Number Of Foxp3 Research Articles

The number of FoxP3+ cells in transbronchial lung allograft biopsies does not predict bronchiolitis obliterans syndrome within the first five years after transplantation.

An important limitation to the success of lung transplantation is the development of bronchiolitis obliterans syndrome (BOS). It has been hypothesized that regulatory T lymphocytes (Tregs) are related to the risk of BOS. We aim to evaluate whether the number of forkhead box P3 (FoxP3+) cells/mm(2) in lung allograft biopsies is a predictor of long-term outcome. A total of 58 consecutive lung transplant patients were included in the study. For 233 routine surveillance biopsy samples, the numbers of FoxP3+ cells/mm(2) were assessed by immunohistochemical staining with antibodies against FoxP3. BOS scores were calculated for the first five yr after transplantation. We determined that acute rejection was related to the time elapsed from transplantation to BOS with hazard ratios of 3.18 (p = 0.02) and 3.73 (p = 0.04) when comparing the levels of acute rejection grade 1 and grade 2/3, respectively, to no rejection. According to a Cox regression analysis, the number of FoxP3+ cells/mm(2) was not predictive of time to BOS. Our data indicate that the number of FoxP3+ cells in the lung allograft did not correlate with BOS-free survival time. Previous studies have been contradictory and included different time points. Our findings emphasize the importance of including a time factor.

Abstract T MP18: In Vivo Expansion of Regulatory T cells with Interleukin-2/Interleukin-2-antibody Complex Protects against Transient Ischemic Stroke

Background and Purpose: Our previous work documents the transfer of regulatory T cells (Tregs) in rodent models of ischemic stroke protects acute ischemic brain injury by regulating poststroke inflammatory response and thereby ameliorating BBB disruption. However, the low number of Tregs restricts the clinical feasibility of Treg transfer. Recently, in vivo expansion of Tregs with IL-2/IL-2-antibody complex (IL-2/IL-2Ab) was validated protective in autoimmune diseases model,renal ischemia reperfusion model and atherosclerosis. Here we investigate the beneficial effect of IL-2/IL-2Ab on ischemic stroke and decipher the underlying mechanisms. Methods: IL-2/IL-2Ab or isotype IgG was ip injected into C57/BL6 mice for 3 consecutive days. The mice are then subjected to 60-minute middle cerebral artery occlusion (MCAO) or sham operation. Brain infarction, inflammation and neurological performance was assessed up to 7 days after reperfusion. Results: Flow cytometry analysis reveals a marked increase of CD4+CD25+Foxp3+ Tregs in the blood, lymph nodes and spleens collected from IL-2/IL-2Ab-treated mice as compared to those from isotype-treated controls. Such Treg elevation could be observed since 3 days after IL-2/IL-2Ab injection and lasts until 7 days after MCAO. Immunochemistry staining confirms the increased number of Foxp3+ cells in the spleen at 3 days after MCAO in IL-2/IL-2Ab-treated mice. IL-2/IL-2Ab promotes function recovery up to 7 days after stroke, as revealed by significantly improved performance in corner test (n=6-9, ***p<0.001), rotarod test (n=8, **p<0.01), cylinder test (n=8, **p<0.01) and adhesive removal test (n=3, *p<0.05). Quantification of TTC staining and microtubule-associated protein (MAP2) staining shows reductions in brain infarct volume at 3 days (n=5-9,*p<0.05) and 7 days (n=7-9,*p<0.01), respectively, after MCAO. Meanwhile, we observed reduced infiltration of peripheral immune cells (CD3+ T cells, MPO+ neutrophils and F4/80+ macrophages) into the ischemic brain. Conclusions: Our finding suggests that IL-2/IL-2Ab treatment is a novel and clinical feasible immune therapy to expand Treg population in vivo, reduce post-stroke inflammatory responses and protect against ischemic brain injury.

