Treg Cell Phenotype Research Articles

IMPS-14PROGNOSTIC ROLE OF REGULATORY T-CELLS IN PRIMARY AND RECURRENT MENINGIOMA

Although aggressive meningiomas WHO II and III are rare tumors, more effective therapies are urgently needed as reflected by a 5-year-recurrence rate of 50-80% in WHO III meningiomas. In the present study we aimed to learn more about the immunological situation of meningioma and thus a potential suitability of immunotherapeutic approaches. Occurrence of tumor-infiltrating lymphocytes (TILs) was studied in 114 primary and recurrent meningiomas WHO°I – III and quantified by multicolor immunofluorescence stainings using the markers CD3, CD8, and FOXP3 by using the semi-automated analysis method TissueFAXS. Although similar levels of TILs (CD3+) were found in all WHO grades, TIL numbers were higher in primary as compared to recurrent meningiomas. Phenotypic subclassification into effector and regulatory T-cells revealed elevated levels of T helper cells (CD8−) but not cytotoxic T-cells (CD8+). In contrast to effector T-cells, we found a significant increase of regulatory (FOXP3+) T-cells, which is mainly based on the CD8+ Treg cell phenotype. In contrast to all other T-cell phenotypes analyzed, solely FOXP3+ and in particular the CD8+ Treg cell subpopulation were increasing when comparing primary and recurrent meningioma and were further found to be correlated with a poor overall survival. Moreover, detection of Treg cells in primary meningioma is more frequently in patients developing recurrent tumors and might therefore serve as a negative prognostic marker. Furthermore, microarray analyses hint to profound changes in the tumor microenvironment with increasing WHO grade and recurrent tumors. Altogether, our results suggest a higher impact of quality than quantity of TILs, highlighting the importance of the microenvironment on the functional immune status, which needs to be considered for developing novel therapy approaches.

T145: Comprehensive flow cytometry tracking of regulatory T cells and other lymphocyte subsets during HD IL-2 therapy for melanoma

High dose IL-2 (HD IL-2) has been extensively used as an immunotherapy against metastatic melanoma However, why HD IL-2 is effective only in a subset of patients and whether predictive biomarkers, before or early during the course of therapy, can be used to improve response rates remain unresolved. In addition, it has been found that IL-2 therapy potently expands CD4+CD25+Foxp3+ T-regulatory cells (Tregs) but how Treg cell levels, phenotype, and function change and whether specific subsets of Tregs are activated and expanded during HD IL-2 therapy is remain unclear. In this study, we performed comprehensive multi-parameter FACS analysis of patient blood before and two days after the last bolus of IL-2 infusion during cycle 1 of HD IL-2 therapy. Two lymphocyte subsets were found to expand the most during the first cycle of IL-2 therapy: CD4+CD25+Foxp3+ Tregs expressing an activation marker, inducible costimulator (ICOS), and CD3-CD56hiCD16loPerforin+ NK cells. ICOS+ Tregs expressed significantly higher levels of CD25, Foxp3 and had a more activated phenotype than ICOS− Tregs as indicated by lower levels of CD45RA and CD127 expression. Further phenotypic characterization revealed a more suppressive phenotype on ICOS+ Treg with higher expression levels of CD39, CD73, and TGF-β/LAP than ICOS− Treg. ICOS+ Tregs were also the predominant Treg cells that secreted IL-10 and have potent T-cell suppressor function. Majority of ICOS+ Tregs from HD IL-2-treated patients were Ki67+ and exhibited an enhanced proliferative response to IL-2 ex vivo relative to ICOS− Tregs. Functional analysis revealed that ICOS+ Tregs secreted little IFN- and IL-2 in comparison to CD4+Foxp3 – cells. Furthermore, analysis on 38 IL-2-treated patients at MD Anderson, we found that non-responders had a significantly higher degree of ICOS+ Treg expansion than responders during the first cycle of IL-2 therapy, while no significant changes in the ICOS− or bulk Treg population. In conclusion, our data suggests that tracking changes in ICOS+ Tregs early during the course of HD IL-2 therapy may be a new predictive biomarker of clinical outcome.

IL-1R1 is expressed on both Helios(+) and Helios(-) FoxP3(+) CD4(+) T cells in the rheumatic joint.

