Tregs In Peripheral Blood Research Articles

Phase I Clinical Trial of CK0801 (cord blood regulatory T cells) in Patients with Bone Marrow Failure Syndrome (BMF) Including Aplastic Anemia, Myelodysplasia and Myelofibrosis

Background: Aplastic anemia is an autoimmune disease of the bone marrow (BM) characterized by defective and decreased regulatory T cells (Tregs) which results in unchecked cytotoxic T cell mediated bone marrow suppression [1]. In a xenogeneic model of induce BM aplasia, adoptive therapy with cord blood (CB) Tregs reverses such a defect (Fig 1A). Furthermore, as compared to peripheral blood (PB), CB Tregs show a higher expression of Helios, a marker of thymic derived Tregs (Fig 1B) and CB Tregs exert a superior suppression of proliferating conventional T-cells in CFSE assay (Fig 1C). In a xenogeneic mouse model of GVHD, CB Tregs can exert suppression across HLA barrier resulting in survival benefit (Fig 1D); and in another xenogenic lupus model, a single dose of CB Tregs results in significant decrease in circulating effector T cells (Fig 1E) up to 8 weeks. Furthermore, under stressful conditions of continued culture in the presence of inflammatory cytokines including IL15 and IL23, CB Tregs do not secrete IL-17 or express RORγt, a concern of plasticity that plagues PB Tregs (Fig 1F). In fact, CB Tregs increase their secretion of the suppressor cytokine, IL-10 in response to inflammatory stress, which supports our hypothesis, that CB Tregs based adoptive therapy can have wide application and can be used to treat inflammatory disorders. Therefore, we hypothesized that adoptive therapy with CB Tregs can be utilized as treatment for the inflammatory bone marrow failure (BMF) disorders including aplastic anemia, hypoplastic myelodysplasia and primary myelofibrosis. Study Design and Methods: We have now launched a phase I clinical trial examining the role of single infusion of CK0801, an allogeneic, fresh CB Treg product, utilizing novel process development that consist of well-defined qualification criteria for the starting material (cord blood units), parameters for manufacturing and culture-expansion; and well-defined analytic testing and lot release criteria, for the treatment of BMF, where 3 doses are examined: i) dose level I: 1x106 cells/kg; ii) dose level II: 3x106 cells/kg and iii) dose level III: 10x106 cells/kg (NCT03773393). The study follows 3+3 phase I design where 3 patients will be treated at dose level I. If no DLT is observed, then the dose will be escalated to dose level II and if no DLT is observed, then the dose will be escalated to dose level III. If 1 DLT is observed at a dose level, then 3 additional patients will be treated at that level. If no additional DLTs, then that dose level will be defined as MTD. If ≥2DLTs are observed at dose level II or III, then prior dose level is defined as MTD. If ≥2DLTs at dose level I, then DSMB will review and evaluate for study continuation. MTD will be decided when 6 patients are treated at a dose level with < 2 DLTs. The eligibility criteria include: i) diagnosis of aplastic anemia, myelodysplastic syndrome or myelofibrosis, ii) Age > 18 years, iii) cord blood unit matched at HLA 3 out of 6 with the patient for generation of CK0801. The primary endpoint includes dose limiting toxicity (DLT) as defined by i) severe (grade 3 or 4) infusion toxicity within 24 hours; ii) regimen related death within 30 days or iii) severe (grade 3 or 4) cytokine release syndrome within 30 days. The secondary endpoints include i) disease-specific response including complete remission, transfusion independence and hematologic improvement and ii) duration of response. The exploratory endpoints include assessment of PB and BM immune reconstitution and inflammatory cytokines at baseline and scheduled follow ups of Days 0, +1, +3, +7, +14, +21, +30, +60, +90, +180 and +365 in the post-treatment setting. These studies will include T cell compartment analysis: Treg, Effector T cells, T-cell anti-viral activity. Serum will be analyzed for inflammatory cytokines: IL1, IL2, IL4, IL6, IL7, IL8, IL10, IL17, IFN-gamma, ST2, REG3a, OPN, Follistatin, Elafin, TGF-beta. Optional exploratory cytokines: SCF, G-CSF, GM-CSF, HGF, VEGF, SDF1a, MCP1, MCP2, TARC, MIP3a, TECK, CTACK, CCL28, FGF, PDGF, EGF, TGF-α, TLR. The first patient is already consented with a planned total of 9 patients.

Increased frequency of regulatory T cells in pediatric inflammatory bowel disease at diagnosis: a compensative role?

Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis. We investigated two main types of Tregs, the CD4+FOXP3+ and IL-10+ Tr1, in pediatric subjects with inflammatory bowel disease (IBD) both at diagnosis and after the clinical remission. Peripheral blood Tregs were analyzed in 16 children with Crohn's disease (CD), 19 with ulcerative colitis (UC), and 14 healthy controls (HC). Two cocktails of fluoresceinated antibodies were used to discriminate between CD4+FOXP3+ and Tr1. We observed in both CD and UC groups a higher frequency of Tr1 at diagnosis compared to controls, which decreased at follow-up compared to diagnosis, in particular in UC. Similarly, in UC patients the percentage of CD4+FOXP3+ Tregs markedly decreased at follow-up compared to the same patients at diagnosis and compared to HC. The expression of CTLA-4 in CD4+FOXP3+ Tregs increased in both groups at clinical remission. This study shows that IBD children present at diagnosis an increased frequency of circulating Tregs, probably as a compensative reaction to tissue inflammation. During the clinical remission, the Treg frequency diminishes, and concomitantly, their activation status increases. Notwithstanding, the high Treg density at diagnosis is not sufficient to counteract the inflammation in the childhood IBD.

Expression of thyroglobulin by regulatory T cells in thyroid tissue.

The follicles are the minimal functional unit of the thyroid; the morphology and the function of each follicle can vary significantly. However, the reasons for the apparent follicular heterogeneity are poorly understood. Some tissue-resident regulatory T cells (Tregs) have a special phenotype that expresses unique molecules related to local tissue and regulates the tissue functions. The aim of this study was to identify the phenotype of thyroid Tregs and the roles of thyroid Tregs in thyroid physiological regulation. Thyroid tissue and peripheral blood samples were obtained from patients with benign thyroid nodules. Microarray-based gene expression, flow cytometry, immunofluorescence microscopy, and functional analysis of thyroid Tregs were performed. Here, we demonstrated that human thyroid Tregs expressed high level of thyroglobulin (Tg), both gene and protein. The immunofluorescence microscopy of thyroid section showed that the FOXP3+Tg+ cells concentrated in some of the thyroid follicles, at the side of the thyroid follicle. The peripheral blood Tregs expressed minimal levels of Tg, and low levels of Tg could effectively induce peripheral blood Tregs to express Tg, which was independent of thyrotropin simulation. Furthermore, the Tg secreted freely from thyroid Tregs that negatively regulated some thyroid-related genes expression. Our results revealed that the thyroid Tregs was a distinct population of Tregs, which expressed high level of Tg. The thyroid Tregs regulate thyroid function by Tg that is paracrine from the cells.

TNF Receptor Type II Is Critical to the Expansion and Suppressive Function of Regulatory T Cells in Malignant Pleural Effusion from Lung Cancer Patients

Background: Tumor-infiltrating Tregs have been found to express high levels of tumor necrosis factor receptor type II (TNFR2), which renders their suppressive function more pronounced. However, the importance of TNFR2 expression on human Tregs from malignant pleural effusion (MPE) and the efficacy of targeting TNFR2 for the treatment of MPE remain unclear. Methods: The characteristics of TNFR2 expression by Tregs in MPE and peripheral blood were analyzed. The effects of TNF/TNFR2 pathway on Treg proliferation and suppressive function were explored. The relationships between effector T cells and Tregs via the TNF/TNFR2 pathway were evaluated. The effects on the percentage of CTL cells and the concentration of IFN-γ were measured after TNFR2 blockade. Findings: The percentages of Tregs in MPE were much higher than in peripheral blood. TNFR2 appears to be a specific proliferative and functional marker for Tregs in MPE. TNF promotes TNFR2+Treg proliferation and the expression of CTLA-4 and PD-L1. There is also a strong positive correlation between the level of TNF in Th17 cells and Foxp3 in CD4+CD25hi T cells, and IL-17 up-regulates the expression of TNFR2 on Tregs in vitro, suggesting Th17 cells may promote Tregs in the proliferation and function via the TNF/TNFR2 pathway. Moreover, the proportions of CTL cells and IFN-γ concentration were significantly increased after blocking TNFR2. Interpretation: Our results provide laboratorial evidence that TNFR2 might be a novel and effective target in the further clinical treatment for lung cancer patients with MPE. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (NNSFC; No. 81973990, No. 81770090 and No. 81470274) and The Graduates' Innovation Fund, Huazhong University of Science and Technology (TGI, HUST; No. 2019YGSCXCY 067). Declaration of Interests: The authors declare no potential conflicts of interests. Ethics Approval Statement: The study protocol was approved by our institutional review board for human studies, and informed consent was obtained from all subjects.

Integral membrane protein expression of human CD25 on the cell surface of HEK293 cell line: the available cellular model of CD25 positive to facilitate in vitro developing assays.

