Tregs In Peripheral Blood Mononuclear Cells Research Articles

Durvalumab and stereotactic ablative body radiotherapy (SABR) in locally advanced (LA) and borderline resectable (BR) pancreatic cancer (PDAC): Long-term outcomes and correlative insights.

4174 Background: Immune checkpoint blockade (ICB) has limited activity in genomically unselected patients (pts) with PDAC. While anti-tumor activity with combination SABR/ICB has been shown, biomarkers of response are poorly defined. We conducted a phase 1/2 study of PD-L1 inhibitor Durvalumab (D) and SABR after induction chemotherapy in LA and BR PDAC (NCT03245541). We report long-term outcomes and correlative biospecimen analyses. Methods: A multi-institutional, single arm phase 1/2 trial was conducted at Cedars-Sinai and Memorial Sloan Kettering. Initial clinical data reported by Tuli et al, ASCO 2023. Pre-treatment tissue assessed for p40 and GATA3 to determine classical vs basal subtype by immunohistochemistry. Tissue interrogated by immunofluorescence for PD-L1 expression and infiltration of immune cell subsets. Multiparametric immunophenotyping of peripheral blood mononuclear cells (PBMCs) by flow cytometry conducted on baseline and on treatment samples. Wilcoxon rank sum test used to determine difference in biomarkers from each timepoint; p-value <0.05 significant. Results: N= 36 enrolled from 08/2017-05/2022; N=31(86%) LA, N=5 (14%) BR. N= 9 (25%) underwent resection; all R0. At median follow up 36.5 months, 6- and 12-month PFS 69%, 36%. Median PFS 8.7m (95% CI 6.1-14), median OS 16m (95% CI 13-26). N=2 (6%) alive without disease progression. Correlative analyses summarized (table). Key findings; Classical/basal subtyping feasible on N=9 (8 classical, 1 basal). Median PD-L1 score not associated with survival. Peri-tumoral, but not intratumoral CD8+ cells associated with PFS >6 months. Flow cytometry feasible for 70 samples from N=35. Frequency of pre-treatment non-naïve CD4+ FoxP3+ Treg associated with PFS >12. CD103+ CD8+ T Cells significantly expanded on treatment only in patients with PFS>12m (p=0.038) and were significantly enriched relative to PFS<12m on treatment. Conclusions: A proportion of pts with LA PDAC achieve durable control with D and SABR after induction chemotherapy. Baseline peri-tumoral CD8+ cells and peripheral CD103+ CD8 T cells and CD4+ Treg in PBMCs at baseline and on treatment may predict for benefit. Further analyses ongoing and validation in larger cohorts is warranted. Clinical trial information: NCT03245541 . [Table: see text]

Differential regulatory T cell signature after recovery from mild COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a range of symptoms in which host immune response have been associated with disease progression. However, the putative role of regulatory T cells (Tregs) in determining COVID-19 outcomes has not been thoroughly investigated. Here, we compared peripheral Tregs between volunteers not previously infected with SARS-CoV-2 (healthy control [HC]) and volunteers who recovered from mild (Mild Recovered) and severe (Severe Recovered) COVID-19. Peripheral blood mononuclear cells (PBMC) were stimulated with SARS-CoV-2 synthetic peptides (Pool Spike CoV-2 and Pool CoV-2) or staphylococcal enterotoxin B (SEB). Results of a multicolor flow cytometric assay showed higher Treg frequency and expression of IL-10, IL-17, perforin, granzyme B, PD-1, and CD39/CD73 co-expression in Treg among the PBMC from the Mild Recovered group than in the Severe Recovered or HC groups for certain SARS-CoV-2 related stimulus. Moreover, Mild Recovered unstimulated samples presented a higher Tregs frequency and expression of IL-10 and granzyme B than did that of HC. Compared with Pool CoV-2 stimuli, Pool Spike CoV-2 reduced IL-10 expression and improved PD-1 expression in Tregs from volunteers in the Mild Recovered group. Interestingly, Pool Spike CoV-2 elicited a decrease in Treg IL-17+ frequency in the Severe Recovered group. In HC, the expression of latency-associated peptide (LAP) and cytotoxic granule co-expression by Tregs was higher in Pool CoV-2 stimulated samples. While Pool Spike CoV-2 stimulation reduced the frequency of IL-10+ and CTLA-4+ Tregs in PBMC from volunteers in the Mild Recovered group who had not experienced certain symptoms, higher levels of perforin and perforin+granzyme B+ co-expression by Tregs were found in the Mild Recovered group in volunteers who had experienced dyspnea. Finally, we found differential expression of CD39 and CD73 among volunteers in the Mild Recovered group between those who had and had not experienced musculoskeletal pain. Collectively, our study suggests that changes in the immunosuppressive repertoire of Tregs can influence the development of a distinct COVID-19 clinical profile, revealing that a possible modulation of Tregs exists among volunteers of the Mild Recovered group between those who did and did not develop certain symptoms, leading to mild disease.

