scholarly journals 840 A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) can also identify immunosuppressive regulatory T (Tregs) cells and down regulate Treg-mediated immunosuppression

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A881-A881
Author(s):  
Kwong Tsang ◽  
Massimo Fantini ◽  
Christopher Cole ◽  
Christina Annunziata ◽  
Philip Arlen
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cancer Patients ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Institutional Review Board ◽  
Human Pbmcs ◽  
Flow Cytometry Analysis ◽  
Institutional Review ◽  
Healthy Donors

BackgroundNEO-201 is an IgG1 mAb reactive against many different human carcinomas expressing the NEO-201 antigen, but not against most normal epithelial tissues. NEO-201 can mediate antitumor activity against tumor cells through multiple mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 immune checkpoint inhibitory pathway. In addition to solid tumors, the NEO-201 target has also been found on human hematopoietic cells. Flow cytometry analysis has demonstrated that 98.9% of CD15+ granulocytes and about 4.6% of CD4+ T cells were positive for NEO-201 staining. No binding was observed with NEO-201 with respect to B cells, NK cells, monocytes, or CD8+ T cells and a majority of CD4+ T cells. This study was designed to characterize the subset of NEO-201+ binding CD4+ T cells and to evaluate the reactivity of NEO-201 to this subset of hematopoietic cells.MethodsPhenotypic analysis of PBMCs from healthy donors and cancer patients were performed by flow cytometry. Reagents used for flow cytometry were antibodies against human CD4, CD127, CD25, CD15s, FOXP3, CD39, CD73 and anti-NEO-201 mAb. Functional assays were performed using a flow cytometry based on CDC assay. Treg cells, isolated from 3 healthy donors using the EasySep™ Human CD4+CD127lowCD25+ Regulatory T (Treg) Cell Isolation Kit were used as target cells.ResultsFlow cytometry analysis revealed that NEO-201+CD4+ T cells were also CD25+/CD127-/FOXP3+/CD15s+ in human PBMCs from both healthy donors and cancer patients. NEO-201 also binds to CD4+/CD25+/CD127-/Foxp3+/CD15s+ cells in Treg cells isolated from human PBMCs using a commercial isolation kit. NEO-201+CD4+ T cells were also CD25+/CD127-/FOXP3+/CD39+. In addition, NEO-201 mAb can kill these isolated Treg cells through CDC.ConclusionsThis study demonstrated that the small subset of NEO-201+CD4+ T cell in human PBMCs are highly suppressive Treg cells and NEO-201 can be used as a novel marker to identify functionally suppressive Treg cells, Furthermore, NEO-201 can kill Treg cells through CDC, presenting an opportunity for therapeutic intervention to increase anti-tumor immunity.Ethics ApprovalThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of National Institutes of Health (NIH). All subjects gave their informed consent for inclusion before they participated in the study.PBMCs from healthy volunteer donors were utilized under the appropriate Institutional Review Board approval (protocol code NCT00001846, first approved Nov 4, 1999; latest update 11/10/2020).PBMCs from cancer patients were utilized under the appropriate Institutional Review Board approval (protocol code NCT03476681, first approved 03/26/2018; latest update 01/08/2020).ConsentInformed consent was obtained from all subjects involved in the study.

IL-10 Contributes to the Development of Immunosuppressive CD14+HLA-DRlow/- monocytes in B-Cell Non-Hodgkin’s Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2979-2979
Author(s):  
Bing Xiu ◽  
Yi Lin ◽  
Deanna Grote ◽  
Steven Ziesmer ◽  
Michael Gustafson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Poor Prognosis ◽  
Underlying Mechanism ◽  
Monocyte Count ◽  
Newly Diagnosed ◽  
Color Flow ◽  
Healthy Donors

Abstract Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients. Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry. Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays. Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell RNA Sequencing Unveils the Hallmarks of Populations at Different Stages of HTLV-1 Infection

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3323-3323
Author(s):  
Yan Huang ◽  
Peifang Jiang ◽  
Jiazheng Li ◽  
Yanxin Chen ◽  
Zhengjun Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Cd4 T Cell ◽  
Expression Level ◽  
Normal People ◽  
Healthy Donors