Synovial fluid from rheumatic joints displays a well-documented enrichment of forkhead box protein 3 (FoxP3)(+) regulatory T cells (tissue Tregs ). However, we have previously demonstrated that the mere frequency of FoxP3 expressing cells cannot predict suppressive function. Instead, extrinsic factors and the functional heterogeneity of FoxP3(+) Tregs complicate the picture. Here, we investigated FoxP3(+) Tregs from blood and synovial fluid of patients with rheumatic disease in relation to Helios expression by assessing phenotypes, proliferative potential and cytokine production by flow cytometry. Our aim was to investigate the discriminatory potential of Helios when studying FoxP3(+) Tregs in an inflammatory setting. We demonstrate that the majority of the synovial FoxP3(+) CD4(+) T cells in patients with inflammatory arthritis expressed Helios. Helios(+) FoxP3(+) Tregs displayed a classical Treg phenotype with regard to CD25 and cytotoxic T lymphocyte-associated antigen (CTLA)-4 expression and a demethylated Treg -specific demethylated region (TSDR). Furthermore, Helios(+) FoxP3(+) T cells were poor producers of the effector cytokines interferon (IFN)-γ and tumour necrosis factor (TNF), as well as of the anti-inflammatory cytokine interleukin (IL)-10. The less abundant Helios(-) FoxP3(+) T cell subset was also enriched significantly in the joint, displayed a overlapping phenotype to the double-positive Treg cells with regard to CTLA-4 expression, but differed by their ability to secrete IL-10, IFN-γ and TNF upon T cell receptor (TCR) cross-linking. We also demonstrate a striking enrichment of IL-1R1 expression in synovial CD4(+) T cells that was restricted to the CD25-expressing FoxP3 population, but independent of Helios. IL-1R1 expression appears to define a tissue Treg cell phenotype together with the expression of CD25, glucocorticoid-induced TNF receptor family-related gene (GITR) and CTLA-4.

PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells.

Regulatory T (Treg) cells act as terminators of T cell immuniy during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8(+) and CD4(+) T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8(+) T cells was necessary for the potent suppression of CD8(+) T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8(+) T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.

CTLA-4⁺ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis.

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4(+)FOXP3(+) intratumoral Treg expressing CTLA-4, CD39, and TGFβ. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4(+)FOXP3(+) Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFβ and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4(+) Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity.

Topical application of sphingosine 1-phosphate receptor 1 prolongs corneal graft survival in mice.

The present study aimed to investigate the effects of topical application of sphingosine 1‑phosphate receptor 1 (S1P1) on allogeneic corneal transplantation in mice. A total of 45 BALB/c mice received corneal grafts from C57BL/6 donors. The recipients were randomly divided into three groups and treated with eye drops containing 0.5% S1P1, 1% cyclosporine A or saline as a negative control. The serum levels of interleukin (IL)‑2, IL‑10, tumor growth factor (TGF)‑β1 and interferon (IFN)‑γ were measured by ELISA. The numbers of CD4+ T cell and T‑regulatory (Treg) cell phenotypes were measured by flow cytometry. The cytokine mRNA expression was analyzed by quantitative polymerase chain reaction. The results demonstrated that corneal graft survival was prolonged in the S1P1 group [mean survival time (MST), 24.11±1.58 days], and 1% cyclosporine A (MST, 25.0±1.91 days) compared with the controls (MST, 13.44±0.48 days; P<0.01). S1P1 and cyclosporine A decreased CD4+ T‑cell levels (P<0.05) in the peripheral blood compared with those of the controls. However, an increase of CD4+ T cells in the spleen was noted in the S1P1 group (P<0.05) and Treg cells were also increased in the cervical lymph nodes in the S1P1 group (P<0.01). TGF‑β1 mRNA transcription in the corneal grafts increased following treatment with S1P1 (P<0.05) and TGF‑β1 in the serum following treatment with S1P1 also increased (P<0.01). In conclusion, S1P1 had a significant effect in corneal allograft rejection inhibition.

