Higher Treg Frequency Research Articles

Cytokine and T cell responses in post-chikungunya viral arthritis: A cross-sectional study.

To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. Participants with chikungunya arthritis were recruited from Colombia from 2019-2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy.

Mechanisms of Resistance and Relapse with Talquetamab in Patients with Relapsed/Refractory Multiple Myeloma from the Phase 1/2 MonumenTAL-1 Study

Introduction: Talquetamab (tal), a novel GPRC5D×CD3 bispecific antibody (BsAb), has shown deep and durable responses with overall response rates (ORRs) &amp;gt;71%, including in high-risk populations, and a clinically manageable safety profile in patients (pts) with relapsed/refractory multiple myeloma (MM) in the MonumenTAL-1 study (NCT03399799/NCT04634552). To better understand mechanisms of resistance and relapse in pts treated with tal, baseline and longitudinal GPRC5D expression and immune profiles were assessed in pts treated with the 0.4 mg/kg weekly (QW) recommended phase 2 dose, including a separate cohort of pts who received prior T-cell redirection therapy (prior TCR; chimeric antigen receptor [CAR]-T cell and BsAb therapies). Methods: MonumenTAL-1 is a first-in-human, phase 1/2, single-arm study of tal monotherapy. Eligible pts had MM and were intolerant to or progressed on established therapies (phase 1) or had ≥3 prior lines of therapy (LOT) including a proteasome inhibitor, immunomodulatory drug, and anti-CD38 antibody (phase 2). Analyses were performed in pts receiving tal 0.4 mg/kg QW who did not receive prior TCR (no prior TCR cohort) and pts receiving tal 0.4 mg/kg QW who received prior TCR (prior TCR cohort). Baseline and on-treatment bone marrow or whole blood were analyzed by flow cytometry for GPRC5D expression and immune cell profiles, respectively. For mechanisms of resistance, samples were analyzed in responders (defined as ≥PR) and nonresponders (defined as stable or progressive disease [PD]); for mechanisms of relapse, samples were analyzed at baseline and at end of treatment (EOT)/PD. Results: Median prior LOT was 6 and 5 in the prior and no prior TCR cohorts, respectively. T-cell counts at baseline were similar between cohorts, whereas pts in the prior TCR cohort had higher expression of single coinhibitory markers (LAG-3, TIM-3, PD-1), dual coinhibitory markers (PD-1/LAG-3 and PD-1/TIM-3), and activation markers (CD38 and CD25) on CD8+ T cells, along with a higher frequency of immunosuppressive regulatory T cells (Tregs) compared with the no prior TCR cohort. GPRC5D was highly expressed on MM cells at baseline; however, expression was variable among pts in both cohorts and was not associated with clinical response. In both cohorts, correlative analyses with response showed that nonresponders vs responders had a more exhausted peripheral immune phenotype at baseline, indicated by lower T-cell counts and higher frequencies of Tregs and expression of coinhibitory markers (LAG-3, TIM-3) on CD8+ T cells. In the first cycle following tal administration, greater and transient activation of T cells was observed in responders vs nonresponders. In addition, greater recovery of CD3+ T cells was observed and sustained in responders within 2 cycles of treatment, whereas persistence of CD38+ expression on CD8+ T cells, higher presence of Tregs, and sustained expression of LAG-3, TIM-3, and PD-1 on CD8+ T cells was observed in nonresponders, consistent with the baseline correlative analysis in the prior and no prior TCR cohorts (Figure). Finally, analysis of progression samples of pts who initially responded and then progressed with tal showed a higher expression of coinhibitory receptors (LAG-3, TIM-3, CD38, PD-1/LAG-3, PD-1/TIM-3) on peripheral CD4+ or CD8+ T cells in both cohorts compared with baseline, indicative of an exhausted T-cell phenotype at relapse. Overall, these correlative data were comparable between prior TCR and no prior TCR cohorts. Conclusions: Baseline immune profiling in heavily pretreated pts from MonumenTAL-1 suggested that compared to pts with prior TCR, pts with no prior TCR had a favorable immune fitness profile (less T-cell dysfunction and immune suppression). In both cohorts, baseline and longitudinal correlative analyses suggest a mechanism of resistance for nonresponders including lower T-cell counts and higher frequency of Tregs and expression of coinhibitory markers on CD8+ T cells, whereas responders show greater T-cell activation and recovery of CD3+ T cells that was sustained longitudinally. Progression data indicated an exhausted T-cell phenotype for pts who relapsed on tal. Approaches to overcome resistance, including combination trials with anti-CD38 monoclonal antibodies (eg, daratumumab) and PD-1 inhibitors as well as alternative dosing regimens, are currently being studied.