Cytokines and regulatory T cells in ankylosing spondylitis.

To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay.

Tislelizumab Rechallenge for Recurrent or Metastatic Nasopharyngeal Carcinoma (RM-NPC) Previously Treated with Another Anti-PD1 Antibody

<h3>Purpose/Objective(s)</h3> To explore the ORR and PFS of Tislelizumab rechallenge in combination with chemotherapy or stereotactic body radiation therapy (SBRT) for recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) previously treated with another anti-PD1 antibody. <h3>Materials/Methods</h3> This was part of a prospective study. RM-NPC patients who showed no response to or progressed after PD-1 blockade were rechallenged with Tislelizumab plus chemotherapy or SBRT. Tislelizumab was given at a fixed dose of 200 mg, repeated every three weeks. Chemotherapy regimen was low dose gemcitabine plus metronomic capecitabine. SBRT was given 4 Gy to 6 Gy in three or five fractions depending on the tumor site. Tislelizumab was delivered one day after the completion of SBRT. The primary endpoint was the ORR. The secondary endpoints included PFS, duration of response (DoR), and safety. Dynamic assessment of T lymphocytes, B lymphocytes, natural killer (NK) cells (TBNK), and regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry every 2-3 months in order to analyze the change patterns following PD-1 blockade. <h3>Results</h3> From March 2019 to February 2022, 25 patients were enrolled in this study. All have previously received high dose platinum-based chemotherapy and PD-1 blockade. The salvage regimen was Tislelizumab rechallenge plus chemotherapy in 21 cases, and Tislelizumab plus SBRT in 4 cases. Altogether, there were 5 (20%) complete response (CR) and 12 (48%) partial response (PR), respectively. Stable disease (SD) and progressive disease (PD) were defined in 5 (20%) and 3 (12%) cases, respectively. The ORR was 17 out of 25 (68%). The disease control rate (DCR) was 22 out of 25 (88%). With a median follow-up of 12 months (4–26 months), three patients terminated immunotherapy due to disease progression, and the remaining 22 patients were still under treatment. The 1-year PFS was 78%, neither the median PFS nor the median DoR has been reached. Of the 12 patients who had regular TBNK and Treg surveillance in PBMCs, all showed an increasing trend in Treg accompanying with tumor progression. On the contrary, responders to Tislelizumab rechallenge demonstrated decrease of Treg in PBMCs. However, no specific regularity could be defined in TBNK. All observed adverse events during Tislelizumab rechallenge were defined as ≤ Grade 2. <h3>Conclusion</h3> Tislelizumab rechallenge combined with chemotherapy or SBRT displayed promising anti-tumor activity in RM-NPC previously undergone PD-1 blockade, with a low toxicity profile. The increase of Treg in PBMCs is always associated with immune tolerance after PD-1 blockade and the decrease of Treg is correlated with benefit from Tislelizumab rechallenge.

Preeclampsia-Derived Exosomes Imbalance the Activity of Th17 and Treg in PBMCs from Healthy Pregnant Women.

The disturbance of maternofetal immune tolerance is identified as one of the important issues in the pathology of preeclampsia (PE). PE exosomes are believed to possess significant roles in immune abnormalities. In this study, to assess the possible effects of PE exosomes in the pathophysiology of preeclampsia patients, exosomes were isolated from the serum of PE patients and incubated with peripheral blood mononuclear cells (PBMCs) of healthy pregnant women. Also, exosomes from healthy pregnant women were utilized as the control. Th17/Treg ratio in PE and healthy pregnant women and the effects of PE exosomes on expression level of Th17 and Treg transcription factors, as well as their related cytokines in PBMCs of healthy pregnant women, were evaluated. A significant decrease in Treg cell number and increase in Th17 cells and Th17/Treg ratio were observed in PE patients. Following PE-exosome intervention, a significant increase in mRNA expression level of RORγt, IL-17, IL-23, IL-1β, and IL-6, and significant decrease in IL-10 and TGFβ were evident. On the other hand, no significant difference in FoxP3 level was detected. Additionally, increased IL-6, IL-17, IL-23, and IL-1β levels and decreased IL-10 level in the supernatant of cultured PBMCs from healthy pregnant women following PE-exosome intervention were exhibited. However, TGF-β level did not change significantly. Based on our findings, PE exosomes are able to alter the activity of Th17 and Treg cells as well as their related gene expression and cytokine profiles. These findings support the probable role of PE exosomes in PE pathogenesis.