Microtransplantation: HLA-Mismatched Allogeneic Cellular Therapy of Acute Myeloid Leukemia (HMMACT)

Background: This clinical trial addressed feasibility and safety of microtransplantation by HLA-mismatched allogeneic cellular therapy (HMMACT) in poor risk acute myeloid leukemia patients. A secondary objective was to estimate complete response rate, infections, GVHD incidence, and induction mortality.Methods: Patients with high risk AML were enrolled: 4 were > 75 yo, 3 with MLL+ ((t(6;11), 11+ and t(10;11)), 3 complex/del7, one FLT3+. Patients received induction chemotherapy with mitoxantrone (10 mg/m2 IV for 3 days) and cytarabine (150 mg/m2 IV for 7 days) and received HLA-mismatched GCSF mobilized PBSCs on day 9 or 10. Family donors were concurrently HLA typed and best available donor underwent GCSF mobilization (5 mg/kg SQ BID x 4 days) and leukapheresis after medical evaluation and safety testing. HLA partial mismatch patients (4- 5/10 antigen) were chosen. Apheresis cells were counted and analyzed by flow cytometry for CD34+ cells; CD3+, CD4+CD25+ and CD8 + cells; and CD3-CD56+ NK cells. Target CD3 cell dose was 1 x 108/kg per cycle; cells cryopreserved as for standard stem cell donors. Family donor HLA-mismatched GCSF mobilized peripheral blood cells were infused fresh on day 9 or 10 (up to day 16), 36-50 hours after end of cytarabine. Bone marrow biopsy was evaluated on day 14 and 28 for AML remission status, and T/NK cell number. Patients achieving a complete response proceeded to consolidation with cytarabine 0.5 -1.0 gram/m2 x 6 doses with fresh or cryopreserved HMMACT cells from their donor for 2 courses a month apart. Patients: 10 patients age 31 – 80 yo consented: 8 received allo cells; 1 screen failure (no haplo family member identified); 1 patient too ill after initiating chemo to collect donor. Eight patients received at least one course of HMMACT; 1 withdrew early due to AML progression, a second for poor performance status. Three patients had de novo AML; 5 patients had secondary AML. Six Patients had2 or more cycles of HMMACT: four achieved CR: Pt 1 and 4 received 3 cycles, Pt 2 and 6 received 2 cycles of cell infusions and all achieved CR. Pt 9 received 2 inductions with cells, and sustained a partial clinical remission for 6 months. Two Patients had 1 cycle of HMMACT:Pt 3: received 1 cycle cells, but response not assessed; Pt 8: received 1 cycle cells, and achieved CR 3+mos but declined further therapy. Safety: Acute reactions to cell infusions were minimal. Delayed reactions attributed to cell infusions included fever grade 1-2, rash grade 1-2, diarrhea grade 1-2 resolving in 7-10 days. Patients with prior MDS or refractory leukemia requiring 2 inductions had a longer time to ANC and platelet recovery. Feasibility: Two patients had no donor; 2 donors only collected enough for 2 cell infusions. Response: 5/8 pts receiving cells had CR/CRi (62.5%) lasting 3 to 10+ months.Conclusions: Although there were the usual significant complications of treating leukemia and infections, the cellular therapy itself was well-tolerated. Patients often had fevers in first week after cell infusions, or experienced transient rash and diarrhea in the same time period, which were self resolving in all cases and may reflect elimination of allogeneic cells by the patient’s immune system. No patient developed GVHD. Feasibility of obtaining family donors was as expected for this diverse US population. Infectious complications were low by AML treatment standards. Complete remission rates are encouraging, but not as durable as hoped, likely reflecting the fact that all of our patients had high risk cytogenetics in contrast to the prior published studies by Chinese investigators. The majority were elderly who would not otherwise be offered intensive therapy. Family donors achieved expected cell targets and tolerated mobilization/collection procedures well. All but one patient receiving 2 to 3 cycles achieved complete remission. Two elderly patients who received one or two cell infusions during induction sustained prolonged survival of 6 months in spite of no further therapy, one achieving a CR and the other a sustained PR. Neutrophil and platelet recoveries tended to follow the pattern of the patient’s prior secondary leukemia or myelodysplastic disorder. Once patients achieved complete remission however, recovery of blood counts was relatively rapid on further cycles of consolidation. We are now assessing Treg, T and NK cell phenotype of patient and donor cells by flow cyAtometry as biological correlates. DisclosuresChaudhary:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

Identification of a Subset of Peripheral T-Cell Lymphoma, Not Otherwise Specified, Characterized By FOXP3-Positive Regulatory T-Cell Phenotype, HTLV-1 Negativity and Poor Outcome