Typically, CD25 is expressed on the cellular surface of regulatory T (Treg) cells. These cells are significant in regulating the self-tolerance and also preventing the immune system from attacking a person's own tissues and cells. They promote the cancer progression by playing an important role in evading the immune system. Thus, the experimental procedures was aimed to clone and express human CD25 in HEK293 cell line, as the available cellular model, for the purpose of developing assays to facilitate and enhance the studies on an available CD25 positive cell. The secondary RNA structure of CD25 was evaluated by in silico analysis. Then, cDNA of human CD25 were synthesized from isolated total mRNA of cultured and stimulated PBMCs from blood donors. After cloning the cDNA of CD25 into a pcDNA3.1(+) plasmid, using the effective transfection of the recombinant pcDNA3.1(+) in HEK293, qRT-PCR and flow cytometry methods were used to quantitatively evaluate CD25 transcripts and protein level. There was a 4.8 fold increase in transcripts and a 76.2% increase in protein levels of CD25 when comparing the transfected and control cell lines. The genetically engineered HEK293 cell line expressing Treg cell surface marker of CD25 was introduced in this study for the first time. This cell line can be used to overcome the problematic issues for studying Treg cells including low population of Tregs in peripheral blood, low recovery methods for Treg isolation, time-consuming and non-cost benefit methods in the conditions of in vitro cell culture experiments for the studies focused on the binding of IL-2 to CD25.

Efficient short-term expansion of human peripheral blood regulatory T cells for co-culture suppression assay

ABSTRACTRegulatory T cells (Tregs) are a small population of CD4+ lymphocytes and play a key role as suppressors of the immune system, a role that can be identified by employing a co-culture suppression assay. Conventional protocol requires a long period of in vitro expansion of Treg numbers; hence, this study describes an establishment of a co-culture suppression assay using a short-term expansion of peripheral blood (PB) Tregs and autologous T cells (Tconvs) IL-2-pre-cultured in parallel for the same length of time, thereby obviating the need of freeze/thawed autologous Tconvs. Tregs and Tconvs were isolated from PB mononuclear cells employing magnetic bead-aided depletion of CD8+ cells followed by cell sorting of CD4+ CD25high+CD127low- (Treg) and CD4+ CD25-CD127+ (Tconv) cell populations. Following a 3-day co-cultivation period under optimized conditions, Treg suppression activity was monitored by comparing using flow cytometry the number of carboxyfluorescein succinimidyl ester-labeled Tconvs to that of Treg-minus control. The assay allowed significant differentiation between Treg suppression activity of patients with active rheumatoid arthritis and those in remission. This method should be more convenient and time-saving than the conventional Treg suppression assay in current use.

Effects of regulatory T cells, natural killer cells, and natural killer T cells on immunosuppression therapy in patients with recurrent embryo implantation failure

Objective: This retrospective study aims to investigate the effects of immunosuppressive therapy on the expression of regulatory T (Treg) cells, natural killer (NK) cells, and natural killer T (NKT) cells in patients with recurrent embryo implantation failure (RIF). Materials and Methods: In vitro fertilization and embryo transplantation (IVF-ET) at Jinhua Municipal Central Hospital from July 2013 to November 2015 of 30 RIF patients were enrolled into RIF group, and peripheral blood Treg, NK, and NKT were detected before entering the embryo transfer cycle. Meanwhile, 20 normal non-pregnant women were enrolled into control group. Twenty-four patients in RIF group were administered prednisone; Treg, NK, and NKT cell contents were detected twice before and after the embryo transfer cycle, respectively. The remaining six patients with high NK cell contents were treated with gamma globulin via intramuscular injection, and Treg, NK, and NKT cell contents were detected twice before the embryo transfer cycle and after treatment. Results: Treg cell content was significantly lower in RIF group than in control group, while NK cell proportion was significantly higher in RIF group than in control group before treatment. There was no statistical difference in NKT cells. After treatment, expression rate of Treg in RIF group was 7.1 ± 1.8%, which compared with that before treatment (2.8±1.6%) was significantly higher (p < 0.01). In addition, NK cell proportion was significantly lower than pretherapy (20.9 ± 3.6% vs. 38.6 ± 8.1%, p < 0.05) and NKT cell percentage did not present the obvious difference before and after treatment. Conclusion: The occurrence of RIF may be related to the decrease in Treg expression and increase of NK cells. Immunotherapy can upregulate the expression of Treg and decrease NK cell proportion, thereby regulating maternal fetal immune tolerance and reducing the rejection effect of NK cells on fetal foreign bodies, which are conducive to embryo implantation.

Circulating Th1, Th2, Th17, Treg, and PD-1 Levels in Patients with Brucellosis.

