The type of responder T-cell has a significant impact in a human in vitro suppression assay

Background

In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4⁺CD25^+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4⁺CD25^low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.

Methods/Principal Findings

We investigated human CD4⁺CD25^low T cells and compared them to CD4⁺CD25^- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4⁺CD25^low T cells divided more rapidly than CD4⁺CD25^- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25^low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25^low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively).

Conclusions/Significance

The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.     

Regulatory Phenotype,PD-1 and TLR3 Expression in T Cells and Monocytes from HCV Patients Undergoing Antiviral Therapy: A Randomized Clinical Trial

Background & Aims

The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment.

Methods

Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4⁺ T-cells or CD8⁺ T-cells and toll-like receptor (TLR) 3 expressing CD14⁺ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls.

Results

Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14⁺ monocytes at baseline had a high rate of cEVR (P<0.05).

Conclusions

Low peripheral TLR3 expressing CD14⁺ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4⁺ or CD8⁺ T-cells.

Trial Registration

Chinese Clinical Trial Registry ChiCTR10001090.     

Y27, a novel derivative of 4-hydroxyquinoline-3-formamide,prevents the development of murine systemic lupus erythematosus-like diseases in MRL/lpr autoimmune mice and BDF1 hybrid mice

Introduction

Naturally occurring CD4⁺CD25⁺ regulatory T (Treg) cells are central to the maintenance of peripheral tolerance. Impaired activity and/or a lower frequency of these cells lead to systemic lupus erythematosus (SLE). Manipulating the number or activity of Treg cells is to be a promising strategy in treating it and other autoimmune diseases. We have examined the effects of Y27, a novel derivative of 4-hydroxyquinoline-3-formamide, on SLE-like symptoms in MRL/lpr autoimmune mice and BDF1 hybrid mice. Whether the beneficial effect of Y27 involves modulation of CD4⁺CD25⁺ Treg cells has also been investigated.

Methods

Female MRL/lpr mice that spontaneously develop lupus were treated orally by gavage with Y27 for 10 weeks, starting at 10 weeks of age. BDF1 mice developed a chronic graft-versus-host disease (GVHD) by two weekly intravenous injections of parental female DBA/2 splenic lymphocytes, characterized by immunocomplex-mediated glomerulonephritis resembling SLE. Y27 was administered to chronic GVHD mice for 12 weeks. Nephritic symptoms were monitored and the percentage of CD4⁺CD25⁺FoxP3⁺ Treg peripheral blood leukocyte was detected with mouse regulatory T cell staining kit by flowcytometry. Purified CD4⁺CD25⁺ Tregs were assessed for immune suppressive activity using the mixed lymphocyte reaction.

Results

The life-span of MRL/lpr mice treated with Y27 for 10 weeks was significantly prolonged, proteinuria and renal lesion severity were ameliorated, and blood urea nitrogen, triglyceride and serum anti-double-stranded DNA antibodies were decreased. Similar results were found in chronic GVHD mice. Administration of Y27 had little impact on percentage of the peripheral blood lymphocyte CD4⁺CD25⁺Foxp3⁺ Treg cells in both groups of mice. In contrast, the suppressive capacity of CD4⁺CD25⁺ Treg cells in splenocytes was markedly augmented in Y27-treated mice ex vivo.

Conclusions

Experimental evidence of the protect effects of Y27 against autoimmune nephritis has been shown. The mechanism may involve enhancement of the suppressive capacity of CD4⁺CD25⁺ Treg cells.     

CD39+ Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

Background

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.

Methods

The numbers of both CD39⁺Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.

Results

Both CD39⁺Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39⁺Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39⁺Tregs could express latency-associated peptide on their surface. When naïve CD4⁺ T cells were cocultured with CD39⁺Tregs, Th17 cell numbers decreased as CD39⁺Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39⁺Tregs.

Conclusions

Therefore, the above results indicate that CD39⁺Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.     

T regulatory cells in cord blood--FOXP3 demethylation as reliable quantitative marker

Background

Regulatory T-cells (Tregs), characterized as CD4+CD25^hi T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4⁺CD25^hi, FOXP3 mRNA, protein expression, and suppressive Treg function.

Methodology

Cord blood mononuclear cells (CBMC) were isolated from 70 healthy neonates, stimulated for 3 days with the microbial stimulus lipid A (LpA), and allergen Dermatophagoides pteronyssinus (Derp1). Tregs (CD4+CD25^hi, intracellular, mRNA FOXP3 expression, isolated cells), DNA methylation of the FOXP3-locus and suppressive Treg function were assessed.