The inflammatory phenotype of the fibrous plate is distinct from the liver and correlates with clinical outcome in biliary atresia

Biliary atresia is an inflammatory cholangiopathy of still undetermined etiology. Correlations between histologic findings and clinical outcome in this disease have largely been based on evaluation of liver parenchyma. This study aimed to characterize the pattern of inflammation within the biliary remnant and identify associations between the type and degree of inflammation and clinical outcome as reflected by the transplant-free interval. The inflammation within the fibrous plates and livers of 41 patients with biliary atresia was characterized using immunohistochemical markers and the cell populations were digitally quantified. The type and quantity of cells within the infiltrate were then correlated with length of time from Kasai portoenterostomy until transplant. Histologic and immunohistochemical features of the biliary remnant allowed stratification of patients into “inflammatory plate” and “fibrotic plate” groups. Overall there was no significant difference in transplant-free interval between the two cohorts; however, there was a trend towards a longer time to transplant among patients in the “fibrotic plate” group. In addition, the composition of the inflammatory infiltrate in the fibrous plate was distinctly different from that present in the liver and only the characteristics of the inflammation in the fibrous plate, in particular the number of Foxp3+ T regulatory lymphocytes correlated with clinical outcome. The results of this study support the view of the extra-hepatic biliary tree as the primary site of injury in BA with the changes seen in the liver as secondary manifestations of outflow obstruction. The association between specific inflammatory cell subtypes within the fibrous plate and the length of transplant-free interval also supports the role of the immune system in the initial process of bile duct damage in biliary atresia.

Increased density of tolerogenic dendritic cells in the small bowel mucosa of celiac patients

To investigate the densities of dendritic cells (DCs) and FOXP3(+) regulatory T cells (Tregs) and their interrelations in the small bowel mucosa in untreated celiac disease (CD) patients with and without type 1 diabetes (T1D). Seventy-four patients (45 female, 29 male, mean age 11.1 ± 6.8 years) who underwent small bowel biopsy were studied. CD without T1D was diagnosed in 18 patients, and CD with T1D was diagnosed in 15 patients. Normal small bowel mucosa was found in two T1D patients. Thirty-nine patients (mean age 12.8 ± 4.9 years) with other diagnoses (functional dyspepsia, duodenal ulcer, erosive gastritis, etc.) formed the control group. All CD patients had partial or subtotal villous atrophy according to the Marsh classification: Marsh grade IIIa in 9, grade IIIb in 21 and grade IIIc in 3 cases. Thirty-nine patients without CD and 2 with T1D had normal small bowel mucosa (Marsh grade 0). The densities of CD11c(+), IDO(+), CD103(+), Langerin (CD207(+)) DCs and FOXP3(+) Tregs were investigated by immunohistochemistry (on paraffin-embedded specimens) and immunofluorescence (on cryostat sections) methods using a combination of mono- and double-staining. Sixty-six serum samples were tested for IgA-tissue transglutaminase (tTG) using a fully automated EliA™ Celikey(®) IgA assay (Pharmacia Diagnostics, Freiburg, Germany). The density of CD11c(+) DCs was significantly increased in CD patients compared with patients with normal mucosa (21.67 ± 2.49 vs 13.58 ± 1.51, P = 0.007). The numbers of FOXP3(+) cells were significantly higher in CD patients (10.66 ± 1.50 vs 1.92 ± 0.37, P = 0.0002) and in patients with CD and coexisting T1D (8.11 ± 1.64 vs 1.92 ± 0.37, P = 0.002) compared with patients with normal mucosa. The density of FOXP3(+) cells significantly correlated with the histological grade of atrophic changes in the small bowel mucosa according to the March classification (r = 0.62; P < 0.0001) and with levels of IgA antibody (r = 0.55; P < 0.0001). The densities of IDO(+) DCs were significantly higher in CD patients (21.6 ± 2.67 vs 6.26 ± 0.84, P = 0.00003) and in patients with CD and coexisting T1D (19.08 ± 3.61 vs 6.26 ± 0.84, P = 0.004) compared with patients with normal mucosa. A significant correlation was identified between the densities of IDO(+) DCs and FOXP3(+) T cells (r = 0.76; P = 0.0001). The mean values of CD103(+) DCs were significantly higher in CD patients (10.66 ± 1.53 vs 6.34 ± 0.61, P = 0.01) and in patients with CD and associated T1D (11.13 ± 0.72 vs 6.34 ± 0.61, P = 0.00002) compared with subjects with normal small bowel mucosa. The mean value of Langerin(+) DCs was higher in CD patients compared with persons with normal mucosa (7.4 ± 0.92 vs 5.64 ± 0.46, P = 0.04). The participation of diverse DC subsets in the pathological processes of CD and the possible involvement of tolerogenic DCs in Tregs development to maintain intestinal immunological tolerance in CD patients are revealed.