Brucella is an intracellular infection bacterium; the pathogenesis of Brucella and chronicity of infection may be related to the immune response of T cells. T lymphocytes mainly participate in cellular immune response. The extent of different T cell subsets imbalanced and their function dysregulated in patients with brucellosis remain not explicit. We grouped patients at different stages (acute, chronic, and convalescent). The frequencies of Th1, Th2, Th17, Treg, and PD-1 (programmed cell death protein 1) in peripheral blood were examined by flow cytometry, and the expressions of T lymphocyte cytokines in serum were detected by cytometric bead array. Th1, Th17, and Treg cell immunity was predominant in the acute stage, while Th2, Th17, and Treg cell immunity was predominant in the chronic stage. The expressions of PD-1 on CD4+ and CD8+ T lymphocytes were significantly different in acute and chronic patients. The percentages of Th1 cells in convalescent patients were still higher than those in healthy controls within one year after withdrawal. The expression of T lymphocyte cytokines in serum was different in patients at different stages. These results indicate that peripheral T lymphocyte immunity was involved in patients with brucellosis and represents a target for the preclinical and clinical assessment of novel immunomodulating therapeutics. The patients' immune function had not completely recovered in a short period of time during convalescence, so long-term follow-up of convalescent patients is needed.

The Dynamic Changes in Soluble CD30 and Regulatory T Cells Before and After Solid Organ Transplantations: A Pilot Study.

Among multiple parameters, applied in the immunologic monitoring of transplantation, the levels of serum soluble CD30 (sCD30) and peripheral regulatory T cells (Tregs) are very promising. These are relatively new biomarkers, considered to reflect immune activation and tolerance in solid organ transplantation. Results are shown here from a preliminary study on the relevance of sCD30 and Tregs in the monitoring of the early post-transplantation period. Sixteen patients with chronic liver or kidney disease were examined. Nine of them were further selected for transplantation. Follow-up of sCD30 and Tregs was carried out during the first month after transplantation. Until day 30 (D30) after transplantation, a progressive decrease in sCD30 levels was observed in all patients. Conversely, the dynamic of Tregs was dependent on the transplanted organ: in liver recipients, an increase of Tregs was detected at day 7 (D7) followed by a gradual decrease until D30, whereas in kidney recipients, a sustained downward trend starting on D7 was observed. In liver recipients, the increase in Tregs preceded albumin normalization, whereas in kidney recipients, sCD30 was found to have predictive significance for the creatinine levels. Our results demonstrated that peripheral blood sCD30 and Tregs are valuable parameters in the immunologic monitoring of transplanted patients.

Activation of the DR3-TL1A Axis in Donor Mice Leads to Regulatory T Cell Expansion and Activation With Reduction in Graft-Versus-Host Disease.

Death receptor 3 (DR3) is a tumor necrosis factor receptor superfamily member (TNFRSF25), which is minimally expressed on resting conventional T cells (though readily inducible upon cell activation), yet highly expressed on resting FoxP3+ regulatory T cells (Treg). We recently demonstrated that activation of DR3 with an agonistic antibody (4C12) leads to selective expansion and activation of Treg in healthy mice and suppression of graft-versus-host disease (GVHD) in recipient mice when donor mice are treated. However, given the long antibody half-life and concomitant safety concerns, along with the lack of a humanized agonistic antibody to DR3, both human and murine fusion proteins incorporating the natural DR3 ligand TL1A (TL1A-Ig) have been developed. Herein, we show that DR3 activation with 4C12 or with TL1A-Ig, with or without the addition of low dose IL-2 to the treatment regimen, led to a significant expansion of murine Treg in spleen, lymph nodes, and peripheral blood. Bioluminescent imaging revealed peak Treg expansion around day 7–8, with return to near baseline after 2–3 weeks. In addition to expansion, all DR3 agonist treatment regimens led to increased activation of Tregs, with significant upregulation of the activation markers ICOS, KLRG-1, PD-1, and CD103, and the proliferation marker Ki-67. The near absence of activated Treg populations in control treated spleens was also detected on tSNE analysis of flow cytometry data. Subtly different patterns of splenic Treg activation by the different DR3 agonists were noted in both tSNE analysis of flow cytometry data and RNA-sequencing analysis. However, upregulation of gene transcripts which play important roles in cell proliferation, trafficking, activation, and effector function were observed regardless of the DR3 agonist treatment regimen used. In the major MHC-mismatch model of hematopoietic cell transplantation, DR3 agonist-mediated expansion and activation of Tregs in donor mice led to a significant improvement in GVHD in recipient mice. These data provide important preclinical information regarding the outcome of DR3 activation with an agonistic antibody or natural ligand and provide insight into the therapeutic use of this approach to reduce GVHD in recipients and improve outcomes of hematopoietic cell transplantation.