Principal Findings

Demethylation of FOXP3 in whole blood was specific for isolated CD4+CD25^hi Tregs. Demethylation of FOXP3 was positively correlated with unstimulated and LpA-stimulated FOXP3 mRNA-expression (p≤0.05), and CD4⁺CD25^hi T-cells (p≤0.03). Importantly, increased FOXP3 demethylation correlated with more efficient suppressive capacity of Tregs (r = 0.72, p = 0.005). Furthermore, FOXP3 demethylation was positively correlated with Th2 cytokines (IL-5, IL-13) following LpA-stimulation (p = 0.006/0.04), with Th2 and IL-17 following Derp1+LpA-stimulations (p≤0.009), but not Th1 cytokines (IFN-γ).

Conclusions

FOXP3 demethylation reliable quantifies Tregs in cord blood. FOXP3 demethylation corresponds well with the suppressive potential of Tregs. The resulting strict correlation with functionally suppressive Tregs and the relative ease of measurement render it into a valuable novel marker for large field studies assessing Tregs as qualitative marker indicative of functional activity.     

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

Background

Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc).

Methods/Principal Findings

Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25^highCD127^- and CD4CD25^lowCD127^high and CD3⁺ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4⁺CD25⁺ and CD25^highFoxP3^highCD127^neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma.

Conclusions/Significance

These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.     

Mycobacterium tuberculosis Specific CD8+ T Cells Rapidly Decline with Antituberculosis Treatment

Rationale

Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8⁺ T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment.Objectives: We sought to determine the relationship of Mtb specific CD4⁺ T cells and CD8⁺ T cells with duration of antituberculosis treatment.

Materials and Methods

We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4⁺ and CD8⁺ T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24.

Results

There was a significant difference in the Mtb specific CD8⁺ T response, but not the CD4⁺ T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8⁺ T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4⁺ T cell during the treatment. The Mtb specific CD4⁺ T cell response, but not the CD8⁺ response, was negatively impacted by the body mass index.

Conclusions

Our data provide evidence that the Mtb specific CD8⁺ T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8⁺ T cell response can detect early treatment failure, relapse, or to predict disease progression.     

Cortisol Patterns Are Associated with T Cell Activation in HIV

Objective

The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.

Methods

We studied 128 HIV-infected adults who were not on treatment and had a CD4⁺ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.

Results

Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4⁺ T cells (r = −0.26, p = 0.006). Participants with lower waking cortisol also showed a trend toward greater activation on CD8⁺ T cells (r = −0.17, p = 0.08). A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4⁺ (r = 0.24, p = 0.018) and CD8⁺ (r = 0.24, p = 0.017) activation.

Conclusions

These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.     

Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers

Background

The development of insulin resistance (IR) in mouse models of obesity and type 2 diabetes mellitus (DM) is characterized by progressive accumulation of inflammatory macrophages and subpopulations of T cells in the visceral adipose. Regulatory T cells (Tregs) may play a critical role in modulating tissue inflammation via their interactions with both adaptive and innate immune mechanisms. We hypothesized that an imbalance in Tregs is a critical determinant of adipose inflammation and investigated the role of Tregs in IR/obesity through coordinated studies in mice and humans.

Methods and Findings

Foxp3-green fluorescent protein (GFP) “knock-in” mice were randomized to a high-fat diet intervention for a duration of 12 weeks to induce DIO/IR. Morbidly obese humans without overt type 2 DM (n = 13) and lean controls (n = 7) were recruited prospectively for assessment of visceral adipose inflammation. DIO resulted in increased CD3⁺CD4⁺, and CD3⁺CD8⁺ cells in visceral adipose with a striking decrease in visceral adipose Tregs. Treg numbers in visceral adipose inversely correlated with CD11b⁺CD11c⁺ adipose tissue macrophages (ATMs). Splenic Treg numbers were increased with up-regulation of homing receptors CXCR3 and CCR7 and marker of activation CD44. In-vitro differentiation assays showed an inhibition of Treg differentiation in response to conditioned media from inflammatory macrophages. Human visceral adipose in morbid obesity was characterized by an increase in CD11c⁺ ATMs and a decrease in foxp3 expression.

Conclusions

Our experiments indicate that obesity in mice and humans results in adipose Treg depletion. These changes appear to occur via reduced local differentiation rather than impaired homing. Our findings implicate a role for Tregs as determinants of adipose inflammation.     

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane,synovial fluid and peripheral blood

Introduction

CD4⁺CD25^+/highCD127^low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.

Methods

Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.

Results

CD4⁺ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO⁺RA^-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.