Targeting focal adhesion kinase reprograms the pancreatic tumor microenvironment and renders pancreas cancer responsive to checkpoint immunotherapy

Checkpoint immunotherapeutics are promising agents with potential to improve patient outcomes in several cancer types. Unfortunately, to date, single agent immunotherapy has achieved limited clinical benefit in patients with pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of the uniquely immunosuppressive tumor microenvironment present in PDACs that creates a barrier to immune surveillance by T cells. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T cell infiltration and high numbers of tumor-associated immunosuppressive cells, such as MDSCs and regulatory T cells (Tregs). To understand which signaling pathways in pancreatic tumor cells might drive this suppressive tumor microenvironment, we analyzed the correlation between hyper-activated signaling molecules and tumor infiltrating leukocytes using 50 human PDAC tumor tissues. Of the pathways evaluated, we found that focal adhesion kinase (FAK) activity is elevated in human PDAC and that FAK activity correlates with highly fibrotic tumors with poor CD8+ T cell infiltration. The oral FAK kinase inhibitor VS-4718, currently in Phase I clinical evaluation, was tested to determine if it could overcome the immunosuppressive tumor microenvironment of PDAC. Single agent VS-4718 dramatically limited tumor progression resulting in a doubling of survival in the p48-CRE/KrasG12D/p53flox/+ PDAC mouse model (KPC mice). This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of FOXP3+ Tregs and tumor-infiltrating myeloid cells, and anti-tumor polarization of tumor-associated macrophages. We postulated that the desirable effects of FAK inhibition on the tumor microenvironment might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 significantly potentiated the efficacy of anti-PD-1 and anti-CTLA4 antibodies in KPC mouse models nearly tripling survival times. We next assessed the mechanism of this potentiation. We found that FAK in tumor cells regulates pro-inflammatory and pro-fibrotic cytokine secretion. Furthermore, we found that shRNA knockdown of FAK in PDAC cells results in failure of PDAC cells to induce pro-fibrotic programs in fibroblasts. Importantly, the FAK inhibitor VS-4718 and FAK shRNA in the tumor cells were each effective in increasing CD8+ cytotoxic T cell infiltration into the PDAC tumors in vivo. Taken together, these data suggest that FAK inhibition increases immune surveillance programs in PDAC tumors by overcoming the fibrotic and inflammatory microenvironment rendering tumors more responsive to immunotherapy. These data provide rationale for clinical evaluation of a FAK inhibitor in combination with a PD-1 or PD-L1 antibody in patients with pancreatic and other cancers.

Expansion of regulatory T cells in theiler’s murine encephalomyelitis virus-infected C57BL/6 mice

Multiple sclerosis (MS), one of the most frequent central nervous system (CNS) diseases in young adults, is a chronic demyelinating disease of unknown etiology and possibly multifactorial causes. Due to clinical and pathological similarities, Theiler’s murine encephalomyelitis (TME) represents a commonly used infectious animal model for the chronic-progressive form of human MS. Inadequate viral clearance in susceptible SJL mice leads to persistent infection of the CNS and immune mediated spinal cord demyelination. In contrast resistant C57BL/6 mice eliminate the virus from the CNS by specific cellular immunity, including effective CD8-mediated cytotoxicity. Previous studies have demonstrated that regulatory T cells (Treg) reduce antiviral immune responses in TME in susceptible mice strains. However, the function of Treg in resistant C57BL/6 mice remains undetermined. Thus the aim of the present study was to investigate the impact of in vivo-expansion of Treg by interleukin-2 immune complexes (Treg-expansion) and antibody-mediated CD8+ T cell depletion (CD8-depletion) upon TME virus-induced disease progression in C57BL/6 mice to get further insights into role of Treg and their interaction with other leukocyte subsets in persistent viral infections. Results of the present study confirmed the central role of antiviral cytotoxicity for TME virus elimination in resistant C57BL/6 mice. In agreement with previous reports simple manipulation of the Treg-compartment showed only minor effect upon the disease course in resistant mice, though Treg-expansion exerted profound inhibitory responses in an CD8-reduced environment. Flow cytometric analyses revealed an increased percentage of Foxp3+ Treg in the blood and spleen of C57BL/6 mice following Treg-expansion and CD8-depletion (combined treated animals) associated with an increased CD4/CD8 ratio. Furthermore, a prolonged effect of Treg-expansion was observed in combined treated mice, indicative of an inhibitory effect of CD8+ T cells upon Treg. Peripheral phenotypical changes were associated with an enhanced recruitment of lymphocytes to the brain and spinal cord of TME virus-infected C57BL/6 mice as determined by histology. Real time polymerase chain reaction confirmed chronic brain infection at 42 days post infection (dpi) in combined treated C57BL/6 mice. Immunohistochemistry revealed a predominent infection of hippocampal neurons and significant infiltrations of CD3+ T cells, CD45R+ B cells and CD107b+ microglia/macrophages till 14 dpi in the cerebrum. Foxp3+ Treg were found in the hippocampus also during the late phase (42 dpi), potentially attributed to termination of inflammatory responses. In the spinal cord CD8-depletion and combined treatment led to prolonged infection and leukomyelitis in mice. Immunohistochemistry revealed a significant loss of myelin basic protein expression at 42 dpi in combined treated mice. Moreover, spinal cord demyelination was associated with an increase of β-amyloid precursor protein-postitive axons predominetely in combined treated mice, indicative of axonal damage. Inflammatory responses in the spinal cord white matter were dominated by CD3+ T cells and CD107b+ macrophages/microglia, suggestive of T cell-mediated immunopathology. In addition, significant numbers of Foxp3+ Treg and CD45R+ B cells were found in the spinal cord white matter during the late phase in combined treated C57BL/6 mice. In conclusion, the present project demonstrates synergistic effects of combined Treg-expansion and CD8-depletion to efficiently reduce antiviral immune responses in TME virus-infected C57BL/6 mice, which renders them susceptible to develop chronic infection, leukomyelitis and myelin loss. In addtion to the well described inhibitory effect of Treg upon CD8+ T cells, also regulatory effects of cytotoxic cells upon Treg or bidirectional interaction of these two T cell subsets, respectively, may occur in infectious CNS disoders. C57BL/6 mice are widely used for genetic modification and several transgenic mice with this genetic background have been established for the use as animal models. Thus, findings will potentially support the development of novel therapeutic strategies for chronic inflammatory disorders and provide a base for future studies upon virus-induced immunopathology using transgenic mice.