High-dimensional immune cell profiling of cerebrospinal fluid from patients with metastatic breast cancer and leptomeningeal disease

Leptomeningeal disease (LMD) is a devastating complication of metastatic breast cancer (MBC). In this non-therapeutic study, we enrolled 12 patients with MBC and known or suspected LMD who were undergoing a lumbar puncture as part of clinical care and collected extra cerebrospinal fluid (CSF) and a paired blood sample from each patient at a single time point. Of the 12 patients, 7 patients are confirmed to have LMD based on positive cytology and/or convincing MRI imaging (LMDpos), and 5 patients are deemed not to have LMD based on similar criteria (LMDneg). Using high-dimensional, multiplexed flow cytometry, we profile and compare the CSF and peripheral blood mononuclear cell (PBMCs) immune populations between patients with LMD and those without. Patients with LMD observe a lower overall frequency of CD45+ cells (29.51% vs. 51.12%, p < 0.05), lower frequencies of CD8+ T cells (12.03% vs. 30.40%, p < 0.01), and higher frequency of Tregs than patients without LMD. Interestingly, the frequency of partially exhausted CD8+ T cells (CD38hiTIM3lo) is ~6.5-fold higher among patients with LMD vs. those without (2.99% vs. 0.44%, p < 0.05). Taken together, these data suggest that patients with LMD may have lower overall immune infiltrates than patients without LMD, suggesting a more permissive CSF immune microenvironment but a higher frequency of partially exhausted CD8+ T cells, which may offer an important therapeutic target.

Immunomodulatory effects of carbon ion radiotherapy in patients with localized prostate cancer

PurposeRadiotherapy is one of the main local treatment modalities for prostate cancer, while immunosuppressive effect induced by radiotherapy is an important factor of radiation resistance and treatment failure. Carbon ion radiotherapy (CIRT) is a novel radiotherapy technique and the immunomodulatory effect of CIRT provides the possibility of overcoming radioresistance and improving efficacy. The aim of this study was to assess the immune response evoked by CIRT in localized prostate cancer patients.MethodsThirty-two patients were treated by CIRT combined with or without hormone therapy and peripheral blood samples were collected before and after CIRT. Investigation of peripheral immune cell frequency, proliferation, and cytokine expression was conducted by flow cytometry, real-time quantitative PCR and ELISA.ResultsThere were no significant differences in the frequencies of CD3 + , CD4 + , CD8 + T cells and NK cells after CIRT. CD4/CD8 ratio increased whereas B cells decreased. All lymphocyte subsets except regulatory T cells (Tregs) displayed increased proliferation and T cells exhibited increased functionality after CIRT, characterized by modestly increased cytokine secretion of TNF. Moreover, higher frequencies of Tregs were shown. Neither monocytic myeloid-derived suppressor cells (MDSCs) nor early MDSCs changed after CIRT. TGF-β1 gene expression decreased while IL-6 showed a non-significant trend towards a decrease. Both IL-10 gene expression and plasma TGF‐β1 level were unchanged.ConclusionCIRT demonstrates the potential to elicit immune activation in localized prostate cancer patients, based on sparing lymphocytes, increased lymphocyte proliferation, enhanced T-cell functionality, together with limited induction of immunosuppressive cells and reduced expression of immunosuppressive cytokines.

Infiltration Profile of Regulatory T Cells in Osteoarthritis-Related Pain and Disability.