Background: T-cell malignancies originating from regulatory T (Treg) cells are almost exclusively confined to human T-cell leukemia virus 1 (HTLV-1) associated adult T-cell leukemia/lymphoma (ATLL), although sporadic cases of other peripheral T-cell lymphomas (PTCLs) with hypothesized Treg derivation have been reported.Patients and methods: We investigated a series of 169 well characterized PTCLs for the expression of Treg-cell phenotypic markers FOXP3, CD25 and CD4 by immunohistochemistry (IHC) using tissue microarray on formalin-fixed paraffin embedded tissue. Clinico-pathological data for patients enrolled were retrieved from the Danish Lymphoma Registry and medical records. Treg tumors were further invetsigated by Affymetrix OncoScan analysis and Illumina Infinium HumanMethylation450 BeadChips.Results: Variable amounts of FOXP3-positive Treg cells were often (99% of the cases) found as part of the non-neoplastic cellular infiltrate. However, in five cases (3%) classified as PTCL, not otherwise specified (PTCL-NOS) the vast majority of what morphologically appeared to be the neoplastic cell population displayed a strong positivity for FOXP3, CD4, CD25 and CD3 suggesting a probable Treg origin of these cells. Cases were HTLV-1 negative and showed monoclonal rearrangements of the T-cell receptor genes. All cases were male older than 60 years and showed an aggressive clinical course with overall survival less than two years, despite all patients had low IPI-risk profile at the time of diagnosis. The PTCL with Treg phenotype showed a complex pattern of chromosomal imbalances. Moreover, as compared to normal T-cell subsets their DNA methylation profiles were mostly related to that of normal Treg cells but significantly differed from that.Conclusions: We suggest that FOXP3-positve PTCL, in the absence of HTLV-1 infection, constitute a distinct entity separated from PTCL-NOS, occurring predominantly in elderly male patients and characterized by a highly aggressive clinical behavior. Further genomic analyses are ongoing. The identification and characterization of these cases may be useful to guide upfront therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

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.

CD4+ Foxp3+ regulatory T cells in autoimmune orchitis: phenotypic and functional characterization.

The phenotype and function of regulatory T (Treg) cells in rats with experimental autoimmune orchitis (EAO) was evaluated. Distribution of Treg cells in draining lymph nodes from the testis (TLN) and from the site of immunization (ILN) was analysed by immunohistochemistry. The number, phenotype and proliferative response (5-bromo-2'-deoxyuridine incorporation) of Treg cells were evaluated by flow cytometry and Treg cell suppressive activity by in vitro experiments. TGF-β expression was evaluated by immunofluorescence. Absolute numbers of Treg cells and BrdU+ Treg cells were increased in LN from experimental compared to normal and control rats. These cells displayed a CD45RC(-), CD62L(-), Helios(+) phenotype. CD4(+) CD25(bright) T cells from TLN of experimental rats were able to suppress T cell-proliferation more efficiently than those derived from normal and control rats. Cells isolated from TLN and ILN expressed TGF-β. Our results suggest that Treg cells with a memory/activated phenotype proliferate extensively in the inflamed testis and LN of rats with EAO exhibiting an enhanced suppressive capacity. TGF-β may be involved in their suppressive mechanism.

Increased production of interleukin-17 over interleukin-10 by treg cells implicates inducible costimulator molecule in experimental spondyloarthritis.

HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic (B27-transgenic) rats develop an inflammatory disorder resembling spondyloarthritis, with accumulation of proinflammatory Th17 cells. Because Treg cells and Th17 cells have opposing effects in inflammatory disorders, we sought to determine whether biased expansion of Th17 cells could result from altered Treg cell frequency and/or function in B27-transgenic rats. We characterized the phenotype and function of Treg cells from B27-transgenic rats in comparison with those from control rats, by examining their expression of cell surface markers, suppressive activity, cytokine production, and differentiation pattern. In B27-transgenic rats, the preferential accumulation of CD4+ Teff cells over Treg cells was not associated with a defect in Treg cell differentiation or suppressive activity. The expression of Treg cell markers was similar between B27-transgenic and control rats, with the exception of the inducible costimulator (ICOS) molecule, which was overexpressed in B27-transgenic rats. High levels of ICOS are considered to be a hallmark of Treg cells with heightened suppressive activity and interleukin-10 (IL-10) expression. Paradoxically, the production of IL-10 by Treg cells was reduced in B27-transgenic rats, whereas the production of IL-17 was enhanced. Moreover, the addition of anti-ICOS monoclonal antibodies during Treg cell differentiation in the presence of dendritic cells from B27-transgenic rats reversed this cytokine profile, restoring the balance between IL-10 and IL-17 in Treg cells from B27-transgenic rats. We observed dysregulated production of IL-10 and IL-17 by Treg cells from B27-transgenic rats, which may contribute to disease development. Moreover, our data highlight a key role for ICOS signaling in the generation of imbalanced production of IL-10 and IL-17 by Treg cells in this experimental model of spondyloarthritis.