Monocarboxylate transporter upregulation in induced regulatory T cells promotes resistance to anti-PD-1 therapy in hepatocellular carcinoma patients.

BackgroundProgrammed cell death-1 (PD-1) immune checkpoint inhibitors are not effective in treating all patients with hepatocellular carcinoma (HCC), and regulatory T cells (Tregs) may determine the resistance to anti-PD-1 therapy.MethodsPatients were divided into two groups based on the clinical efficacy of anti-PD-1 therapy. Flow cytometry was used to determine the phenotype of CD4+, CD8+, and Tregs in peripheral blood mononuclear cells (PBMCs). CD4+CD45RA+T cells were sorted to analyze Treg differentiation and function.ResultsNo significant differences were found between resistant and sensitive patients in the percentage of CD4+ T cells and Tregs in PBMCs or the differentiation and function of induced Tregs (iTregs). However, iTregs from resistant patients presented higher monocarboxylate transporter (MCT) expression. Lactate induced more iTregs and improved OXPHOS levels in the resistant group. MCT1 and MCT2 were highly expressed in tumor-infiltrating Tregs, and patients with higher MCT1 expression had worse clinical outcomes. Combinatorial therapy with MCT antibody and anti-PD-1 therapy effectively inhibited tumor growth.ConclusionMCT and its downstream lactate signal in Tregs can confer anti-PD-1 resistance and may be a marker of poor prognosis in HCC.

Low-Dose Decitabine Restores Immune Tolerance in ITP By Modulating Regulatory T Cells

Immune thrombocytopenia (ITP) is an aquired autoimmune disease triggered by increased platelet destruction and impaired platelet production. Loss of immune tolerance is considered to be the pivotal mechanism of ITP. Decitabine, a hypomethylating agent promoting cell differentiation at low dose, has been applied in the treatment of myelodysplastic syndrome (MDS) with a considerable platelet response. Our previous and current studies demonstrated that low-dose decitabine could significantly increase the number of mature polyploidy megakaryocytes and had a long-term efficacy that could not be explained by its role on promoting platelet production in ITP patients. Recent reports have demonstrated that decitabine could decrease the production of interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-a), and increase immune suppressive regulatory T cells. Thus, low-dose decitabine might correct the loss of immune tolerance to induce a more durable response in patients with ITP.CD4+CD25+Foxp3+ regulatory T cells (Tregs), which play an important role in maintenance of peripheral tolerance, were reported to be decreased in number and function in ITP (Stasi R et. al. Blood 2008). In the in vitro study, peripheral blood mononuclear cells (PBMCs) were isolated and cultured with 100nM decitabine or PBS for 72h. We performed flow cytometry to determine the percentage of Tregs. It was shown that the percentage of Tregs in PBMCs and CD4+ T cells were significantly elevated after decitabine treatment. We also sorted the CD4+CD25+CD127- Tregs from ITP patients and co-cultured them with CFSE-labeled CD4+CD25-T cells to evaluate the suppressive activity of Tregs. The results indicated that the inhibitory function of Tregs after decitbine treatments was significantly elevated compared with controls.As compared to healthy individuals, patients with ITP also had increased number of TH1, TH17, TH22, Bregs and CD16+ monocytes. We subsequently investigated the effects of low-dose decitabine on those cells in patients with active ITP. Three cycles of decitabine were administered intravenously at a dose of at 3.5 mg/m2 for 3 days per cycle with a 4-week interval between cycles. After decitabine treatment, there was a significant increase in the relative number of CD25+Foxp3+ Tregs in the CD4+ T cell population. We also observed an increase in Tregs function after the decitabine treatment. In contrast, decitabine could significantly decrease the percentage of CD4+IFN-γ+TH1 in the CD4+T cell population and the percentage of CD16+monoyctes. However, we have not observed the effect of decitabine on the CD4+IFN-γ-CD17+TH17, CD4+IFN-γ-CD22+TH22, and CD19+CD24+CD38+Bregs by now.We further established the murine ITP model by transferring the splenocytes of C57BL/6 CD61 knockout mice, which were immunized against platelets from wild-type syngeneic C57BL/6 mice into severe combined immunodeficient (SCID) mice. We treated the ITP mice model with a low-dose range of decitabine (0.01, 0.03 or 0.10 mg/kg) intravenously three times a week. Our data showed a significant effect on PLT increase and the expansion of the Tregs population and the suppression of TH1 cells and CD16+ monocytes .In addition, programmed death-1 (PD-1) is a co-receptor that is expressed predominantly by T cells as well as B cells, monocytes and was found to be lower on the PBMCs and in serum of ITP patients (Zhong J et. al. Hematology. 2016). And research has reported that PD-1 signaling pathway is important in the pathogenesis of autoimmune diseases by inhibition of self-reactive T cells and increasing the development and functions of Tregs. Furthermore, it was reported that the expression of PD-1 is enhanced by decitabine in MDS (Yang H et. al. Leukemia 2014). In our current studies, decreased PD-1 expression on CD4+T cells of ITP patients was observed and decitabine could elevate the PD-1 expression on CD4+T cells after the decitabine treatment in ITP patients. Therefore, decitabine may correct the loss of immune tolerance by upregulating the Expression of PD-1 and be a promising therapeutic strategy for the management in ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumour immune evasion mechanism in non-small cell lung cancer by inducing Blimp-1 in CD4+CD25+Foxp-3+ T regulatory cells