Emerging evidence indicates that regulatory T cells (Treg) intervene in the inflammatory processes that drive osteoarthritis (OA). However, whether polarized Tregs affect clinical features of the disease in the short- or long-term, and if so, what their role in OA-related pain and functional disability really is, remains elusive. Thus, the aim of the current study was to characterize the infiltration profile of Tregs in systemic (peripheral blood) and joint-derived (synovial fluid and synovial membrane) samples from patients with knee OA in relation to OA-induced symptoms. To this end, Treg infiltration (CD4+CD25+/high CD127low/−) was analyzed in matched samples of peripheral blood (PB), synovial fluid (SF) and synovial membrane (SM) from a total of 47 patients undergoing elective knee arthroplasty using flow cytometry. At the same time, knee pain and function were assessed and correlated with Treg proportions in different compartments (PB, SF, SM). Interestingly, matched-pair analysis revealed significantly higher Treg proportions in joint-derived samples than in PB, which was mainly attributed to the high Treg frequency in SF. Moreover, we found significant associations between infiltrating Tregs and OA-related symptoms which indicate that lower Treg proportions—especially in the SM—are related to increased pain and functional disability in knee OA. In conclusion, this study highlights the importance of local cellular inflammatory processes in OA pathology. Intra-articular Treg infiltration might play an important role not only in OA pathogenesis but also in the development of OA-related symptoms.

Human liver stem cells alleviate Con-A induced liver injury by regulating the balance of Treg/Th17 cells

BackgroundLiver injury is a serious threat to human health that has become a worldwide problem. To date, there is still no effective treatment strategy. In the present study, we examined the protective effects of Human liver stem cells (HLSCs) against concanavalin A (Con A)-induced acute liver injury. MethodsIsolated HLSCs were characterized by microscopy, functional assays, and gene expression. HLSCs or HLSCs culture medium were transplanted in mice for 12 h and subsequently challenged with Con A via tail-vein injection. The effects were evaluated through survival rate, histology, blood tests, TUNEL assay, quantitative RT-PCR and flow cytometry. CellTracker™ CM-Dil labled HLSCs were tracked by fluorescence microscope. ResultsTransplantation of HLSCs reduced the mortality rate, reduced the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL), narrowed the area of liver necrosis, and inhibited hepatocyte apoptosis induced by Con A. Injection of HLSCs culture medium could also alleviate Con A-induced liver injury. Of note, HLSCs-transplanted mice exhibited lower frequencies of Th17 cells and higher frequencies of Tregs in their liver and spleen following Con A injection. Moreover, transplantation of HLSCs significantly reduced the expression of IL-17A, IL-17F and ROR-γt induced by Con A, while reversed Con A-induced downregulation of Foxp3 expression and IL-10. ConclusionsHLSCs protect mice from immune-mediated liver injury by regulating the balance of Treg/Th17 cells, suggesting that transplantation of HLSCs is a potential and effective therapeutic method for amelioration of liver injury.

Determination of a CD4+CD25+ Foxp3+T cells subset in Egyptian Colorectal Cancer Patients

Human tumors including colorectal cancers (CRC) are often infiltrated by immune cells predominantly T lymphocytes especially regulatory T (Treg) cells expressing the forkhead box protein 3 (Foxp3). It has been suggested that CD25+CD4+Foxp3+ regulatory T cells (Tregs) might hamper effective immunosurveillance of emerging cancer cell. The aim of this study was to measure the frequency of total CD4+CD25+ Tregs & CD4+CD25+Foxp3+ subset of Treg cells in peripheral blood of Egyptian CRC patients and their correlation with the tumor stage, histopathology of the tumor and lymph node affection. A total of 31 CRC patients were enrolled in the study. The tumor was categorized using a TNM staging system. Peripheral blood samples were collected within the first 24 h of surgery. The frequency of total CD4+CD25+ Tregs & CD4+CD25+ Foxp3+ subset of Treg cells in peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry and absolute count was determined. High frequency of Tregs was detected in cancer patients with distal margin involvement (44-48 cells/μL) compared with those with free distal margin (5-32 cells/μL). Similarly, higher frequency of Tregs were detected (16-44 cells/μL) in cancers with lymph node involvement compared with cancers without lymph node involvement (5-32 cells/μL). Higher frequency of CD4+CD25+Foxp3+ Tregs were found in mucinous adenocarcinomas than in other histopathological types, although both observations were statistically insignificant. The median value for total absolute lymphocyte count/ μL was 639, out of which CD4+CD25+ subset constituted 35 cells, and about half of this subset were Foxp3+Tregs. In conclusion, CD4+CD25+Foxp3+ Tregs may be a useful marker for predicting invasion, metastasis, and prognosis of colorectal cancer in Egyptian patients.