Abstract Background Adult T-cell leukemia-lymphoma (ATL) is an aggressive mature T-cell neoplasm caused by human T -cell leukemia virus type 1 (HTLV-1). Up to 5% of infected individuals develop to ATL. HTLV-1 preferentially infects CD4 + T cells, and stimulates cell proliferation and prevents cell death by apoptosis. The viral oncogene-encoded proteins, Tax and HBZ, play important roles in viral infection and cell immortalization. However, the host factor of developing from carrier to patient is not clear. Results To characterize the heterogeneity of ATL patients, we performed single-cell RNA-sequencing (10x Genomics) analysis on single cell suspensions isolated from PBMCs of nine samples, including three ATL patients, three HTLV-1 asymptomatic carriers as well as three healthy donors (HD). We acquired 82977 high-quality cells with a median of 1718 genes detected per cell. Unsupervised clustering using Seurat followed by visualization in t-Stochastic Neighbor Embedding (t-SNE) identified 29 distinct cell clusters (Figure 1A). The single cell profiling of ATL patients were significantly different from that of carriers and healthy donors, while the latter two had little difference (Figure 1B). Based on singleR packages and marker genes of each cluster, 4 major cell populations (T cells, NK cells, B cells and myeloid cells) and other rare cell types were annotated, such as erythrocyte cluster and eosinophils cluster. We observed an enrichment of CD4 + T cell from patients in 4 cluster (Figure 1C), which proportion of cells was higher than that of carriers and healthy donors. According to cell type annotation, cells from cluster 11 were CD4 + CD25 + Foxp3 + Treg cells. Based on the increasing proportion of cluster 11 in healthy people, carriers and patients, without significant statistical differences, we assumed that Foxp3 + Treg cells were involved in the evolution of ATL tumor cells. That was identical with published literatures. To investigate the differences between tumor and normal CD4 + T cell, the gene expression was compared among 7 clusters of CD4 + T cell from three groups. Using a threshold of p value &lt; 0.05 and | fold change| &gt;2. Through integrated analysis, we identified 26 commonly upregulated genes (gene expression level: patients &gt; carriers &gt; HD) and 9 downregulated genes (gene expression level: patients &lt; carriers &lt; HD. To further analyze the biological function of the common DEGs, gene ontology (GO) analysis showed that these genes could be mainly categorized into plasma membrane and protein binding. Subsequently, we validated the mRNA expression level of upregulated common DEGs among three groups by qRT-PCR. The isolated CD4 + T cell using CD4 microbeads of a total of 6 patients, 3 carriers and 9 normal specimens were included. The result showed that the mRNA expression levels of gene CADM1 and RGS13 in patients were higher than those in carriers and healthy donors, although there was no statistical difference between patients and carriers, and the expression levels of carriers tended to be higher than those in normal people (Figure 1D and E). Previously, CADM1 has been revealed to be highly expressed in HTLV-1-infected CD4 + T cells. Our study confirmed this result by single-cell profiling. RGS13, a member of the regulators of G protein signaling (RGS) family, participates in cellular communication. The role of RGS13 in ATL needs to be investigated. Conclusions This study is the first time to analyze the single-cell RNA level of ATL patients, HTLV-1 virus carriers and normal people. The peripheral blood cell composition of the patient is significantly different from that of the carriers and healthy donors, while it is similar between carriers and normal people. CD4 + T cells are the main cell population of patients. The proportion of CD4 + CD25 + Foxp3 + Treg cells increased gradually in healthy people, carriers and patients. DEGs analysis showed that CADM1 and RGS13 were highly expressed in CD4 + T cells of patients, followed by carriers, validated by 18 clinical samples. However, the molecular mechanism of RGS13 in the process from HTLV-1 infection to ATL needs to be further studied. Figure 1 Figure 1. Disclosures Hu: Astellas Pharma, Inc.: Research Funding.

