD25⁻ Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4⁺ CD25⁻ Treg cells over CD4⁺ CD25⁺ Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4⁺ CD25⁻ Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4⁺ CD25⁻ Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.