Characteristics of T-cell responses in COVID-19 patients with prolonged SARS-CoV-2 positivity - a cohort study.

ObjectiveSARS‐CoV‐2 has caused a worldwide pandemic of COVID‐19. The existence of prolonged SARS‐CoV‐2 positivity (PP) has further increased the burden on the health system. Since T cells are vital for viral control, we aimed to evaluate the characteristics of T‐cell responses associated with PP.MethodsWe established a PP cohort and two age‐ and sex‐matched control cohorts: a regular clinical recovery (CR) cohort and a healthy donor (HD) cohort. The mean time for RNA negativity conversion in the PP cohort was markedly longer than that in the CR cohort (66.2 vs 25.3 days), while the time from illness onset to sampling was not significantly different. T‐cell responses in the PP cohort were assayed, analysed and compared with those in the CR and HD cohorts by flow cytometry and ELISpot analysis of peripheral blood mononuclear cells.ResultsCompared with the CR cohort, the proliferation, activation and functional potential of CD8+ and CD4+ T cells in the PP cohort were not significantly different. However, the frequencies and counts of Teff and Tem in CD8+ but not in CD4+ T cells of the PP cohort were prominently lower. Moreover, a weaker SARS‐CoV‐2 N protein‐specific IFN‐γ+ T‐cell response and a higher frequency of Tregs were detected in the PP cohort.ConclusionSuppressed CD8+ T‐cell differentiation is associated with PP and may be an indicator for the prediction of prolonged SARS‐CoV‐2 positivity in COVID‐19 patients. The association between suppressed CD8+ T‐cell differentiation and elevated Tregs warrants studies in the future.

Marrow-Infiltrating Regulatory T Cells Correlate with the Presence of Dysfunctional CD4+PD-1+ Cells and Inferior Survival in Patients with Newly Diagnosed Multiple Myeloma.

Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy. We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival. We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1-expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction. BM-infiltrating T-cell subsets, specifically Tregs and PD-1-expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.

Maturation of Gut Microbiota and Circulating Regulatory T Cells and Development of IgE Sensitization in Early Life.

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium. These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed.

The interplay between intrahepatic lymphocyte populations and Hepatitis B virus antigens related to liver damage in chronic hepatitis

