Potential role of plasmacytoid dendritic cells for FOXP3 regulatory T cell development in human colorectal cancer and tumor draining lymph node

FOXP3⁺ regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. Compared with conventional myeloid dentritic cells (mDCs), plasmacytoid dendritic cells (pDCs) exhibit poor immunostimulatory ability, and their interaction with T cells often promotes the development of Tregs. The aim of this study was to determine FOXP3⁺ Tregs and CD123⁺pDCs infiltration in colorectal cancer and tumor draining lymph node (TDLN), and to evaluate the clinical significance and relationship between pDCs infiltration and Tregs development in the CRC tolerogenic milieu. An immunohistochemical assay was conducted to assess FOXP3⁺Tregs and CD123⁺pDCs infiltration in tumor tissue and in metastatic-free TDLN (mfTDLN) and metastatic TDLN (mTDLN). The results showed that FOXP3⁺ Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa (P < 0.001). FOXP3⁺Tregs infiltration was associated with advanced TNM stage and lymph node metastasis (P < 0.01 and P < 0.01 for TNM stage and lymph node metastasis, respectively). Different from FOXP3⁺Tregs, CD123⁺pDCs frequencies were lower in most CRC tumor tissues, whereas the positive rate of CD123 expression in CRC was significantly higher than in adjacent normal mucosa tissue (P < 0.01). Compared to mfTDLN, mTDLN was significantly enriched in FOXP3⁺ Tregs (P < 0.01) and increased in pDC/mDC ratio (P < 0.01). The statistical analysis demonstrated a significant correlation in both Tregs and pDC/mDC ratio in mTDLN. These results suggest that there are more FOXP3⁺ Tregs with a stronger prognostic significance which might promote tumor tolerance, and that CD123⁺pDCs might contribute to Tregs development in the CRC tolerogenic milieu.     

PDCD5 negatively regulates autoimmunity by upregulating FOXP3+ regulatory T cells and suppressing Th17 and Th1 responses

Maintenance of FOXP3 protein expression is crucial for differentiation and maturation of regulatory T (Treg) cells, which play important roles in immune homeostasis and immune tolerance. We demonstrate here that PDCD5 interacts with FOXP3, increases acetylation of FOXP3 in synergy with Tip60 and enhances the repressive function of FOXP3. In PDCD5 transgenic (PDCD5tg) mice, overexpression of PDCD5 enhanced the level of FOXP3 protein and percentage of CD4⁺CD25⁺FOXP3⁺ cells. Naïve CD4⁺ T cells from PDCD5tg mice were more sensitive to TGF-β-induced Treg polarization and expansion. These induced Tregs retained normal suppressive function in vitro. Severity of experimentally-induced autoimmune encephalomyelitis (EAE) in PDCD5tg mice was significantly reduced relative to that of wild-type mice. The beneficial effect of PDCD5 likely resulted from increases of Treg cell frequency, accompanied by a reduction of the predominant pathogenic Th17/Th1 response. Activation-induced cell death enhanced by PDCD5 was also linked to this process. This is the first report revealing that PDCD5 activity in T cells suppresses autoimmunity by modulating Tregs. This study suggests that PDCD5 serves as a guardian of immunological functions and that the PDCD5-FOXP3-Treg axis may be a therapeutic target for autoimmunity.     

Inflammatory bowel disease

Systemic sclerosis (SSc) is a generalized connective tissue disorder, characterized by a wide spectrum of microvascular and immunological abnormalities, leading to a progressive thickening and fibrosis of the skin and other organs, such as the lungs, GI tract, heart and kidneys. SSc is thought to be an autoimmune disease owing to the presence of high affinity antibodies and possible clinical overlap with other autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Autoimmune diseases arise because of a breakdown in immunological self tolerance. Self tolerance is maintained via multiple regulatory mechanisms within the immune system, including the thymic deletion of self-reactive T cells and mechanisms of peripheral tolerance. In recent years, the presence of CD4⁺CD25⁺FOXP3⁺ Tregs has been identified as a major mechanism of peripheral tolerance, and accumulating evidence indicates that alterations in Treg frequencies and/or function may contribute to autoimmune diseases. Here, we will review recent data on the percentage, function and phenotype of CD4⁺CD25⁺ Tregs in rheumatic disease, and discuss how recent developments may guide research in this area in SSc.     

IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16% ± 1.8 vs. 5.53% ± 1.2, p < 0.05). Despite observing a higher total number of dead CD4⁺ cells in the IDO group, there was a more abundant live CD4⁺CD25⁺ subpopulation in this group. Further analysis reveales that these CD4⁺ CD25⁺ cells have the capacity to expand in the presence of IDO expressing DFs. Greater number of CTLA-4⁺ cells and high expression of TGF-β and IL-10 were found in CD4⁺ cells of the IDO group compared to those of the controls. This finding confirmed a suppressive functionality of the expanded Tregs. Furthermore, CD4⁺ CD25⁺ cells isolated from the IDO group showed an alloantigen specific suppressive effect in a mixed lymphocyte reaction assay. These results confirm that IDO expressing dermal fibroblasts can expand a population of suppressive antigen specific Tregs. In conclusion, IDO expressing dermal fibroblasts have the capacity to stimulate the expansion of a subset of Tregs which can be used to generate antigen-specific immune tolerance.     