PD-1 regulates extrathymic regulatory T-cell differentiation.

Treg cells and the programed death-1/programed death ligand-1 (PD-1/PD-L1) pathway are both critical for maintaining peripheral tolerance to self-Ags. A significant subset of Treg cells constitutively expresses PD-1, which prompted an investigation into the role of PD-1/PD-L1 interactions in Treg-cell development, function, and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD-1-deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD-1 deficiency. The suppressive function of PD-1(-/-) Treg cells was similar to their PD-1(+/+) counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD-1(-/-) conventional CD4(+) T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD-1 is dispensable for thymic Treg-cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that Ab blockade of the PD-1/PD-L1 pathway may augment T-cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells.

Adipose tissue‐resident regulatory T cells: phenotypic specialization, functions and therapeutic potential

Foxp3(+) CD4(+) regulatory T (Treg) cells, recognized to be one of the most important defences of the human body against an inappropriate immune response, have recently gained attention from those outside immunology thanks to the compelling evidence for their capability to exert non-canonical immune functions in a variety of tissues in health and disease. The recent discovery of the differences between tissue-resident Treg cells and those derived from lymphoid organs is affecting the mindset of many investigators now questioning the broad applicability of observations originally based on peripheral blood/lymphoid organ cells. So far, the best characterized 'Treg flavour' comes from studies focused on their role in suppressing adipose tissue inflammation and obesity-driven insulin resistance. Adipose tissue derived Treg cells are distinct from their counterparts in lymphoid organs based on their transcriptional profile, T-cell receptor repertoire, and cytokine and chemokine receptor expression pattern. These cells are abundant in visceral adipose tissue of lean mice but their number is greatly reduced in insulin-resistant animal models of obesity. Interestingly, peroxisome-proliferator-activated receptor γ expression by visceral adipose tissue Treg cells is crucial for their accumulation, phenotype and function in the fat and surprisingly necessary for complete restoration of insulin sensitivity in obese mice by the anti-diabetic drug Pioglitazone. This review surveys recent findings relating to the unique phenotype and function of adipose tissue-resident Treg cells, speculates on the nature of their dynamics in lean and obese mouse models, and analyses their potential therapeutic application in the treatment of type 2 diabetes.

Liver injury in acute hepatitis A is associated with decreased frequency of regulatory T cells caused by Fas-mediated apoptosis

ObjectiveFoxp3+CD4+CD25+ regulatory T cells (Tregs) control immune responses, but their role in acute viral hepatitis remains elusive. Herein, we investigated alteration in the peripheral blood Treg population during acute hepatitis...

Highly heterogeneous, activated, and short-lived regulatory T cells during chronic filarial infection.

The mechanisms underlying the increase in the numbers of regulatory T (Treg) cells in chronic infection settings remain unclear. Here we have delineated the phenotype and transcriptional profiles of Treg cells from 18 filarial-infected (Fil(+) ) and 19 filarial-uninfected (Fil(-) ) subjects. We found that the frequencies of Foxp3(+) Treg cells expressing CTLA-4, GITR, LAG-3, and IL-10 were significantly higher in Fil(+) subjects compared with that in Fil(-) subjects. Foxp3-expressing Treg-cell populations in Fil(+) subjects were also more heterogeneous and had higher expression of IL-10, CCL-4, IL-29, CTLA-4, and TGF-β than Fil(-) subjects, each of these cytokines having been implicated in immune suppression. Moreover, Foxp3-expressing Treg cells from Fil(+) subjects had markedly upregulated expression of activation-induced apoptotic genes with concomitant downregulation of those involved in cell survival. To determine whether the expression of apoptotic genes was due to Treg-cell activation, we found that the expression of CTLA-4, CDk8, RAD50, TNFRSF1A, FOXO3, and RHOA were significantly upregulated in stimulated cells compared with unstimulated cells. Taken together, our results suggest that in patent filarial infection, the expanded Treg-cell populations are heterogeneous, short-lived, activated, and express higher levels of molecules known to modulate immune responsiveness, suggesting that filarial infection is associated with high Treg-cell turnover.