Background: Chronic hepatitis B virus (HBV) infection is still a global health problem. Even though it is well known that adaptative immune response plays an important role in the pathogenesis of chronic infection, the impact of each immune cell population in liver damage and viral surveillance is still a matter of debate. Our aim was to elucidate the interplay between the immune response and viral activity in the context of liver damage. Methods & Materials: Immunostaining was performed in 23 liver biopsies from CHB treatment naïve patients (40% HBeAg-positive) to:1) characterize liver infiltrate [Th (CD4+), Th1 (Tbet+), Th17 (IL-17A+), Treg (Foxp3+), and CTL (CD8+)] [portal quantification: immunostained/total lymphocytes; lobular quantification: immunostained lymphocytes in 10 fields; (400x)], 2) evaluate the Hepatitis B surface antigen (HBsAg) expression (presence was determined). Inflammatory activity and fibrosis were assessed using the modified Knodell scoring system (Histological Activity Index, HAI) and METAVIR. Results: All studied populations were observed in portal/periportal infiltrates with predominance of Th {Th [0.65 (0.33-0.77)] > CTL [0.54 (0.16-0.74)] > Treg [0.09 (0.00-0.25)] > Th1 [0.07 (0-0.20)] > Th17 [0.05 (0-0.20)]}. However, only CTL were present in the intralobular area [2.77 (0.01-0.25)]. Concerning liver damage, Th17 frequency was increased in cases with more severe fibrosis (p = 0.04), while Th in cases with severe hepatitis (p = 0.02) mostly due to Treg (p = 0.02). The frequency of intralobular CTL also depicted association with severe hepatitis (p = 0.02). Regarding HBV antigen profile, only 62% of cases showed presence of HBsAg, being its absence associated with a higher number of Treg (p = 0.02) and severe hepatitis (p = 0.01). Moreover, HBe Ag-positive cases exhibited higher Treg and Th frequencies (p = 0.0002 and p = 0.037; respectively). Conclusion: Although Treg population was highly represented in the infiltrate, they were not related to liver damage. In contrast, intrahepatic Th17 mediated fibrosis severity. The presence of CTL in the intralobular area may indicate a relationship between them and hepatocytes denoting their contribution to hepatitis severity. Finally, our results suggest that the presence of a regulatory microenvironment enhances viral replication. This hypothesis is supported by the fact that a higher Treg frequency was observed in the context of absence HBsAg as well as among HBeAg-positive cases.

Dihydrotestosterone levels at birth associate positively with higher proportions of circulating immature/na\xefve CD5+ B cells in boys

Boys present with higher proportions of immature/naïve CD5+ B cells than girls up to 3 years of age. Boys also have higher fractions of regulatory T cells (Tregs) in early infancy, but the mechanisms for these sex-related differences are unknown. In the prospective FARMFLORA follow-up study of 23 boys and 25 girls, we investigated if these immunological differences remained at 8 years of age. We also examined if testosterone or dihydrotestosterone (DHT) levels at birth and at 8 years of age were associated with immune maturation. Immunological variables and androgen levels were examined and measured in blood samples obtained at birth, 3–5 days and at 8 years of age. Boys had higher proportions of CD5+ and immature/transitional CD24hiCD38hi B cells, whereas girls had higher fractions of B cells with a memory phenotype at 8 years of age. School-aged boys also presented with higher frequencies of Tregs, and a greater capacity to produce T-cell-associated cytokines. Among boys, higher cord blood DHT levels were associated with higher proportions of CD5+ B cells in early infancy and at 8 years of life. These results suggest that DHT actions in utero might be involved in the mechanism for delayed peripheral B-cell maturation in boys.

Altered profile of regulatory T cells and associated cytokines in mild and moderate dengue.

The pathogenesis of dengue is immune-mediated. Regulatory T cells suppress immune response and may contribute to better prognosis. The present study evaluates Tregs and cytokines in dengue patients in the context of disease severity, time of sampling and immune status. The cohort included 90 patients (51 mild, 39 moderate) and 27 healthy controls. Frequencies of Tregs, CD4(+)CD25(-)Foxp3(+) T cells and CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) T cells were enumerated by flow cytometry. Circulating levels of 15 cytokines/chemokines were measured using Luminex technology and mRNA levels of Foxp3, IL-10 and TGF-β were assessed by real-time polymerase chain reaction (PCR). Significantly higher frequencies of Tregs were observed in mild cases, especially during post-defervescence. The difference between mild and moderate cases was more evident in secondary infections. Frequencies of T cells were higher in mild cases but during pre-defervescence. On the other hand, the levels of IL-6, IL-7, IL-8, TNF-α and IL-10 were significantly higher in moderate cases. IL-6 and IL-8 levels correlated negatively with Treg frequencies during post-defervescence and in secondary infections. Higher levels of IL-10 and TGF-β in moderate cases were not reflected by their corresponding mRNA levels. Platelet counts correlated positively with Treg frequencies and TGF-β levels, and negatively with IL-10 levels. Higher Treg frequencies may favour a beneficial outcome in dengue. Higher cytokine levels may indirectly contribute to disease severity by exerting an inhibitory influence on Tregs. The dichotomy between mRNA and proteins levels for IL-10 and TGF-β is suggestive of increased translational efficiency.