FoxP3-Positive T-Regulatory Cells in Lymph Nodes with Mycosis Fungoides and Sézary Syndrome

Mycosis fungoides and Sézary syndrome are indolent cutaneous T-cell lymphomas, with skin-associated peripheral lymph nodes being the most frequent extracutaneous site of involvement. Acquisition of functional properties of regulatory T-cells by malignant T-cells in advanced disease may contribute to immunosuppression. Whereas previous studies examining FoxP3 protein expression in mycosis fungoides and Sézary syndrome have focused on skin specimens, little data are available on lymph nodes from patients with these conditions. In this study we examined FoxP3+ regulatory T-cells in lymph nodes from 26 patients with mycosis fungoides and Sézary syndrome and correlated the findings with clinical data, molecular assays for T-cell clonality, and flow cytometry. Except for one case of Sézary syndrome in which malignant T-cells expressed FoxP3 protein, a significantly lower number of FoxP3-expressing cells occurred in lymph nodes that were clearly involved with lymphoma as compared to uninvolved nodes. Cox proportional hazards model showed that lymph node rating and histological evidence of transformation, but not number of FoxP3+ cells, were factors significantly associated with adverse prognosis. We speculate that modulation of FoxP3+ cells in lymph nodes involved with lymphoma might play a role in disease progression. Attainment of a regulatory T-cell phenotype by a subset of lymphoma cells might signal a poor prognosis.

IKKα is required for the homeostasis of regulatory T cells and for the expansion of both regulatory and effector CD4 T cells.

It was reported that TNF receptor type II signaling, which has the capacity to stimulate CD4+ forkhead box P3+ (Foxp3+) regulatory T cells (Tregs), activated the noncanonical NF-κB pathway in an IKKα-dependent manner. Therefore, we studied the role of IKKα in the homeostasis of Treg population. To this end, we generated a mouse strain with conditional knockout of IKKα in CD4 cells (Ikkα(f/f):CD4.Cre) that showed a >60% reduction in the number of Tregs in the thymus and peripheral lymphoid tissues, whereas the number of Foxp3- effector T cells (Teffs) remained at a normal level. The function of Tregs deficient in IKKα was examined using Rag1(-/-) mice cotransferred with naive CD4 cells (nCD4s). Although wild-type (WT) Tregs inhibited colitis induced by transfer of WT nCD4s, IKKα-deficient Tregs failed to do so, which was associated with their inability to reconstitute Rag1(-/-) mice. Furthermore, nCD4s deficient in IKKα also failed to reconstitute Rag1(-/-) mice and were defective in proliferative responses in vitro and in vivo. Thus, our study reveals a novel role of IKKα in the maintenance of a normal Treg population and in the control of expansion of CD4 T cells. These properties of IKKα may be exploited as therapeutic strategies in the treatment of major human diseases.