Abstract Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth. The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs). We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain). Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients. Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta. Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.

Luteolin Regulates the Differentiation of Regulatory T Cells and Activates IL-10-Dependent Macrophage Polarization against Acute Lung Injury.

Objectives Inflammatory disease characterized by clinical destructive respiratory disorder is called acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Studies have shown that luteolin exerts anti-inflammatory effects by increasing regulatory T cells (Tregs). In this study, we aimed to determine the effects of luteolin on ALI/ARDS and Treg differentiation. Methods In this paper, we used cecal ligation puncture (CLP) to generate an ALI mouse model to determine the effects of luteolin on ALI/ARDS. Lung tissues were stained for interleukin- (IL-) 17A and myeloperoxidase (MPO) by immunohistochemical analysis. The levels of Treg-related cytokines in serum and bronchoalveolar lavage fluid (BALF) of mice were detected. The protein levels of NF-κB p65 in lung tissues were measured. Macrophage phenotypes in lung tissues were measured using immunofluorescence. The proportion of Tregs in splenic mononuclear cells and peripheral blood mononuclear cells (PBMCs) was quantified. Furthermore, in vitro, we evaluated the effects of luteolin on Treg differentiation, and the effects of IL-10 immune regulation on macrophage polarization were examined. Results Luteolin alleviated lung injury and suppressed uncontrolled inflammation and downregulated IL-17A, MPO, and NF-κB in the lungs of CLP-induced mouse models. At this time, luteolin upregulated the level of IL-10 in serum and BALF and the frequency of CD4+CD25+FOXP3+ Tregs in PBMCs and splenic mononuclear cells of CLP mice. Luteolin treatment decreased the proportion of M1 macrophages and increased the proportion of M2 macrophages in lungs of CLP-induced mouse models. In vitro, administration of luteolin significantly induced Treg differentiation, and IL-10 promoted the polarization of M2 macrophages but reduced the polarization of M1 macrophages. Conclusions Luteolin alleviated lung injury and suppressed uncontrolled inflammation by inducing the differentiation of CD4+CD25+FOXP3+ Tregs and upregulating the expression of IL-10. Furthermore, the anti-inflammatory cytokine IL-10 promoted polarization of M2 macrophages in vitro. Luteolin-induced Treg differentiation from naïve CD4+ T cells may be a potential mechanism for regulating IL-10 production.