Frequency of CD4+CD25+Foxp3+ cells in peripheral blood in relation to urinary bladder cancer malignancy indicators before and after surgical removal.

Tumor cells communicate with stromal cells, including cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), to form microenvironment inhibiting immune responses. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) stimulate immune tolerance and facilitate tumor progression. We analyzed the changes in Treg frequencies assessed using flow cytometry in the peripheral blood of patients with urothelial bladder cancer before and after tumor-removal. Changes in Treg frequency were investigated in relation to clinicopathomorphological indicators of tumor malignancy and expression of RCAS1 on CAFs and TAMs. Higher Treg frequencies were observed in early phase of tumor growth (pTa-pT2), in larger tumors, with more aggressive type of invasion, and with expression of RCAS1. The later phase of tumor development, accompanied by a nonclassic differentiations and pT3-pT4 advancement, had lower number of tumor infiltrating lymphocytes (TILs) and lower Treg frequency. Furthermore, in pT2-pT4 tumors, a decreased post-surgery Treg frequency was associated with poorer prognosis: patients with the lowest frequency of Tregs died first. These findings strongly suggest that the Treg frequencies at later phase of tumor growth, associated with a low anti-tumor response, represent a new and important prognostic indicator in urinary bladder cancer.

Analysis of dendritic cell subpopulations in follicular lymphoma with respect to the tumor immune microenvironment

The immune cell composition of the follicular lymphoma (FL) tumor microenvironment is increasingly recognized as an important determinant for clinical outcome. Here, we explored frequency and distribution of dendritic cell (DC) subtypes in relation to regulatory T cells (Treg) by immunohistochemistry in lymph node biopsies from patients with de novo FL. We found that neoplastic follicles contained lower DC and higher Treg frequencies than hyperplastic follicles in control lymph nodes. Treg numbers particularly correlated with the subset of conventional CD11c+ DCs. Additionally, both a high intra- to interfollicular ratio of CD11c+ DCs and increased intrafollicular Treg frequencies were associated with decreased overall survival. This suggests that functional interactions between these cells may be relevant for FL progression/recurrence. The presence of CD11c+ DCs in the tumor microenvironment may assist tumor infiltration by Tregs, thus contributing to the suppression of an otherwise beneficial T-cell-dominated FL microenvironment.

Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease

AbstractThe paucity of regulatory T cells (Tregs) limits clinical translation to control aberrant immune reactions including graft-versus-host disease (GVHD). Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4+FoxP3+ Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model. αDR3 induced selective proliferation of functional Tregs. CD4+ T cells isolated from αDR3-treated mice contained higher numbers of Tregs and were less proliferative to allogeneic stimuli. In vivo GVHD studies confirmed that Tregs from αDR3-treated donors expanded robustly and higher frequencies of Tregs within donor CD4+ T cells were maintained, resulting in improved survival. Conventional T cells derived from αDR3-treated donors showed reduced activation and proliferation. Serum levels of proinflammatory cytokines (IFNγ, IL-1β, and TNFα) and infiltration of donor T cells into GVHD target tissues (gastrointestinal tract and liver) were decreased. T cells from αDR3-treated donors retained graft-vs-tumor (GVT) effects. In conclusion, a single dose of αDR3 alleviates acute GVHD while preserving GVT effects by selectively expanding and maintaining donor Tregs. This novel strategy will facilitate the clinical application of Treg-based therapies.