Induction of regulatory T cell–resistant helper CD4+ T cells by bacterial vector

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1404-1412 ◽  
Author(s):  
Hiroyoshi Nishikawa ◽  
Takemasa Tsuji ◽  
Elke Jäger ◽  
Gabriel Briones ◽  
Gerd Ritter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Th1 Cells ◽  
High Avidity ◽  
T Helper 1 ◽  
Bacterial Vector ◽  
Healthy Donors

Abstract Salmonella typhimurium engineered to deliver cancer/testis antigen NY-ESO-1 through type III secretion (S typhimurium–NY-ESO-1) was shown to be an efficient cancer vaccine construct in mice and to stimulate NY-ESO-1–specific CD8+/CD4+ T cells in vitro in patients with cancer with NY-ESO-1 spontaneous immunity. We also showed that individuals without spontaneous immunity to NY-ESO-1 had specific CD4+ T-cell precursors with high avidity to NY-ESO-1 under tight control by CD4+CD25+ regulatory T (Treg) cells. We now found that in healthy donors and patients with melanoma without NY-ESO-1 spontaneous immunity, S typhimurium–NY-ESO-1 elicits CD4+ T helper 1 (Th1) cells in vitro recognizing naturally processed antigen from these high-avidity NY-ESO-1–specific naive precursors. In contrast to peptide stimulation, induction of specific Th1 cells with S typhimurium–NY-ESO-1 did not require in vitro depletion of CD4+CD25+ Treg cells, and this prevailing effect was partially blocked by disruption of interleukin-6 or glucocorticoid-induced TNF receptor (GITR) signals. Furthermore, S typhimurium–induced Th1 cells had higher GITR expression than peptide-induced Th1 cells and were resistant to suppression by CD4+CD25+ Treg cells in a GITR-dependent fashion. We propose that S typhimurium–NY-ESO-1 induces antigen-specific T-cell responses that are resistant to suppression by CD4+CD25+ Treg cells.

scholarly journals Insufficient Phosphorylation of STAT5 in Tregs Inhibits the Expression of BLIMP-1, Leading to a Lack of Tregs in Pediatric Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2183-2183
Author(s):  
Lifen Huang ◽  
Junbin Huang ◽  
Chengming Zhu ◽  
Hongman Xue ◽  
Chi Kong Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Low Expression

Abstract Aplastic anemia (AA) is a group of bone marrow failure diseases characterized by three-line blood cell reduction and decreased myeloproliferation. It is believed that T cell immune disorder is the leading cause of the disease, especially the number and functional damage of regulatory T cells (Tregs). BLIMP-1 is a transcription factor encoded by PRDM1 gene, which is indispensable for Tregs. The expression of BLIMP-1 is mainly induced by the IL-2/STAT5 signaling pathway. However, the level of phosphorylation of STAT5 and the expression of BLIMP-1 in Tregs from patients with AA has not been revealed, and the mechanism of Tregs damage in AA has not yet been clarified. In the present study, we collected peripheral blood from 10 newly diagnosed AA children and 10 age-matched healthy donors. We observed that the ratio of Tregs/lymphocytes and Tregs/CD4 + T cells decreased significantly in AA patients, compared with healthy controls by flow cytometry. In addition, we found significantly elevated levels of inflammatory cytokines IL-2, IL-6, and IFN-γ, but decreased levels of anti-inflammatory cytokines IL-10 and TGF-β in plasma of children with AA, compared with healthy controls. Quantitative real-time PCR showed decreased transcriptional level of BLIMP-1 in peripheral blood mononuclear cells (PBMC) from children with AA, compared with healthy donors. We used flow cytometry to detect the protein level of BLIMP-1 in Tregs and found that the level of BLIMP-1 in Tregs in the peripheral blood of children with AA was significantly lower than that of healthy donors. The correlation analysis showed that the percentage of BLIMP-1 + Tregs was positively correlated with the ratio of Tregs/CD4 + T cells (r=0.829, p＜0.001), the plasma level of IL-10 (r=0.492, p=0.027), and TGF-β (r=0.482, p=0.030), suggesting that low expression level of BLIMP-1 in Tregs may lead to decreased number of Tregs in peripheral blood and declined levels of IL-10 and TGF-β in children with AA. When stimulated IL-2, the level of pSTAT5 in CD4 + T cells of children with AA was significantly reduced compared with that of healthy donors. The level of pSTAT5 in CD4 + T cells was also positively correlated with the ratio of Tregs/CD4 + T cells (r= 0.575, p= 0.008) and the expression of BLIMP-1 in Tregs (r=0.693, p＜0.0001)，suggesting that STAT5 signal is poorly activated in pediatric AA, and it may be an important cause for the low expression of BLIMP-1 in Tregs and the decrease in the number of Tregs in children with AA. Furthermore, we constructed an AA mouse model by co-administering IFN-γ and busulfan for 10 consecutive days. These mice exhibited decreased ratio of Tregs/CD4 +T cells in the spleen and lower BLIMP-1 in Tregs, compared with controls. Also, we isolated Tregs with immunomagnetic beads from spleens of mice and observed that the level of IL-2-stimulated pSTAT5 was lower in isolated Tregs from AA mice than controls. These phenotypes were partially rescued by intervention of IL-2-JES6-1, an antibody complex tends to promote the proliferation of Tregs in mice, while inhibiting the proliferation of effector T cells. IL-2-JES6-1 increased the level of pSTAT5 and the expression of BLIMP-1 in Tregs from spleen of the AA mice, and elevated the ratio of Tregs/CD4 + T cells in the spleen. In conclusion, we found that Tregs from AA patients have impaired phosphorylation of STAT5 and insufficient expression of BLIMP-1, which may contribute to the pathogenesis of AA. Disclosures No relevant conflicts of interest to declare.