Mono-allergic and poly-allergic rhinitis patients have comparable numbers of mucosal Foxp3+CD4+ T lymphocytes

Background: We previously found that allergic rhinitis patients with an isolated pollen sensitization responded more strongly to a nasal provocation with grass pollen (GP) than patients who had an additional house dust mite (HDM) sensitization. To elucidate this phenomenon, we investigated the dynamics of Foxp3+CD4+ T lymphocytes in allergic rhinitis patients with distinct allergen sensitizations. Methods: Three groups of allergic rhinitis patients with skin prick test confirmed allergic sensitizations were investigated and compared to 14 healthy controls: 14 subjects with an isolated grass pollen sensitization (Mono-GP); 9 subjects with isolated housedust mite sensitization (Mono-HDM); 29 subjects with grass pollen and house dust mite sensitization (poly-sensitized). Subjects in the Mono-GP group were challenged with grass pollen extract, subjects in the Mono-HDM group were challenged with house dust mite extract, subjects in the poly-sensitized group and the healthy controls were randomly challenged with either grass pollen or house dust mite. Nasal biopsies were taken before and after nasal provocation. We compared the distribution of FoxP3+CD4+ cells in nasal biopsies before and after nasal provocation using immunohistochemistry. Results: There was no difference in the number of FoxP3+CD4+ cells between healthy and the three allergic groups at baseline.Nasal provocation did result in an increase in eosinophils in the three allergic groups, but did not result in a change in the number of FoxP3+CD4+ cells in any of the groups or induced differences between any of the groups. Conclusion: Clinical differences in the response between mono-GP and multiple-sensitized allergic individuals are not related to differences in the number of regulatory T cells in the nasal mucosa.

The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2.

The number of Foxp3+ regulatory T (Treg) cells must be tightly controlled to efficiently suppress autoimmunity while not impairing normal immune responses. Here we show that the adapter molecule TRAF3 is intrinsically required for restraining lineage determination of thymic Treg cells. T cell-specific TRAF3 deficiency resulted in a 2-3 fold increase of Treg cell frequency, due to more efficient transition from T precursors to Foxp3+ Treg cells. TRAF3 dampened interleukin-2 (IL-2) signaling by facilitating recruitment of T cell protein tyrosine phosphatase (TCPTP) to the IL-2 receptor complex, resulting in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of Jak-STAT5 signaling. Our results identify a role for TRAF3 as an important negative regulator of IL-2 receptor signaling that impacts Treg cell development.

Fundamentally Different Roles of HDAC1 and HDAC2 in Control of Foxp3+ T-Regulatory (Treg) Cell Function.

Understanding the molecular mechanisms by which Foxp3+ Tregs function is key to their pharmacologic regulation and/or use in cell therapy of transplant (Tx) recipients. The histone deacetylases (HDAC), HDAC1 and HDAC2, are highly homologous (>80%) and essential epigenetic enzymes that function in multiprotein complexes (Sin3, NuRD, CoREST) to regulate gene transcription. We undertook the first analysis of the effects of their targeting on alloresponses by conditionally deleting HDAC1 or 2 in Tregs, through mating Foxp3-cre and floxed HDAC1 or HDAC2 mice. In each case, HDAC deletion had no effect on thymic Treg development or on the numbers of Foxp3+ Tregs in peripheral lymphoid tissues. Expression profiling of peripheral Tregs showed that whereas HDAC2 deletion induced significant increases in CTLA4, IL-10 and TGF-b transcription, HDAC1 deletion resulted in significant increases in IL-2 and IFN-g transcription. When activated in vitro with CD3/CD28, and cultured in the presence of IL-2 (50 U/ml) and IL-6 (50 ng/ml), Foxp3 levels were unaffected in both populations, but HDAC1-/- Treg produced more IL-17 and IFN-g. Consistent with these data, the in vitro suppressive function of HDAC2 KO increased, but that of HDAC1 KO was decreased, compared to WT Tregs. To test in vivo Treg function, we transplanted BALB/c cardiac allografts into Rag1-/- C57BL/6 recipients and adoptively transferred 1 million WT conventional T cells and a half million WT or HDAC1-/- Treg cells. WT Treg maintained allograft survival for >100 days post-Tx, whereas use of HDAC1-/- Treg led to acute rejection within 2 weeks of engraftment. Likewise, allograft tolerance was readily attained by costimulation blockade therapy in WT recipients, but was ineffectual in HDAC1-/- recipients. In contrast, whereas even WT Treg could not promote allograft survival when transferred at a 4:1 ratio of T cells to Tregs, this ratio of HDAC2-/- Treg to WT conventional T cells significantly extended allograft survival. In summary, despite evidence that HDAC1 and 2 have complimentary functions in other cell types, they showed differential functions in Tregs. HDAC1 enhances Treg function by inhibiting IL-2 and IFN-g production, whereas HDAC2 inhibits Treg function by down-regulating CTLA4, IL-10 and TGF-b expression. These data provide a powerful rationale for the use of selective HDAC inhibitors to regulate the phenotype and functions of Tregs post-Tx.