Conclusions

Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L^-CD69⁺), whereas peripheral Tregs are resting central memory cells (CD62L⁺CD69^-).     

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective

We investigated whether the frequency, phenotype, and suppressive function of CD4⁺FOXP3⁺ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls.

Design and Methods

Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4⁺CD25^bright were co-cultured with autologous CD4⁺CD25⁻ target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function.

Results

Despite a lower frequency of CD4⁺ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3⁺ Tregs among CD4⁺ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3⁺, and CCR4⁺CCR6⁺ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4⁺CD25⁻ T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group.

Conclusions

The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4⁺ T cells.     

Mesenchymal stromal cell exosome–enhanced regulatory T-cell production through an antigen-presenting cell–mediated pathway

Background aims

The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production.

Cigarette Smoking Promotes Inflammation in Patients with COPD by Affecting the Polarization and Survival of Th/Tregs through Up-Regulation of Muscarinic Receptor 3 and 5 Expression

Background

CD4⁺ T cells in the lung are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), although CD4⁺ T cell subsets and the direct effect of smoking on these cells, especially the expression of MRs, have not been comprehensively examined.

Methods

First, circulating CD4⁺ T cell subsets in healthy nonsmokers, patients with SCOPD and patients with AECOPD were evaluated by flow cytometry. Then, differentiation experiments were carried out using RT-PCR, and Ki-67/Annexin V antibodies were used to measure proliferation and apoptosis. We also explored the impact of CSE on the differentiation and survival of CD4⁺Th/Tregs and examined the expression of MRs in healthy nonsmokers and patients with SCOPD.

Results

We found the percentages of circulating Th1 and Th17 cells were increased in patients with AECOPD, while the percentage of Th2 cells was decreased in patients with SCOPD. The percentages of Th10 cells were decreased in both patients with SCOPD and patients with AECOPD, while the percentages of Tregs were increased. In addition, the percentages of CD4⁺α-7⁺ T cells were decreased in patients with SCOPD and patients with AECOPD. However, only the decrease observed in patients with AECOPD was significant. In vitro studies also revealed MR expression affected the polarization of T cells, with different CD4⁺ T cell subtypes acquiring different MR expression profiles. The addition of CSE facilitated CD4⁺ T cell polarization towards pro-inflammatory subsets (Th1 and Th17) and affected the survival of CD4⁺ T cells and Treg cells by up-regulating the expression of MR3 and 5, resulting in an imbalance of CD4⁺ T cell subsets.

Conclusions

Our findings suggest an imbalance of circulating CD4⁺ T cell subsets is involved in COPD pathogenesis in smokers. Cigarette smoking may contribute to this imbalance by affecting the polarization and survival of Th/Tregs through the up-regulation of MR3 and MR5.     

Heat Shock Protein 60 in Eggs Specifically Induces Tregs and Reduces Liver Immunopathology in Mice with Schistosomiasis Japonica

Background

Parasitic helminths need to suppress the host immune system to establish chronic infections. Paradoxically, immunosuppression induced by the worm also benefits the host by limiting excessive inflammation and tissue damage, which remains the major cause leading to serious morbidity and mortality. Regulatory T cells (Tregs) are key immune regulators of this mutualism. The successive rise in Tregs during schistosome infection plays a critical role in immunoregulation. We and others previously showed that Schistosoma japonicum (S. japonicum) egg antigens (SEA) induce Tregs both in vitro and in vivo. In addition, we identified that SjHSP60 derived from SEA significantly induces Tregs in vivo and in vitro. However, the contribution of SjHSP60 in SEA to Treg induction and the related mechanisms of the Treg induction have not yet been identified.

Methodology/Principal Findings

In this study, we showed that S. japonicum stress protein HSP60 (SjHSP60) was constitutively and extensively expressed in eggs of S. japonicum. SjHSP60 specially induced Tregs in vivo and in vitro without inducing other CD4⁺ T sub-populations including Th1, Th2 and Th17 cells. Furthermore, we showed that the SjHSP60-depleted SEA almost lost the ability in vitro and displayed a significant impaired ability to induce Tregs in vivo. Finally, our study illustrated that the mechanisms of SjHSP60-mediated induction of Tregs are through both conversion of CD4⁺CD25^- T cells into CD4⁺CD25⁺Foxp3⁺ Tregs and expansion of preexisting CD4⁺CD25⁺Foxp3⁺ Tregs in a TLR4-dependent manner.