Connecting blood and intratumoral Treg cell activity in predicting future relapse in breast cancer.

Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA-FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.

Abstract LB-139: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer

Abstract Background: Regulatory T cells (Tregs) are potent immunosuppressive cells of the immune system. They facilitate the survival and progression of tumor through their ability to suppress the antitumor immunity. The expression of tumor necrosis factor receptor type II (TNFR2) on Tregs is reported to identify the maximally suppressive Treg population in both mice and human. According to our previous research, expression of TNFR2 appeared to correlate better than CD25+ and CD127− with FoxP3 expression. Aim: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer are needed to be studied. Methods: The expression and phenotype of TNFR2 + Tregs in peripheral blood and tumor tissues of 33 cases of gastric cancer patients and in peripheral blood of 15 normal controls were detected by flow cytometry. The ratio of Foxp3+Tregs and TNFR2+Tregs of 145 cases of gastric cancer patients was analyzed by multicolor immunofluorescence assay. We analyzed their relationship with gastric cancer stage, metastasis and other clinical indicators by clinical and pathological data. Results: Flow cytometry showed that the expression of TNFR2 in human peripheral blood Tregs was significantly higher than that in conventional T cells (Tconvs) in peripheral blood CD4 + T cell subsets of gastric cancer patients and healthy individuals (P<0.05). The proportion of TNFR2 + cells in peripheral blood Tregs cells was significantly higher in patients with gastric cancer than in normal subjects (P <0.05). Tregs and Tconvs cells expressed very low level of CTLA-4 and TIM-3 in both normal controls and gastric cancer patients. Multicolour immunofluorescence showed that the ratio of Foxp3 + Tregs and TNFR2 + Tregs was increased in the tumor tissue, and TNFR2 + Tregs cells showed a tendency to increase with tumor progression and lymphatic metastasis. Higher levels of infiltration of TNFR2+ Tregs cells are significantly associated with patients’ OS (P<0.05) and DFS (P<0.05). Conclusion: Our findings revealed that TNFR2 could be a promising molecular marker associated with poor prognosis. The mechanism by which TNFR2+Tregs promote tumor survival and progression in gastric cancer is still needed further research. Citation Format: Yang Qu, Gang Zhao, Hui Li. The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-139.

Abstract 4492: Humanized anti-EGFR antibody panitumumab inhibits tumor growth of inflammatory breast cancer by inducing antitumor immunity

Abstract Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no approved targeted therapies specifically for IBC have been approved. The epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC, with a reported pathological complete response rate of 42%. The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Therefore, delineating the cross-talk between EGFR-targeted therapy and TME components could lead to more efficient combination regimens and novel clinical trial designs for IBC. Because an immunocompetent IBC animal model is not available for our study, we established an IBC SUM149 immunocompetent mouse model (SUM149-hu-NSG-SGM3) for such studies. Here we report the effects of panitumumab (PmAb), a humanized monoclonal anti-EGFR antibody, on tumor growth and on the TME in the SUM149-hu-NSG-SMG3 mouse model. Methods: SUM149 cells were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with human hematopoietic stem cells. SUM149 tumor growth in mice treated with either isotype IgG2 or PmAb was measured. We used flow cytometry to measure the percentages of human immune cells in the peripheral blood and tumor tissues from mice treated with IgG2 and PmAb. The effects of PmAb treatment on the expression and distribution of TME components in tumor tissues was examined by multiplex immunohistochemical staining. Cytokine expression in tumor tissues from IgG2- and PmAb-treated mice was measured by cytokine antibody array, and the changes in cytokines expression were validated with RT-PCR. Results: PmAb treatment, compared with IgG2 treatment, significantly reduced SUM149 tumor growth. Flow cytometry analysis showed infiltration of lymphocytes in SUM149 tumors with an observed increase in the level of CD8+ T cells and a reduced level of Tregs in both peripheral blood and tumor tissues from PmAb-treated mice compared with IgG2-treated mice. Cytokine array and RT-PCR showed that PmAb treatment increased expression of cytokines that function as a chemoattractant for T cells, natural killer cells, and monocytes, including CXCL10, CCL5, CCL4, and CXCL9. In contrast, PmAb treatment reduced expression of cytokines involved in antitumor immune suppression, including osteoprotegerin (OPG), CCL2, CXCL5, and CCL20. Conclusion: PmAb treatment reduced IBC tumor growth by increasing cytotoxic T cells and reducing immunosuppressive Treg cells. Our data also suggest that the combination of PmAb with an immune checkpoint inhibitor may potentiate the efficacy of anti-EGFR therapy in IBC. A study of the therapeutic efficacy of the PmAb and anti-PD-L1 combination in the SUM149-hu-NSG-SMG3 mouse model is in progress. Citation Format: Xiaoping Wang, Shan Shao, Takashi Semba, Troy Pearson, James M. Re, Debu Tripathy, Naoto Ueno. Humanized anti-EGFR antibody panitumumab inhibits tumor growth of inflammatory breast cancer by inducing antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4492.