Novel biomarkers of hidden inflammation and implantation

Successful implantation of dental implants depends not only on the implant material, but is also significantly affected by the immunologic status of the organism. The most problematic here is chronic low grade inflammation which increases with the age (i.e., immunaging). This type of inflammation differs from classical acute inflammation by the absence of clinical symptoms and specific activation of monocytes in the circulation and tissue macrophages. Highly specific and sensitive biomarkers for detection of this inflammatory condition are urgently needed. In our laboratory, we have identified several biomarkers that are expressed by macrophages differentiated in the microenvironment typical for chronic low grade inflammation. These markers include receptors Stabilin-1 and IL17RB, and transcription factor FoxQ1. Stabilin-1 is a multifunctional macrophage receptor responsible for scavenging of unwanted self products and for sorting of newly synthesized ligands and facilitating their secretion. These functions of Stabilin-1 indicate that it is not only a marker, but also actively involved in the regulation of inflammatory reactions. IL17RB is a receptor for IL17E – the sole member of IL17 family that is not involved in regulation of type 1 inflammation, but rather mediating low grade type 2 inflammation. FoxQ1 is a transcription factor of forkhead family. The entire family is actively involved in regulation of innate and adaptive immune reactions including the development of regulatory T-cell phenotype. Our data indicate that FoxQ1 is typical for macrophages showing increased adhesion and migration capabilities, making them active players in low grade inflammation in tissues. All above mentioned biomarkers are expressed by monocytes/macrophages upon stimulation with IL-4 or in combination with TGFβ and/or IFNγ- cytokines playing important role in chronic low grade inflammation. Association of Stabilin-1, FoxQ1 and IL17RB with chronic low grade inflammation makes them not only viable personalized diagnostic markers, but also suggests their usefulness as targets for development of novel personalized therapeutic options. We propose to use novel biomarkers of chronic inflammation to predict individual reactions of patients to implants and as a reference for development of immunostabilizing therapies to prevent implant failure.

A8.26 Inducible IL-10 secreting CD49b +Treg cells as cell based-therapy for rheumatoid arthritis

BackgroundAdoptive transfer of regulatory T cells (Tregs) is a promising approach to restore tolerance in autoimmune diseases. However based on the heterogeneity of the Tregs, we need to precisely establish...

Interleukin‐6 Receptor Blockade Enhances CD39+ Regulatory T Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti-IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti-IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. Mice with collagen-induced arthritis (CIA) were treated with a mouse anti-IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. In MR16-1-treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell-sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti-IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the RhD protein in HLA-transgenic mice.

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.

Skin manifestations of adult T‐cell leukemia/lymphoma: Clinical, cytological and immunological features

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells caused by human T-cell leukemia virus type I. The endemic areas include Japan, especially in Kyushu, the Caribbean, Papua New Guinea, South America and Africa. Approximately 50% of ATLL patients exhibit skin manifestations. Cytologically, ATLL tumor cells are characterized by CD4(+) CD25(+) regulatory T-cell phenotype, high expressions of CCR4, and programmed cell death (PD)-1 and PD-ligand 1. The skin eruptions are categorized into six types: patch, plaque, multipapular, nodulotumoral, erythrodermic and purpuric. The overall survival of the eruption-bearing patients was poorer than that of the non-eruption-bearing patients in acute, chronic and smoldering types, but the survival levels of both groups were comparable in lymphoma type. The prognosis was poor in the order of: erythrodermic, nodulotumoral, multipapular/purpuric, plaque, then patch. Multivariate analysis revealed that the eruption type is an independent prognostic factor for ATLL. Patients may have other skin manifestations, secondary and infective lesions.