Depot Formulation of Interleukin-2 as an Immunotheraphy for B-Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1398-1398
Author(s):  
Sofia Grille ◽  
Andreina Brugnini Lic ◽  
Esteban Corley ◽  
Martha Nese ◽  
Jose Alejandro Chabalgoity ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
Interleukin 2 ◽  
B Cell Lymphoma ◽  
Treg Cells ◽  
Depot Formulation

Abstract Relapses occurring in most lymphoma patients after treatment highlight the need for effective immunotheraphy approaches. Induction of tumor-specific adaptive immunity in cancer patients is an ideal approach because its selectivity and memory for the same tumors eventually prevent the relapse after conventional therapies. Vaccination with cytokines and tumor cells represents an attractive therapeutic approach for patients with B-cell lymphoma. Interleukin-2 (IL-2) has a wide range of immunologic effects, including the activation of cytotoxic T cells and natural killer cells (NK). The therapeutic use of IL-2 has generated substantial interest based on its ability to induce the regression of metastatic renal cell carcinomas and malignant melanomas tumors in humans. However, toxicity associated with systemic administration of large doses of cytokine is a major drawback for clinical application. In this work, we investigated whether in vivo vaccination with a cytokine-based immunotherapy using IL-2 adsorbed in alum, as a depot system, with lysed lymphoma cells could stimulate lymphoma-specific immunity and improve survival. We developed a reproducible syngenic model of B-cell lymphoma in Balb/c mouse based upon the A20 cell line. In this model animals died between day 35 and 40 after tumor cells inoculation. Mice were first injected subcutaneously with 1 x 106 A20 cell in right flank. Four groups of mice received at days 3 and 7 after tumor cell inoculation by subcutaneous injections one of the following: lysed A20 cells with IL-2 in alumn (A20-IL-2); Lysed A20 cells in alumn (A20); IL-2 in alumn (IL-2); and phosphate-buffered saline (PBS) as control. Mice were followed for survival and immune response was evaluated. At day 22, 17% of A20-IL-2 group had systemic disseminated disease in comparison with 33% A20 group, 83% for IL-2 group and 100% of PBS group (p=0.009). Mice vaccinated with A20-IL-2 had longer survival compared with mice vaccinated with A20 or PBS (p=0.0019). Prolonged survival was related with a marked increase in the number of intratumoral CD4+ T cells (p=0.0001), CD8+ T cells (p=0.001) and NK cells (p=0.001). Aditionally, at day 22 groups of mice vaccinated with A20 showed a significantly lower percentages of intratumoral CD4+ CD25+ CD127- Treg cells in the tumor as compared with PBS group (p=0.0001). At day 39 the percentage of intratumoral Treg cells increased in the groups vaccinated with IL-2 (A20-IL-2 and IL-2 groups). Intratumoral CD4+, CD8+, NK and Treg cells were evaluated by flow cytometry. Interestingly, intracellular cytokine analysis showed a greater number of intratumoral INF-γ producing CD4+ T cells in mice vaccinated with A20-IL-2. Proliferation assays by flow cytometry (using carboxy-fluorescein diacetate, succinimidyl ester and propidium iodide) showed enhanced proliferation upon stimulation with irradiated A20 cells in splenocytes from A20-IL-2 vaccinated mice (p=0.004). In conclusion, the results of this study indicate that vaccination with A20 antigens combined with a depot formulation of IL-2 elicits strong anti-tumor specific immunity and extended survival. This approach may be an interesting strategy to promote systemic immunity against B-cell lymphoma with therapeutic value.