Conclusions/Significance

Collectively, our findings identify SjHSP60 as a major parasitic contributor of Treg induction in S. japonicum egg antigens, which not only contributes to the better understanding of the mechanism of immunoregulation during helminth infection, but also suggests its potential as a therapeutic target for control of immunopathology, allergic and autoimmune diseases.     

Prognostic Impact of FoxP3+ Regulatory T Cells in Relation to CD8+ T Lymphocyte Density in Human Colon Carcinomas

Background

T-lymphocyte infiltration into colon carcinomas can influence clinical outcome, and interactions among T cell subsets may be more informative than either subset alone. Our objective was to examine the prognostic impact of tumor-infiltrating FoxP3⁺ regulatory T cells (Tregs) in relation to cytotoxic CD8⁺ T lymphocytes in patients with colon carcinomas characterized by DNA mismatch repair (MMR) status who participated in adjuvant chemotherapy trials.

Methods

FoxP3⁺ and CD8⁺ densities in tumor epithelial and stromal compartments were analyzed by immunohistochemistry and quantified in resected, stage II and III colonic carcinomas (N = 216). Immune marker density was dichotomized at the median and categorized as high vs low. MMR status was classified as MMR deficient (dMMR) or proficient (pMMR). Cox models were adjusted for age, stage, and tumor grade.

Results

The density of FoxP3+ infiltration was similar in tumor stroma and epithelia, whereas CD8+ was higher in stroma. The prognostic impact of FoxP3+ and CD8+ T cell infiltration was stronger in stroma vs epithelia, and the density of each marker in stroma was independently associated with improved overall survival (OS). However, the impact of FoxP3+ on survival was dependent upon CD8+ density (P interaction  = .040). Among CD8+_low tumors, FoxP3+_high cases had significantly improved OS compared to FoxP3+_low cases after adjustment for covariates (hazard ratio 0.43; 95% confidence interval 0.19 to 0.95; P = .030). In contrast, FoxP3+ was not prognostic among CD8+_high tumors. FoxP3+ remained prognostic in CD8+_low tumors after further adjustment for MMR or BRAF ^V600E mutation status. Additionally, these immune markers identified a pMMR subgroup with a similarly favorable OS as for dMMR tumors.

Conclusions

The prognostic impact of FoxP3+ and CD8+ T cell density are inter-dependent, whereby FoxP3+ exerts a favorable influence on survival only in colon cancers with low CD8+ infiltration.     

Depletion of Regulatory T Lymphocytes Reverses the Imbalance between Pro- and Anti-Tumor Immunities via Enhancing Antigen-Specific T Cell Immune Responses

Background

The regulatory T cells (Tregs) can actively suppress the immune responses. However, literature about detailed changes of host effective and suppressive immunities before and after depletion of Tregs in ovarian carcinomas, is rare.

Materials and Methods

Ovarian cancer patients and the ascitogenic animal model were employed. Immunologic profiles with flow cytometric analyses, immunohistochemistric staining, RT-PCR, ELISA, and ELISPOT assays were performed. In vivo depletion of Treg cells with the mAb PC61was also performed in the animal model.

Results

The cytokines, including IL-4 (p = 0.017) and TNF-α (p = 0.046), significantly decreased while others such as TGF-β (p = 0.013), IL-6 (p = 0.016), and IL-10 (p = 0.018) were elevated in ascites of ovarian cancer patients, when the disease progressed to advanced stages. The ratio of CD8⁺ T cell/Treg cell in ascites was also lower in advanced diseases than in early diseases (advanced 7.37±0.64 vs. early 14.25±3.11, p = 0.037). The kinetic low-dose CD25 Ab depletion group had significantly lower intra-peritoneal tumor weight (0.20±0.03 g) than the sequential high-dose (0.69±0.06 g) and sequential low-dose (0.67±0.07 g) CD25 Ab deletion groups (p = 0.001) after 49 days of tumor challenge in the animal. The kinetic low-dose CD25 Ab depletion group generated the highest number of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups (p<0.001).

Conclusions

The imbalance between effective and suppressive immunities becomes more severe as a tumor progresses. The depletion of Treg cells can correct the imbalance of immunologic profiles and generate potent anti-tumor effects. Targeting Treg cells can be a new strategy for the immunotherapy of ovarian carcinoma.     

The Ratio of Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune Thrombocytopenia

Background

Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4⁺ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio.

Methods

Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4⁺CD25^hiFoxp3⁺ Treg cells and CD3⁺CD4⁺IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry.

Results

The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity.

Conclusion

The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.     

Effects of natalizumab treatment on Foxp3+ T regulatory cells

Background

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology

A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal Findings

Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25^highCD127^lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions

We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.