TLR-2 expression and dysregulated human Treg/Th17 phenotype in Aspergillus flavus infected patients of chronic rhinosinusitis with nasal polyposis

BackgroundT helper (Th)17 and regulatory T (Treg) cells with toll-like receptor (TLR)-2 have been acknowledged to play a critical role in chronic rhinosinusitis with nasal polyposis (CRSwNP). However, its pathogenesis has been perplexed by conflicting reports on the role of Th17/Treg cells in patients of distinct ethnicities. We attempted to understand the role of Th responses induced during host defense against Aspergillus flavus.ResultsThe percentages of Th17 (CD4+CD161+IL23R+) and Treg (CD4+CD25+FoxP3+) cell populations and various cytokine profiles in peripheral blood mononuclear cells (PBMCs) challenged by A. flavus antigens were characterized from 50 CRSwNP cases, before and after treatment, and in 50 healthy controls. TLR-2 expression was analyzed in tissues of cases and controls for disease co-relation. The major pathogen identified in our study was A. flavus by mycological investigations. A marked immune imbalance was noted with elevated Th17 and decreased Tregs in PBMCs of CRSwNP patients after A. flavus stimulation. Comparatively, interleukin (IL)-17 and IL-10 levels were increased, with low transforming growth factor (TGF)-β levels in A. flavus stimulated PBMC supernatants of patients. The mRNA expression of TLR-2 in polyps of CRSwNP patients indicated significant (p = 0.001) upregulation in comparison to the controls.ConclusionsOur data highlights the excessive expression of TLR-2 in nasal polyps contributing to the imbalance in Th17/Tregs population in patients. After therapy, recovery of Tregs cells indicates restoration and tissue homeostasis, though high circulating CD4+CD161+ Th17 cells may continue to be a threat to patients predisposed to future recurrences. The constant exposure and tendency of A. flavus to colonize nasal cavities can lead to a Th17 driven airway inflammation. Dysregulated Th17 with TLR-2 promote resistance to treatment and progression to the chronicity of the disease.

Increased frequency of regulatory T cells in the peripheral blood of patients with endometrioid adenocarcinoma.

The aim of the present study was to investigate the frequency of cluster of differentiation (CD)4+CD25+CD127- regulatory T cells (Tregs) in the peripheral blood mononuclear cells (PBMCs) of patients with endometrioid adenocarcinoma (EA). A total of 82 female patients with EA were recruited. The PBMCs were flow cytometrically analyzed to determine the percentage of CD4+CD25+CD127- Tregs within the CD4+ T cell population. The associations between the prevalence of Tregs in PBMCs and defined clinical prognostic parameters were evaluated. To study the immunoregulatory capacity of Tregs, the level of specific cytokines were detected by ELISA, and the proliferation of cells was analyzed by incorporation of 3H-thymidine. It was revealed that Treg/CD4+ ratio in the peripheral blood of patients with EA was 4.89±1.42%, significantly higher than Treg/CD4+ ratio in healthy women. No correlation was observed between Treg frequency and stage, grade of differentiation, menopausal status or age. CD4+CD25+CD127- Tregs secreted large amounts of IL-10 but not IFN-γ. The level of IL-10 secreted by Tregs from patients with EA and healthy controls was not significantly different. In addition, there was no significant difference in the suppressive activity of Tregs in patients with EA compared with that of the healthy controls. These findings demonstrate that the increased frequency of immunosuppressive Tregs in patients with EA may be responsible for immune tolerance in endometrial cancer.

Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer

Abstract Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naïve CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p&amp;lt;0.001) and 82% (IQR: 38% to 99%; p=0.004) of Treg from ovarian tumor-infiltrating lymphocytes (TIL) and ascites, respectively. Surface NRP1 was detected on 0.1% (IQR: 0% to 1%) of Treg in PBMC from healthy donors, compared with 11.1% (IQR: 1.1% to 60%; p=0.01) on ovarian TIL and 82% (IQR: 14% to 82%; p=0.0015) on ascites. Comparing expression of intracellular NRP1 on Treg from N=2 benign ovarian tissues versus N=14 malignant ovarian tumors, there was a trend towards increased NRP1 expression (median 9.3% versus 54.5%, respectively; p=0.044). Finally, Treg isolated from ascites fluid were capable of suppressing CD8+ T cells ex vivo proliferation in a dose-dependent manner. Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD8 cells ex vivo. Future studies will evaluate whether blockade of NRP1 on human Treg limits either the survival or suppressive capacity of Treg. Note: This abstract was not presented at the conference. Citation Format: Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali. Neuropilin-1 expression on regulatory T cells in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A23.