Abstract P5-04-01: Breast cancer related immune suppression in the sentinel lymph node can be effectively countered by combined CpG-B administration and JAK2/STAT3 inhibition

Abstract Background Increasing evidence suggests that immune regulated pathways influence both breast cancer (BrC) development and response to (neo) adjuvant chemotherapeutic treatment. The sentinel lymph node (SLN) is the first site of BrC induced suppression of immune effector subsets, rendering them unable to mount an effective anti-tumor response. Since detailed knowledge of the functional status of these immune effector subsets is lacking, we compared the immune status of BrC SLN with healthy lymph nodes (HLN) using viable cell samples and correlated our findings to several clinico-pathological characteristics. Additionally we investigated if ex vivo conditioning with the Toll-like receptor-9 ligand CpG-B with or without simultaneous inhibition of JAK2/STAT3 signaling (a downstream signaling pathway implicated as the ‘master switch’ in tumor induced immune suppression) could overcome the observed immune suppression in BrC SLN. Methods Viable SLN cells were obtained from 40 clinically node negative BrC patients. Axillary HLN were obtained from 17 BRCA-1 or -2 patients undergoing a prophylactic mastectomy. Frequencies and activation state of dendritic cell (DC) subsets and regulatory T cells (Treg) were determined by extensive multi-color flowcytometric analyses. Additionally, SLN cells from 12 BrC patients were cultured in absence and presence of CpG-B (PF-3512676) and the combination of CpG-B with a JAK2-STAT3 inhibitor (AG-490). Results Of the 40 BrC SLN 11 contained metastasis. We found clear evidence of BrC-related immune suppression, as significantly higher Treg frequencies were observed in metastasis negative BrC SLN as compared to HLN. These frequencies further increased in metastasis positive BrC SLN. Activation state (by expression of the co-stimulatory molecules CD40, CD83 and CD86) of lymph node resident (LNR)-DC subsets (both CD11c+ myeloid DC and CD123+BDCA2+ plasmacytoid DC), but not of CD1a+ migratory subsets, was significantly lower in BrC SLN as compared to HLN. Additionally, in BrC SLN, activation state of these LNR-DC subsets was further reduced upon metastatic involvement and in non-luminal BrC subtypes (triple negative and HER-2+) as compared to luminal BrC subtypes (ER and/or PR positive). We previously showed in early-stage melanoma patients that local treatment with CpG-B led to the preferential activation of LNR-DC subsets. Similarly, ex vivo targeting of BrC SLN with CpG and CpG + JAK2/STAT3 inhibition resulted primarily in significantly enhanced activation of LNR-DC subsets, with superior effects of combined CpG and JAK2/STAT3 targeting evidenced by significantly higher CD83 expression. Conclusion BrC induced immune suppression in SLN seems to be primarily mediated by hampered activation of LNR-DC subsets, which was most effectively overcome (ex-vivo) with the immune activating compound CpG-B in combination with a small-molecule inhibitor of the immune suppressive JAK2/STAT3 signaling pathway (AG490). This combined immune targeting strategy might be of clinical benefit in enhancing effectiveness of conventional (neo)adjuvant chemotherapeutic treatment, especially in -more immune suppressed- non-luminal BrC subtypes with known poor prognosis. Citation Format: Kim M van Pul, Ronald JCLM Vuylsteke, Sinéad M Lougheed, Lisette EA te Velde, Petrousjka van den Tol, Emiel J Th Rutgers, Hein BAC Stockmann, Tanja D de Gruijl. Breast cancer related immune suppression in the sentinel lymph node can be effectively countered by combined CpG-B administration and JAK2/STAT3 inhibition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-01.

CD4+/CD25+ T cells suppress autologous CD4+/CD25- lymphocytes and secrete granzyme B during acute and chronic hepatitis C

We aimed to verify whether CD4(+)/CD25(+) T cells suppress CD4(+) T cells and secrete Granzyme B (GZB) during acute and chronic hepatitis C (CHC) infection. We enrolled 50 subjects: 20 patients with CHC (Group A), 15 healthy individuals (Group B), 10 patients with acute hepatitis C later evolved to persistent infection (Group C) and five patients who resolved hepatitis C virus infection during acute phase (Group D). We analysed, on enrolled subjects CD4(+)/CD25(+) T cells and related GZB production as well as Annexin V activity. Patients from Groups A and C had higher frequency and function of peripheral Treg cells than healthy individuals. Groups A and C showed an increase in spot-forming colonies (SFCs) of GZB compared with Group B (P < 0.01, Mann-Whitney U-test). CD4(+)/CD25(+) T cells in Group D had a lower number of GZB SFCs compared with Groups A and C but higher number than Group B (P < 0.01 Mann-Whitney U-test). Annexin V production was higher in Groups A and C than B or D. Patients having acute and chronic hepatitis C have a higher Treg frequency and function in peripheral blood than healthy controls or those resolving the infection in acute phase secreting GZB, probably inducing apoptosis.