Intravenous Immunoglobulin Attenuates Experimental Autoimmune Arthritis by Inducing Reciprocal Regulation of Th17 and Treg Cells in an Interleukin‐10–Dependent Manner

Intravenous immunoglobulin (IVIG) is used as a therapeutic agent in various autoimmune diseases. The aims of this study were to investigate the therapeutic effects of IVIG on collagen-induced arthritis (CIA) and identify the mechanism responsible for any therapeutic effects. IVIG was administered to mice with CIA, and the in vivo effects were determined. Th17 and Treg cell frequencies were analyzed by flow cytometry, and cytokine levels in the supernatant were measured by enzyme-linked immunosorbent assay. Subpopulations of T cells and B cells in the spleen were assessed by confocal microscopy. The arthritis severity score and incidence of arthritis were lower in mice treated with IVIG compared with untreated mice. Histopathologic analysis showed less joint damage in mice treated with IVIG. The expression of proinflammatory cytokines, specific type II collagen antibodies, and osteoclast markers was significantly reduced in mice treated with IVIG. Administration of IVIG induced increased FoxP3 expression and inhibited Th17 cell development. The number of FoxP3+ Treg cells was increased, and the number of Th17 cells was decreased in the spleens of mice treated with IVIG. The number of FoxP3+ follicular helper T cells was increased, and subsequent maturation of germinal center B cells was inhibited by IVIG. In addition, IVIG up-regulated interleukin-10 (IL-10) and Fcγ receptor IIB expression. The treatment effects of IVIG on arthritis were lost in IL-10-knockout mice. These results showed that IVIG has therapeutic effects by modulating CD4+ T cell differentiation. The therapeutic effects of IVIG are dependent on IL-10.

Expression of costimulatory molecules B7-H1, B7-H4 and Foxp3+ Tregs in gastric cancer and its clinical significance

Immune escape plays an important role in tumor progression. In the present study, the expression of B7-H1, B7-H4 and Foxp3 involved in immune escape in gastric carcinoma was investigated and the corresponding clinical significance was evaluated. Immunohistochemistry was used to detect the expression of B7-H1, B7-H4 and Foxp3 in 100 gastric cancer specimens, and 30 paracarcinoma tissues were used as the control. Both B7-H1 and B7-H4 showed high expression levels in gastric cancer tissues (65.0 and 71.0 %, respectively), and the expressions of B7-H1 and B7-H4 were positively correlated with the depth of tumor invasion, lymph node metastasis and American Joint Committee on Cancer (AJCC) stage (P < 0.05). The number of Foxp3(+) Tregs was much higher in gastric cancer tissues than control tissues, which was positively correlated with lymph node metastasis (P < 0.05). Similarly, a positive correlation between B7-H1 or B7-H4 expression and the number of Foxp3(+) Tregs was observed. The median overall survival rate of patients with high expression of B7-H1, B7-H4 and Foxp3 was significantly poorer than that of patients with low expression of these proteins (P < 0.05). Cox regression multivariate analysis confirmed that lymph node metastasis, AJCC stage, and B7-H1 and Foxp3 overexpression were independent prognostic factors. B7-H1, B7-H4 and Foxp3 were overexpressed in gastric cancer tissues. B7-H1 and Foxp3 are negative prognostic factors for patients with gastric cancer.