Comparison of the Percentage of Regulatory T cells and their p-STAT5 Expression in Allergic and Non-Allergic Common Variable Immunodeficiency Patients

ABSTRACTBackground: Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by an immunologic deficiency in immunoglobulin production. Regulatory T cells (Tregs) play a key role in preventing the development allergic disorders. p-STAT5 is a known factor for the function and survival of Tregs. This study aimed to investigate the number of Tregs and their p-STAT5 expression in allergic and non-allergic CVID patients.Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 10 healthy volunteers, 10 allergic patients, and 16 CVID patients (allergic and non-allergic) using Ficoll density centrifugation. The percentage of Tregs in PBMCs was analyzed by flow cytometry. Tregs were also isolated from participants using an immunomagnetic separation method and p-STAT5 expression was evaluated in Tregs using flow cytometry.Results: The results revealed that Treg percentage was significantly lower in the CVID patients than the control groups (healthy and allergic individuals) (p<0.001). There was a significant reduction in Treg percentage in allergic patients compared to healthy subjects (p<0.05). No significant difference in Treg percentage between allergic and non-allergic CVID patients was observed. The expression of p-STAT5 in Tregs was significantly lower in CVID patients than the control groups (p<0.001). In addition, the expression of p-STAT5 in Tregs of allergic patients was significantly decreased compared to healthy subjects (p<0.001). However, the deference of p-STAT5 level was not statistically significant between allergic and non-allergic CVID patients.Conclusion: These findings suggest that p-STAT5 signaling defect and decreased Treg number may not participate in the development of allergy in CVID patients.

Imbalance of Th17 and Treg in peripheral blood mononuclear cells of active tuberculosis patients

ObjectiveMaintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB). This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls) were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs) in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. ResultsThe percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31±1.35% vs. 1.58±0.71% or 1.15±0.49%, p<0.05; Treg: 11.44±2.69% vs. 7.54±1.56% or 4.10±0.99%, p<0.05). The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85±9.68 vs. 7.23±5.19 or 8.21±5.51pg/mL, p<0.05; IL-10: 28.70±11.27 vs. 20.25±8.57 or 13.94±9.00pg/mL, p<0.05). ConclusionsOur study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

Safety and Feasibility of Ultra-Low Dose IL-2 As Graft Versus Host Disease Prophylaxis in Haplo-Identical Stem Cell Transplantation- a Proof of Concept Pilot Study

INTRODUCTIONHaploidentical allogeneic stem cell transplantation (haplo-SCT) incurs a risk of bidirectional immune reactions with either severe acute graft versus host disease (aGVHD) or graft rejection. Induction of immune tolerance with post-graft cyclophosphamide dramatically improves the outcomes of haplo-SCT. However the optimal duration and the combination of systemic immunosuppressive agents in haplo-SCT remain controversial. Ultra-low dose interleukin 2 (ULD IL-2) preferentially expands regulatory T cells (Tregs) and natural killer (NK) cells, promoting both GVHD prevention and graft versus leukemia (GVL) effects. These properties suggest that ULD IL-2 could play a useful role in haplo-SCT. Here we report the outcomes of a pilot study to determine the safety and feasibility of ULD IL-2 as GVHD prophylaxis in haploidentical allo-SCT (14-H-0180, Clinical Trials.gov ID: NCT02226861) METHODSTen subjects with high risk hematological malignancies received a myeloablative conditioning regimens of fludarabine 120mg/m2 (day -10 to day -8) and total body irradiation (TBI; 600-1200 cGy, day -10 to day -6). Thereafter the subjects received donor lymphocyte infusion (DLI) products (2x108 CD3+/kg) on day -6, followed by post-DLI cyclophosphamide 120mg/kg on days -3 and -2. CD 34+ selected, peripheral blood stem cell product was infused on day 0. Sirolimus was initiated on day -1 with goal trough level of 5-12ng/mL until day+60. ULD-IL2 (aldesleukin, 100,000 IU/m2) was given subcutaneously daily for 12 weeks starting day +1. Peripheral blood mononuclear cells (PBMC) and plasma samples were collected at days 14, 28, 60, 84, 100 post-transplant. Multi-color flow cytometry immunophenotyping assay were performed to characterize the subsets of memory T cells, Tregs, NK cells, and monocytes with various functional markers. Plasma levels of biomarkers (ST2, Reg3α, sTNFR1, ANG1, IL-6) were measured using a multiplex microfluidic channel assay. RESULTSThe median age at transplant was 35 years (range 20-66). Most subjects had a high risk EBMT score (median 4, range 2-7) and HCT co-morbidity index (median 4, range 2-7). All subjects achieved successful engraftment (neutrophil >500/uL; median 13 days, platelet >20k/uL; median 15 days) and rapid full donor myeloid and lymphoid chimerism by day 21. At median follow up of 6 month, the overall survival was 71%. One subject died of hepatic veno-occulusive disease (VOD) on day 32 and one subject died of relapse on day 178. All evaluable subjects tolerated ULD-IL2 without significant toxicities. Four subjects experienced either de-novo or rapid exacerbation of acute GVHD after discontinuation of ULD IL-2, resulting in the cumulative incidence of grade II-IV aGVHD of 61% and grade III-IV aGVHD of 36% (Figure A). ULD IL-2 expanded and maintained Helios+FoxP3+Tregs population (pre-transplant, 4.7%±3.1%; day 30, 36.2%±23.1%; day 84, 17.4%±10.6%) as well as CD56brightNK cells population (pre-transplant, 10.7%±13.7%; day 30, 49.7%±10.8%; day 84, 26.1%±6.8%). However on discontinuation of ULD IL-2 both populations decreased to low levels within one week. The timing of aGVHD correlated with a fall of %Tregs in PBMC and a sharp increase of ST2 level in plasma (Figure B). CONCLUSIONULD IL-2 can be safely administered as GVHD prophylaxis after haplo-SCT. Rebound GVHD after discontinuation of ULD IL-2 implies that donor-derived Tregs acquired dependency to exogenous ULD IL-2. Our study is proof of principle that ULD IL-2 induces immune tolerance through Tregs expansion in haplo-SCT, inspiring further clinical and basic researches in human IL-2 biology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The role of regulatory B cells (Bregs) in the Tregs-amplifying effect of Sirolimus