Dexamethasone upregulates FOXP3 expression without increasing regulatory activity

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are “truly” Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4⁺CD25^high cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25^high cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4⁺CD25^high cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.     

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.     

Isolation and expansion of thymus-derived regulatory T cells for use in pediatric heart transplant patients

Regulatory T-cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP-compatible protocol for expanding sorted thymus-derived CD3⁺CD4⁺CD25⁺CD127^– (Tregs) as well as CD3⁺CD4⁺CD25⁺CD127^–CD45RA⁺ (RA⁺Tregs) cells. We aimed to understand whether thymic RA⁺Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA⁺ Tregs. We show that both Tregs and RA⁺Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN-γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA⁺Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.     

Sirolimus vs mycophenolate moftile in Tacrolimus based therapy following induction with Antithymocyte globulin promotes regulatory T cell expansion and inhibits RORγt and T-bet expression in kidney transplantation

BackgroundAccumulating evidence suggests that regulatory T cells (Tregs) have a crucial role in immune tolerance and long-term graft survival. However, the influence of immunosuppressive drugs on the level of Tregs has not been fully understood. Therefore we prospectively compare the effect of two different calcineurin inhibitor (CNI)-based immunosuppression protocols on Tregs frequencies and expression of regulatory and effector T cell-related genes in renal transplant recipients.MethodsThe study included 24 renal transplant recipients who received induction therapy (Antithymocyte globulin) and were on triple immunosuppressive therapy so that one group was on Tacrolimus (Tac), mycophenolate moftile (MMF) and prednisolone (P) whereas another group was on Tac, Sirolimus (SRL) and P. The frequency of circulating Treg cells was analyzed by flow cytometry before and 4 months after transplantation. Also, the mRNA expression of FOXP3, T-bet, GATA3 and RORγt was examined by quantitative RT-PCR before and 4 months after transplantation.ResultsCompared to baseline, the frequency of CD4⁺ CD25⁺ FOXP3⁺ Treg cells was significantly increased in the all patients following transplantation. Patients who received Tac/MMF had significantly higher CD4⁺ CD25⁺ FOXP3⁺ Treg cells compared to patients who received Tac/SRL. There was no a significant difference in the frequency of CD3⁺CD8⁺ CD28^? Tregs between two different calcineurin inhibitor (CNI)-based immunosuppression protocols. FOXP3 mRNA levels in the patients who received Tac/MMF were increased 4 months after transplantation and the expression was significantly higher than patients who received Tac/SRL. On the other hand, T-bet and RORγt expression levels were significantly lower in the Tac/SRL group in comparison to Tac/MMF group. We did not observe any significant difference in GATA3 mRNA level between the two groups.ConclusionsOur results suggest that although Tac/MMF-containing immunosuppressive regimen could significantly increase the frequency of CD4⁺ CD25⁺ FOXP3⁺ Tregs, unlike to Tac/SRL-containing regimen, it could not significantly decrease the expression levels of RORγt and T-bet.     

Identification of FOXP3-negative regulatory T-like (CD4(+)CD25(+)CD127(low)) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disorder caused by mutations in the FOXP3 gene, which plays a key role in the generation of CD4⁺CD25⁺regulatory T (Treg) cells. We selected CD127 as the surface marker of Treg cells to illustrate the development and function of Treg cells in IPEX syndrome. CD4⁺CD25⁺FOXP3⁺ T cells, the putative Treg cells, were almost completely absent in all patients. Importantly, a substantial number of CD4⁺CD25⁺CD127^low T cells were observed in 3 IPEX patients with hypomorphic mutations in the FOXP3 gene. We demonstrated that CD4⁺CD25⁺CD127^low T cells isolated from these 3 patients exhibited an appreciable suppressive activity on effector T cell proliferation, although less than that displayed by Treg cells from healthy controls. These results suggest that genetically altered FOXP3 can drive the generation of functionally immature Treg cells, but that intact FOXP3 is necessary for the complete function of Treg cells.     

B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas

Objective

To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro.

Methods

B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8⁺T, CD4⁺T, and FOXP3⁺ (Forkhead Box P3) T lymphocytes and their levels of IFN-γ and TGF-β₁ production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA.

Results

B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8⁺T lymphocytes and their IFN-γ production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3⁺T and CD4⁺T cells or TGF-β1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4⁺T and CD8⁺T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4⁺T and CD8⁺T cells decreased, but CD4⁺T/CD8⁺T and the proportion of CD4⁺CD25⁺Foxp3⁺T cells increased. In addition, concentrations of IL-10 and TGF-β1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-γ decreased. B7-H4 had no significant effect on apoptosis of the T cells.

Conclusion

B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8⁺T lymphocytes and therefore less IFN-γ production. In vitro, B7-H4 inhibits the proliferation of CD4⁺T and CD8⁺T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-β1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8⁺T cell in microenvironments of cervical 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.     

Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritis in an interferon-gamma knockout model

Maintaining an appropriate balance between subsets of CD4⁺ helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4⁺T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-β) or Th17-polarizing (anti-CD3/CD28, TGF-β, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-β-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-β-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3⁺ Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.     

Analysis of FOXP3<Superscript>+</Superscript> regulatory T cell subpopulations in peripheral blood and tissue of patients with systemic lupus erythematosus

Regulatory T cells (Tregs) are critical mediators of immune tolerance, yet their involvement in the autoimmune disease systemic lupus erythematosus (SLE) is incompletely understood. We analyzed CD4⁺ T cell subpopulations with Treg-related phenotypes and their association with disease activity in peripheral blood (PB) and tissues of patients with SLE. In detail, we quantified subpopulations regarding CD25, FOXP3, CD62L, CCR6, CD27, CD45RA, and CD45RO expression in PB from 31 patients with SLE divided into two disease activity groups and 32 healthy controls using flow cytometry. CD4⁺ and FOXP3⁺ T cells in skin and kidney biopsies of patients with SLE were quantified by immunohistochemistry. CD4⁺CD25^+/++FOXP3⁺ and CD4⁺CD25⁺CD45RA^?/CD45RO⁺ T cell frequencies were significantly higher in PB from patients with active compared to inactive SLE. The fraction of CD4⁺CD25⁺⁺FOXP3⁺ Tregs and CD4⁺CD25⁺CD45RA⁺/CD45RO^? naïve Tregs was not significantly different between these groups. CD4⁺CD25⁺⁺ Tregs from active SLE patients comprised significantly less CD27⁺ cells and more CCR6⁺ cells compared to patients with inactive SLE. The percentage of CD4⁺FOXP3⁺ T cells among inflammatory infiltrates in skin and kidney biopsies of SLE patients was not different from other inflammatory skin/kidney diseases. In conclusion, although CD4⁺FOXP3⁺ T cell frequencies in the inflamed tissues of SLE patients were comparable to other inflammatory diseases, distinct T cell subpopulations appeared misbalanced in PB of patients with active SLE. Here, cells phenotypically resembling activated T cells, but not Tregs, were increased compared to patients with inactive SLE. Within Tregs of patients with active SLE, markers related to Treg function and homing were altered.     

Aging and human CD4 regulatory T cells

Alterations in immunity that occur with aging likely contribute to the development of infection, malignancy and inflammatory diseases. Naturally occurring CD4⁺ regulatory T cells (Treg) expressing high levels of CD25 and forkhead box P3 (FOXP3) are essential for regulating immune responses. Here we investigated the effect of aging on the number, phenotypes and function of CD4⁺ Treg in humans. The frequency and phenotypic characteristics of CD4⁺, FOXP3⁺ T cells as well as their capacity to suppress inflammatory cytokine production and proliferation of CD4⁺, CD25⁻ T cells (target cells) were comparable in young (age ≤40) and elderly (age ≥65) individuals. However, when CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells were co-cultured at a ratio of 1:1, the production of anti-inflammatory cytokine IL-10 from CD4⁺, CD25⁻ T cells was more potently suppressed in the elderly than in the young. This finding was not due to changes in CTLA-4 expression or apoptosis of CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells. Taken together, our observations suggest that aging may affect the capacity of CD4⁺, FOXP3⁺ T cells in regulating IL-10 production from target CD4⁺ T cells in humans although their other cellular characteristics remain unchanged.     

Predictive and prognostic factors in locally advanced breast cancer: effect of intratumoral FOXP3+ Tregs

This study aimed to investigate the prognostic and predictive effect of FOXP3+ Tregs together with clinicopathologic factors in locally advanced breast cancer (LABC) patients. The medical records of 101 LABC patients who received neoadjuvant chemotherapy (NAC) between 2005 and 2012 were evaluated retrospectively. The density of intratumoral FOXP3+ lymphocytes in paraffin-embedded tissues was assessed by immunohistochemical analyses in appropriate cases. The relationship with clinicopathologic features, prognosis and chemotherapy response was investigated. HR(–) and HER2(+) tumors tended to have higher pre-chemotherapy Tregs than HR(+) tumors, and significantly higher pathologic complete response (PCR) rates were observed in these patients. Treg decline after NAC was associated with better pathological response rates. Lower intratumoral infiltration of FOXP3+ Tregs after NAC (<3.4/HPF) was significantly associated with higher PCR rates for breast, and close to the significance limit for total (or both for breast and axillary) PCR rates (PCR for breast: 25 vs. 2.9 % for low vs. high Treg, p = 0.001; PCR for breast + axillary tissue: 13.9 vs. 0 %, p = 0.05). Despite better PCR rates, patients with high intratumoral Treg infiltrates (≥11.5/HPF) before chemotherapy had significantly shorter overall survival than patients with low Treg infiltrates (<11.5/HPF). Cox multivariate regression analyses demonstrated that the density of Treg infiltration before chemotherapy was the strongest predictor for survival. This study established the predictive and prognostic effect of intratumoral FOXP3+ Tregs in LABC patients. To predict clinical outcome, evaluation of FOXP3+ Tregs in tumoral tissues before and after NAC should be considered for these high-risk patients.