Enhancing the antitumor effects of adoptive cell transfer immunotherapy using preconditioning thermal ablation (TUM7P.956)

Abstract A major challenge facing T cell based immunotherapeutic strategies is the limited trafficking of cytotoxic T cells at vascular checkpoints within the tumor microenvironment. Here, we report the use of radiofrequency ablation (RFA; 90°C, 1min), a cytoreductive strategy used clinically to treat unresectable tumors, as a pre-conditioning regimen to overcome deficient CD8 T trafficking across tumor vasculature. RFA pre-treatment caused a concomitant decrease in the number of Foxp3+CD4+CD25+ regulatory T cells while bolstering CD8 T cells in tumor draining lymph nodes and the non-ablated region within the tumor microenvironment. Taking advantage of the immunostimulatory activities of RFA, adoptive transfer of antigen-specific T cells was performed 6 hours post-RFA in implantable murine tumors (B16.F10 melanoma or CT26 colorectal tumors). Increased trafficking of adoptively transferred T cells was observed in both the tumor draining lymph node and tumor microenvironment. Improved trafficking was associated with a robust increase in antigen-restricted apoptosis of tumor cell targets and significantly enhanced overall survival without recurrence. Further investigation revealed that adoptive T cell transfer following RFA elicited a durable tumor-specific memory response. Taken together, these findings suggest that thermal ablative regimens may have clinical potential as an adjuvant to augment the efficacy of T cell-based cancer immunotherapies.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Expression and clinical significance of costimulatory molecules B7-H1, B7-H4 and forkhead box P3+ regulatory T cells in gastric carcinoma

Objective To investigate the expression of B7-H1,B7-H4 and forkhead box P3 (Foxp3) in gastric carcinoma and its clinical significance.Methods The expression of B7-H1,B7-H4 and Foxp3 was detected by using IHC in 100 gastric carcinomas,and the relationship between the expression of B7-H1,B7-H4 and Foxp3 with clinicopathological features was statistically analyzed.Results Both B7-H1 and B7-H4 showed high expression in gastric carcinoma tissues (65.0％ and 71.0％).The expression of B7-H1 and B7-H4 proteins was positively correlated to the depth of tumor invasion,lymph node metastasis and TNM stage.The positive expression rate of B7-H1 and B7-H4 in the patient with less than 5-year survival was obviously higher than that in the patients with more than 5-year survival (P ＜ 0.05).High expression of Foxp3 + regulatory T cells (Treg) cells in gastric cancer tissues was correlated to lymph node metastasis (P ＜ 0.05).Statistical analysis also showed that the expression of B7-H1 and B7-H4 protein was positively correlated to the number of Foxp3 + Treg cells (r =0.437,P ＜ 0.05 ; r =0.456,P ＜0.05).Conclusion B7-H1,B7-H4 and Foxp3 + Treg cells were up-regulated in gastric cancer tissues,and may be correlated to gastric carcinoma metastasis and prognosis. Key words: Gastric cancer; Costimulatory molecule; Forkhead box P3

Assessment of tissue FoxP3+, CD4+ and CD8+ T‐cells in active and stable nonsegmental vitiligo

The exact etiology of vitiligo remains obscure. Studies have indicated a role for cellular immunity in the pathogenesis of vitiligo. The aim of this study is to assess tissue FoxP3+ natural regulatory T-cells (Tregs), as well as CD4+ and CD8+ T-cells in active vs. stable nonsegmental vitiligo. Immunohistochemical double-staining for expression of CD4+ and CD8+ T-cells with immunostaining for expression of FoxP3 in lesional, marginal, and nonlesional skin of nonsegmental vitiligo was used to evaluate the abundance of Tregs among CD4+ and CD8+ T-cells in active and stable cases of vitiligo. A significant increase in the number of CD4+ and CD8+ T-cells and a highly significant reduction in the number of FoxP3-expressing Tregs were detected in marginal skin in both stable and active vitiligo cases. FoxP3+ cells were decreased in tissue of patients with vitiligo compared with healthy controls. The number of CD8+ T-cells was increased in the epidermis and dermoepidermal junction (DEJ) in comparison with the number of CD4+ T-cells. Tregs were mostly present at the DEJ. The reduction in the number of FoxP3+ cells in the marginal skin suggests that this is the site where regulatory activity is needed to suppress the activity of helper and cytotoxic T-cells that are actively contributing to depigmentation.