Sirolimus can significantly amplify regulatory T cells (Tregs) in vivo and in vitro, but the specific mechanism of this has not been well documented. The role of regulatory B cells (Bregs) in the Tregs-amplifying effect of Sirolimus was investigated in peripheral blood mononuclear cells (PBMCs) in vitro in this study. The results showed that the percentages of both CD19+CD24+CD38+TGF-β1+ Bregs and CD19+CD24+CD38+IL-10+ Bregs to B cells were elevated by Sirolimus in PBMCs including B cells. Sirolimus significantly enhances the cytokine production of transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) in PBMCs with B cells, and the enhancement significantly decreased in PBMCs without B cells. The percentage of CD4+CD25+Foxp3+ Tregs to T cells was also elevated by Sirolimus in PBMCs including B cells. The elevation of Tregs percentage decreased in PBMCs without B cells and recovered when additional TGF-β1 and IL-10 were added. The amplification of Tregs by Sirolimus was partially inhibited when either TGF-β1 or IL-10 was neutralized, and it even disappeared when these two cytokines were both neutralized. These results indicate that Sirolimus can amplify Bregs and Tregs in PBMCs in vitro, and Bregs may be the why Sirolimus amplifies Tregs.

CD4⁺ T cells expressing latency-associated peptide and Foxp3 are an activated subgroup of regulatory T cells enriched in patients with colorectal cancer.

Latency-associated peptide (LAP) - expressing regulatory T cells (Tregs) are important for immunological self-tolerance and immune homeostasis. In order to investigate the role of LAP in human CD4+Foxp3+ Tregs, we designed a cross-sectional study that involved 42 colorectal cancer (CRC) patients. The phenotypes, cytokine-release patterns, and suppressive ability of Tregs isolated from peripheral blood and tumor tissues were analyzed. We found that the population of LAP-positive CD4+Foxp3+ Tregs significantly increased in peripheral blood and cancer tissues of CRC patients as compared to that in the peripheral blood and tissues of healthy subjects. Both LAP+ and LAP− Tregs had a similar effector/memory phenotype. However, LAP+ Tregs expressed more effector molecules, including tumor necrosis factor receptor II, granzyme B, perforin, Ki67, and CCR5, than their LAP− negative counterparts. The in vitro immunosuppressive activity of LAP+ Tregs, exerted via a transforming growth factor-β–mediated mechanism, was more potent than that of LAP− Tregs. Furthermore, the enrichment of LAP+ Treg population in peripheral blood mononuclear cells (PBMCs) of CRC patients correlated with cancer metastases. In conclusion, we found that LAP+ Foxp3+ CD4+ Treg cells represented an activated subgroup of Tregs having more potent regulatory activity in CRC patients. The increased frequency of LAP+ Tregs in PBMCs of CRC patients suggests their potential role in controlling immune response to cancer and presents LAP as a marker of tumor-specific Tregs in CRC patients.