Regulatory T cells in peripheral blood and cerebrospinal fluid of syphilis patients with and without neurological involvement.

BackgroundSyphilis, a sexually transmitted disease caused by spirochetal bacterium Treponema pallidum, can progress to affect the central nervous system, causing neurosyphilis. Accumulating evidence suggest that regulatory T cells (Tregs) may play an important role in the pathogenesis of syphilis. However, little is known about Treg response in neurosyphilis.Methodology/Principal FindingsWe analyzed Treg frequencies and Transforming Growth Factor-β (TGF-β) levels in the blood and CSF of 431 syphilis patients without neurological involvement, 100 neurosyphilis patients and 100 healthy donors. Suppressive function of Tregs in peripheral blood was also assessed. Among syphilis patients without neurological involvement, we found that secondary and serofast patients had increased Treg percentages, suppressive function and TGF-β levels in peripheral blood compared to healthy donors. Serum Rapid Plasma Reagin (RPR) titers were positively correlated with Treg numbers in these patients. Compared to these syphilis patients without neurological involvement, neurosyphilis patients had higher Treg frequency in peripheral blood. In the central nervous system, neurosyphilis patients had higher numbers of leukocytes in CSF compared to syphilis patients without neurological involvement. CD4+ T cells were the predominant cell type in the inflammatory infiltrates in CSF of neurosyphilis patients. Interestingly, among these neurosyphilis patients, a significant decrease in CSF CD4+ CD25high Treg percentage and number was observed in symptomatic neurosyphilis patients compared to those of asymptomatic neurosyphilis patients, which may be associated with low CSF TGF-β levels.ConclusionsOur findings suggest that Tregs might play an important role in both bacterial persistence and neurologic compromise in the pathogenesis of syphilis.

Incomplete Normalization of Regulatory T-Cell Frequency in the Gut Mucosa of Colombian HIV-Infected Patients Receiving Long-Term Antiretroviral Treatment

IntroductionTo evaluate the effect of late initiation of HAART and poor immune reconstitution on the frequency of regulatory T-cells (Treg) in the peripheral blood and gut of HIV-infected patients, we studied Colombian HIV-infected patients who had been on suppressive HAART for at least one year. They had undetectable viremia but were either immunological responders (HIR); (CD4 counts >500 cells/µl) or non-immunological responders (NIR); (CD4 T-cell count <300 cells/µl). Untreated HIV-infected patients and uninfected controls from the same region were also evaluated.MethodsFrequency and phenotype of regulatory T-cells (Treg) were analyzed in gut biopsies and blood samples. The functional effect of Treg depletion on CMV and HIV responses was determined. Markers of immune activation and circulating LPS levels were quantified.ResultsUntreated patients exhibited high Treg frequency in PBMC and gut, and their Treg express high levels of CTLA-4 and PD-1. Although HAART significantly decreased mucosal Treg frequency, it did not normalize it in any of the treated groups (HIR and NIR patients). Treg normalization was observed in the blood of HIR patients following HAART, but did not occur in NIR patients. Treg from HIV-infected patients (treated or not) suppressed HIV and hCMV-specific T-cells from gut and blood. Plasma LPS levels and percentage of HLA-DR+CD38+ T-cells were significantly elevated in all infected groups compared to controls.ConclusionsThese findings suggest that control of viral replication is not sufficient to normalize gut Treg frequency in patients, independent of their response to HAART. Furthermore, persistence of functional Treg in the gut appears to be associated with the failure of HAART to repair mucosal damage.