Role of Treg and TH17 Cells of the Gastric Mucosa in Children With Helicobacter pylori Gastritis

The aim of the present study was to examine the expression of FOXP3, interleukin (IL)-10, transforming growth factor (TGF)-β1, IL-17A, and T helper 17 (TH17) cells/FOXP3+ regulatory T (Treg) cells balance in the gastric mucosa of children with Helicobacter pylori infection, in relation to the gastric histopathology. Antral mucosal biopsies were obtained from 20 children with H pylori(+) gastritis and 20 age- and sex-matched normal controls. Histopathology was assessed by the updated Sydney classification. Gene expression of FOXP3, IL-10, and TGF-β1 was analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining for FOXP3+ Treg and TH17 cells was performed. The gene expression levels of FOXP3, TGF-β1, and IL-10 messenger RNA (mRNA) and the number of FOXP3+ Treg were significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.01). FOXP3 mRNA levels were correlated positively with TGF-β1 and IL-10 mRNA levels in the H pylori(+) gastritis group (P < 0.05). Furthermore, FOXP3 mRNA levels were correlated positively with the bacterial density, infiltration of polymorphonuclear cells, and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). The number of TH17 cells was significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.05). In addition, the number of TH17 cells was correlated negatively with the bacterial density and positively with the inflammatory scores of polymorphonuclear cells and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). A negative correlation between the TH17 cells/FOXP3+ Treg ratio and the bacterial density was demonstrated in the H pylori(+) gastritis group (P < 0.05). This study suggested that a TH17/Treg balance toward a Treg-biased response favors the persistence of bacteria, causing chronic active gastritis.

Mono-allergic and poly-allergic rhinitis patients have comparable numbers of mucosal Foxp3+CD4+ T lymphocytes

We previously found that allergic rhinitis patients with an isolated pollen sensitization responded more strongly to a nasal provocation with grass pollen (GP) than patients who had an additional house dust mite (HDM) sensitization. To elucidate this phenomenon, we investigated the dynamics of Foxp3+CD4+ T lymphocytes in allergic rhinitis patients with distinct allergen sensitizations. Three groups of allergic rhinitis patients with skin prick test confirmed allergic sensitizations were investigated and compared to 14 healthy controls: 14 subjects with an isolated grass pollen sensitization (Mono-GP); 9 subjects with isolated housedust mite sensitization (Mono-HDM); 29 subjects with grass pollen and house dust mite sensitization (poly-sensitized). Subjects in the Mono-GP group were challenged with grass pollen extract, subjects in the Mono-HDM group were challenged with house dust mite extract, subjects in the poly-sensitized group and the healthy controls were randomly challenged with either grass pollen or house dust mite. Nasal biopsies were taken before and after nasal provocation. We compared the distribution of FoxP3+CD4+ cells in nasal biopsies before and after nasal provocation using immunohistochemistry. There was no difference in the number of FoxP3+CD4+ cells between healthy and the three allergic groups at baseline.Nasal provocation did result in an increase in eosinophils in the three allergic groups, but did not result in a change in the number of FoxP3+CD4+ cells in any of the groups or induced differences between any of the groups. Clinical differences in the response between mono-GP and multiple-sensitized allergic individuals are not related to differences in the number of regulatory T cells in the nasal mucosa.

T2 enhances in situ level of Foxp3+ regulatory cells and modulates inflammatory cytokines in Crohn's disease

Acting via IL-10 and transforming growth factor-β (TGF-β), t regulatory cells (Tregs) that express the Forkhead Box P3 (Foxp3) play a vital role in maintaining intestinal immune homeostasis. Many studies have found correlation between Foxp3(+) Treg cells and Crohn's disease (CD). T2, extracted from the medicinal plant Tripterygium wilfordii Hook F, has already been proved to be therapeutically effective in inducing the remission of CD. However, the mechanisms in human studies remain largely unknown. We aimed to explore the effect of T2 on the in situ levels of inflammatory cytokines and the number of Foxp3(+) Tregs in inflamed mucosa of CD. Mucosal biopsies from 20 patients treated with T2 were taken by colonoscopy. The changes of Foxp3(+) Tregs as well as TNF-α and IL-10 in diseased tissue were visualized by immunochemistry. Western blot and ELISA were used to quantify levels of Foxp3 protein expression and inflammatory cytokines. T2 treatment ameliorated the pathological inflammation of CD. We observed that the significantly elevated Foxp3(+) Tregs and IL-10 levels in the mucosa of CD patients after T2 treatment concurred with the down-regulation of proinflammatory TNF-α. We confirmed the efficacy of T2 treatment in CD and showed that microscopic inflammation was attenuated by the modulation of in situ levels of inflammatory cytokines. The therapeutic mechanisms might involve the up-regulation of Foxp3(+) Tregs.