Role of immune dysfunction in pathogenesis of type 1 diabetes mellitus in children

ObjectivesTo investigate the function of cytokines, chemokines, and regulatory T cells (Tregs) in the pathogenesis of type 1 diabetes mellitus (T1DM) in children. MethodsA total of 35 children with T1DM and 30 healthy controls were enrolled in this study. Levels of serum cytokines (IL-1α, IL-6, IL-10, IL-12, and TNF-α) and chemokines (MIP-1α, MIP-1α and MCP-1) were detected by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) were isolated and culture supernatant of phytohaemagglutinin (PHA)-stimulated PBMCs was subjected to ELISA for levels of cytokines (IL-1α, IL-6, IL-10, IL-12 and TNF-α) in T1DM and control group. Furthermore, flow cytometry was used to determine the percentage of Tregs in PBMCs of two groups. ResultsLevels of serum cytokines including IL-1α, IL-6, IL-10 and TNF-α as well as chemokines, such as MIP-1α and MIP-1α in children with T1DM children were significantly higher than those in healthy controls (P<0.05, respectively). PBMCs with PHA stimulation in T1DM group secreted more IL-1α and TNF-α (P<0.05, respectively), but less IL-10 (P<0.05), as compared with control group. Furthermore, the proportion of CD4+, CD25+, Foxp3+, Tregs in PBMCs isolated from children with T1DM was obviously lower than those in healthy controls (P<0.05). ConclusionsImmune dysfunction, with upregulation of inflammatory factors such as IL-1α, IL-6, TNF-α and MIP-1α, downregulation of IL-10 and Tregs, plays an important role in the pathogenesis of T1DM in children.

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4+ T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8+ T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8+ T cell effectors when used in the inflammatory environment of vaccination.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-013-1397-7) contains supplementary material, which is available to authorized users.

Abstract 4749: Cyclophosphamide induces dose dependent apoptosis of CD4+FoxP3+ regulatory T cells relative to CD4+FoxP3- effector T cells in breast cancer patients.

Abstract CD4+FoxP3+ regulatory T cells (Tregs) accumulate at tumor sites and within the peripheral blood of patients with a variety of cancers. Tregs suppress antigen-specific immune responses, and represent a major barrier to the efficacy of cancer immunotherapy. We previously conducted a dose-ranging study of low dose Cyclophosphamide (0, 200, 250, and 350 mg/m2) given one day prior to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting HER-2+ breast cancer vaccine in 28 patients with metastatic breast cancer. We found that vaccine-induced HER-2-specific immunity was best optimized by 200 mg/m2 CY, and that doses of CY higher than 200 mg/m2 suppressed antigen-specific immunity. To further explore the pharmacodynamic impact of CY on immune priming, we analyzed Tregs and effector CD4+ T cells present in peripheral blood mononuclear cells (PBMC) collected pre- and post-vaccination with or without CY by flow cytometry. We found that the percentage of CD4+FoxP3+ Tregs in PBMC did not change with vaccination alone. Conversely, CY at doses of either 200 or 250 mg/m2 selectively induced the apoptosis of CD4+FoxP3+ Tregs relative to CD4+FoxP3− effector T cells, and CY at 350 mg/m2 induced apoptosis of both CD4+FoxP3+ Tregs and CD4+FoxP3− effector T cells. Finally, CY at all doses selectively decreased CCR7−CD45RA− effector memory Tregs and increased CCR7+CD45RA+ naïve Tregs relative to vaccine alone. Collectively, these data suggest that low dose CY selectively induces apoptosis of CD4+FoxP3+ Tregs relative to CD4+FoxP3− effector T cells, providing a window for more efficient induction of vaccine-induced HER-2-specific immunity. Citation Format: Gang Chen, James M. Leatherman, Melek Erdinc Sunay, Leisha A. Emens. Cyclophosphamide induces dose dependent apoptosis of CD4+FoxP3+ regulatory T cells relative to CD4+FoxP3- effector T cells in breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4749. doi:10.1158/1538-7445.AM2013-4749