The effect of maternal use of telbivudine on neonatal CD4+CD25+ regulatory T cells for the prevention of mother-to-child transmission of hepatitis B virus

Antiviral treatment could block mother-to-child transmission (MTCT) of hepatitis B virus (HBV) effectively. We examined whether maternal use of telbivudine (LdT) could decrease the proportion of CD4+CD25+ regulatory T cells and explored the immunological mechanism. A total of 89 pregnant women with HBsAg positive were enrolled, where 30 pregnant women with HBeAg negative (viral load<106 IU/ml) and the other 59 pregnant women with HBeAg positive (viral load≥106 IU/ml) were followed in the study. The women with high viral load were divided to the LdT-treated group where they were prescribed with 600mg LdT daily (29 cases) during the third trimester of pregnancy or to the non-treated group (30 cases) on a voluntary basis. Samples of neonates were taken for analyzing CD4+CD25+ Tregs with flow cytometric techniques. A more significant decrease in the proportion of CD4+CD25+Tregs in neonatal peripheral blood had been observed with maternal use of telbivudine (2.8%±1.1%) than those without any treatment (7.0%±1.6%, P< 0.01). None of the infants in the LdT-treated group were HBsAg positive at 7 months of age. In addition, neonates whose mothers received telbivudine had a significant improvement in cellular immune function, as indicated by the proportion of CD8+ T cells. For HBV carriers with high viral load, maternal use of LdT may be useful in regulating neonatal immune function involved in mother-to-child transmission of hepatitis B virus.

Regulatory T cells and IgE expression in duodenal mucosa of Strongyloides stercoralis and human T lymphotropic virus type 1 co-infected patients.

BackgroundStrongyloides stercoralis is an intestinal nematode unique in its ability to replicate in the human host, allowing ongoing cycles of autoinfection, persisting for decades within the same host. Although usually asymptomatic, overwhelming infections can occur in Strongyloides and HTLV-1 co-infected individuals (SS/HTLV-1). Regulatory T cells (Tregs) are able to blunt specific Th2 responses necessary to control the parasite. We previously reported that peripheral blood Tregs are increased in SS/HTLV-1 and correlate with low Th2 responses. We hypothesized that Tregs are also increased at the site of infection in duodenal mucosa.MethodsParaffin embedded duodenal biopsies were obtained from 10 SS/HTLV-1 patients, 3 controls with non-parasitic chronic duodenitis, and 2 healthy controls. Immunohistochemistry was performed using monoclonal antibodies against human CD3, CD8, IgE and FoxP3. The number of cells were counted using a conventional light microscope. The number of CD3+, CD8+, FoxP3+ and IgE positive cells per 0.35 mm2 was measured using ImagePro Plus software comparing areas adjacent or distant from parasite material.ResultsIn patients with SS/HTLV-1, T lymphocyte counts and CD8+ cells were lower in areas adjacent to the parasite compared to non-adjacent areas (CD3+: adjacent: 6.5 [Interquartile range (IQR: 2.8–12.3)]; non-adjacent: 24.5 [IQR: 20.9–34.4]; Mann-Whitney p = 0.0003; CD8+: adjacent: 4.5 [IQR: 2.3–11.8]; non-adjacent: 21 [IQR: 15.3–42.9]; Mann-Whitney p = 0.0011). Tregs cells in the intestines (FoxP3+ expressing cells) were increased in patients with SS/HTLV-1 compared with patients with chronic duodenitis (SS/HTLV-1: 1.5 [IQR: 0.7–2.3]; duodenitis controls: 0 [range 0–0.7]; healthy controls: 0; Mann-Whitney p = 0.034). There was also a trend towards fewer eosinophils adjacent to the parasites. Among SS/HTLV-1 patients the number of IgE expressing cells was increased for in areas not adjacent to the parasite compared to non-adjacent areas (ANOVA, p = 0.001).ConclusionsOur data shows increased Treg cell numbers localized adjacent to the parasites in the duodenum SS/HTLV-1 patients. In addition, other T lymphocytes and IgE expressing cells were decreased adjacent to the parasites, suggesting an important role for Tregs in down-regulating local parasite effector responses.

PB2049 DETECTION OF TREGS BY FLOW CYTOMETRY IN THE DIAGNOSIS OF HODGKIN LYMPHOMA

Background:Regulatory T (Tregs) cells constitute a subset of CD4+ lymphocytes that play a key role in the regulation of self‐tolerance and the maintenance of tissue homeostasis. It seems well established that in cancer patients, accumulation of Tregs both in tissue and peripheral blood (PB) is associated with the suppression of anti‐tumor immune effector functions, tumor progression and poor prognosis. Flow cytometry (FC) is an effective and rapid tool to quantify Tregs and to evaluate the immunological characteristics of the individual patient.Aims:We wanted to verify the usefulness of FC in quantifying Tregs in the diagnosis of the Hodgkin lymphoma (HL), where the contribution of the FC is limited, and to demonstrate if this subset T CD4+ could help to define the microenvironment of the HL characterized by a low number of malignant cells surrounded by an abundant reactive infiltrate of immune cells rich of T lymphocyte.Methods:We investigated Tregs in the involved lymph nodes (LN) of 15 HL patients and 15 patients with reactive lymph‐nodes. Peripheral presence of Tregs was analysed in PB obtained from the same patients and 15 age‐matched healthy donors (controls). Analyses were performed using FACSCanto flow cytometer (BD Biosciences) and FACSDiva software. The Tregs were calculated both as percentage of total CD4 T lymphocytes. Tregs were analyzed according to the commonly used Treg definitions FOXP3+ CD4highCD25high, characterized by low expression of CD127 (IL‐7 receptor) and negativity for CD45RA (to discriminate activate cells from naïve cells). To improve the specificity of the analysis, the gating strategy considered Tregs positive for CD39 (marker of homing to inflamed regions), positive for CD62L (L‐selectin) and negative for CD26. Differences were considered statistically significant at with p‐value of &lt; 0.05. Statistical analysis was performed using PRISM‐Graph Pad software version 6.3.Results:The percentage of Tregs was significantly higher in biopsy of HL patients compared with reactive lymph nodes’ patients: median (3,400 ± 0,8800) vs (1,130 ± 0,1647) p = 0,0430. Instead no statistically significant differences were found comparing circulating Tregs values, between the HL patients group vs patients with reactive lymph‐nodes and vs healthy controls: median (0,2857 ± 0,05728) vs (0,192 ± 0,02215) vs (0,2500 ± 0,052) p = 0,3722. To identify potential differences in proportions of Tregs between patients with HL, reactive and healthy controls, we included detailed analysis of Treg immunophenotypes.Summary/Conclusion:: Up to now, Tregs were evaluated mostly in solid cancer, while studies evaluating the numbers of Tregs in patients with lymphomas are few and with controversial results in fact it may be associated with either negative or positive outcome. Our work was focused on patients with HL where we found an increased number of Tregs that seems to be context‐dependent. In fact in HL lymph nodes the number of Tregs were higher compared to reactive ones. Otherwise no statistical differences were found in percentages of Treg among circulating CD4+ lymphocytes in patients compared with the control group. So, detecting Tregs during diagnosis in LN can help to discriminate HL from reactive ones. Our data could suggest a routine application of the FC in the diagnosis of HL to characterize specifically the microenvironment. Moreover we highlight the need to request a confirmation in larger patient population to confirm our data in PB.image

Soluble glucocorticoid-induced tumor necrosis factor receptor regulates Helios expression in myasthenia gravis

BackgroundHelios is important for functional and phenotype stability of regulatory T cells (Tregs). However, the role of Helios in autoimmune diseases and its regulation remains unclear. This study aimed to investigate the role of Helios+ Tregs in myasthenia gravis (MG) and glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) in the modulation of Helios.MethodMulticolor flow cytometry was performed to analyze Helios+ Tregs in peripheral blood from MG patients and healthy donors (HDs). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of soluble GITRL/GITR in plasma. Tregs were isolated via magnetic separation and treated with recombinant GITRL and GITR-Fc. Membrane GITRL on Tregs and expression of Helios and other markers (FOXP3, CD25, CD39, CTLA-4, PD-L1 and IL-10) involved in immunosuppressive activity were determined by flow cytometry.ResultBoth Helios+ Tregs and soluble GITR were decreased in generalized MG (GMG) patients (n = 14), compared with HDs (n = 14) and ocular MG (OMG) patients (n = 16). Helios+ Tregs possessed greater immunosuppressive capacity compared to Helios− Tregs. Further analysis indicates soluble GITR was negatively correlated with quantitative MG score and promoted Helios expression and enhanced function of Tregs independently of membrane GITRL.ConclusionThis work demonstrates abnormal changes in Helios+ Tregs and soluble GITR in MG, as well as direct regulation of Helios by GITR in the context of Tregs. This work provides new insight into the role of GITR in the regulatory pathway of Helios and pathogenesis of MG.

Roles of regulatory T cells in the pathogenesis of pediatric aplastic anemia

The pathogenesis of aplastic anemia (AA) in children is not clear. This study was conducted to investigate the changes in the proportion and function of regulatory T cells (Tregs) in pediatric AA. The proportion of Tregs, mRNA levels of transcription factors, and concentrations of cytokines were measured by flow cytometry, reverse transcription-PCR, and enzyme-linked immunosorbent assay, respectively. Tregs were co-cultured with effector T cells (Teff) to evaluate the function of Tregs. The proportion of Tregs after immunosuppressive therapy (IST) in pediatric AA was monitored dynamically. Compared to the control, the proportions of Tregs in peripheral blood and bone marrow lymphocytes of the untreated AA group were lower (1.31% ± 0.73% vs. 3.16% ± 0.92%, 1.49% ± 0.81% vs. 3.06% ± 0.82%, respectively, p < 0.001). The mRNA levels of FOXP3 and STAT3 in the AA group were lower (p = 0.014; p < 0.001). However, the mRNA levels of T-BET did not significantly differ between groups. The concentration of interferon-γ and interleukin-17 in the AA group were higher (p = 0.004; p = 0.003), whereas the concentration of TGF-β decreased (p = 0.044). The immunosuppressive function of Tregs was impaired in the AA group. After IST, the proportion of Tregs was significantly lower than that in the control. The proportion of Tregs at the time of diagnosis in the nonresponsive group was lower than that in the responsive group, but the difference was not significant. Treg levels were significantly decreased and were functionally impaired at the time of diagnosis of pediatric AA. However, there was no significant change in Tregs at the resolution of AA.

Interleukin-37 Inhibits the Imbalance Between T Helper 17 Cells and Regulatory T Cells in Hand, Foot, and Mouth Disease.

The aim of this study was to explore the role of interleukin-37 (IL-37) in imbalance of T helper (Th)17/regulatory T cells (Tregs) in hand, foot, and mouth disease (HFMD). The proportions of CD4+ IL-17A+ Th17 cells and CD4+ CD25+Foxp3+ Tregs in peripheral blood or peripheral blood mononuclear cells (PBMCs) from HFMD patients and healthy controls were measured by fluorescence activated cell sorter. The level of IL-37, IL-10, IL-17A, IL-23, and transforming growth factor β1 (TGF-β1) in serum or PBMCs of HFMD patients and control subjects were detected using enzyme-linked immunosorbent assay. Results showed that Th17 cells proportion and IL-17A and IL-23 levels were highly increased, whereas Tregs proportion and IL-10 and TGF-β1 levels were significantly decreased in HFMD patients. Moreover, IL-37 stimulation elevated Tregs proportion but reduced Th17 cell proportion in subjects with HFMD. On the contrary, we found methylprednisolone pulse therapy/methylprednisolone combinated with intravenous gamma globulin inhibits Th17/Treg imbalance through upregulation of IL-37 in HFMD. In conclusion, IL-37 inhibits the imbalance of Th17/Tregs in HFMD.

Effect of montelukast combined with methylprednisolone for the treatment of mycoplasma pneumonia.

ObjectiveTo study the effect of the leukotriene receptor agonist montelukast combined with methylprednisolone on inflammatory response and peripheral blood lymphocyte subset content in children with mycoplasma pneumonia.MethodsSeventy-four children were enrolled and randomly divided into a standard treatment group and a montelukast plus methylprednisolone group. Serum levels of inflammatory cytokines and corresponding cytokines of T lymphocyte subsets were measured, and peripheral blood was collected to determine the T cell subset content.ResultsAt 3 days and 7 days after treatment, serum MCP-1, PCT, ICAM-1, CXCL8, CRP, IFN-γ, and IL-17 levels and peripheral blood Th1 and Th17 content were significantly decreased in both groups, while serum IL-4 and TGF-β levels and peripheral blood Treg and Th2 content were significantly increased. However, serum MCP-1, PCT, ICAM-1, CXCL8, CRP, IFN-γ, and IL-17 levels and peripheral blood Th1 and Th17 content were significantly lower while serum IL-4 and TGF-β levels and peripheral blood Treg and Th2 content were significantly higher in the montelukast plus methylprednisolone group compared with the control group.ConclusionMontelukast combined with methylprednisolone for the treatment of mycoplasma pneumonia can inhibit inflammatory responses and regulate levels of Th1/Th2 and Th17/Treg cells.