scholarly journals DOP85 The role of fibroblasts in the pathogenesis of Crohn’s disease-associated fistulas and in mesenchymal stem cell therapy

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S125-S125
Author(s):  
R S Bruckner ◽  
M C Barnhoorn ◽  
H Mei ◽  
S M Kiełbasa ◽  
T J Harrijvan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Differentially Expressed Genes ◽  
Treg Cells ◽  
Differentially Expressed ◽  
Flow Cytometry Analysis ◽  
Normal Colon ◽  
Mrna Sequencing

Abstract Background Perianal fistulas are a severe and frequent complication in Crohn’s disease (CD) patients, significantly affecting their quality of life. High recurrence rates, incomplete fistula healing and non-responding patients make the treatment challenging. Despite some novel insights, current knowledge about the pathogenesis of fistula formation is still limited. Fibroblasts are abundantly present in CD fistulas and were recently reported to regulate Th1 cell activity in inflammatory bowel disease (IBD) (Beswick EJ et al. 2018). We hypothesised that fibroblasts act as the key drivers of fistula pathogenesis by regulating inflammatory cell recruitment. Methods Fibroblasts were isolated from curettage material of perianal CD fistulas, non-CD-associated fistulas, and the normal colon (obtained at surgery for colorectal cancer at least 10 cm away from the primary tumour). For all experiments, fibroblast populations were identified by qPCR and flow cytometry analysis first. Then gene expression profiling via mRNA sequencing of cultured fistula- and colon-derived fibroblasts was performed. The characterisation was complemented by flow cytometry analysis. Furthermore, the interaction of fistula-derived fibroblasts with regulatory T cells (Tregs) was studied in co-culture experiments. Results mRNA sequencing analysis revealed no significant differences in gene expression between perianal CD fistulas and non-CD-associated fistulas. However, comparing fistula-derived samples to fibroblasts derived from the normal colon, we found over 6000 significantly differentially expressed genes. Among the 20 most significant differentially expressed genes between CD fistulas and colon fibroblasts were genes related to epithelial-mesenchymal transition, cell migration or the NF-κB signalling pathway. Furthermore, co-culture experiments with CD fistula-derived fibroblasts, revealed increased proliferation rates of CD25+FoxP3+ Treg cells. In addition, more CD4+ T cells differentiated into Treg cells after exposure to CD fistula fibroblasts. Conclusion Our data indicate a pathogenic role of fibroblasts located in the fistula tract. Their gene expression profile markedly differed from fibroblasts derived from the normal part of the colon. Based on our data, there is no difference between CD-associated and non-CD-associated fistula fibroblasts. CD fistula-derived fibroblasts seem to increase both the proliferation of Treg cells, and the differentiation of CD4+ T cells into Treg cells, indicating an important immunoregulatory role of fibroblasts. Ongoing experiments should reveal which cytokines and chemokines are involved in this process.

scholarly journals Subpopulations of regulatory T-lymphocytes in the peripheral blood of patients with rheumatoid arthritis

2016 ◽  
Vol 71 (2) ◽  
pp. 148-153 ◽  
Author(s):  
P. N. Kravchenko ◽  
G. A. Zhulai ◽  
A. V. Churov ◽  
E. K. Oleinik ◽  
V. M. Oleinik ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Chemokine Receptor ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Expression Level ◽  
Blood Samples ◽  
Healthy Donors ◽  
Chemokine Receptor Ccr4

Background: Rheumatoid arthritis (RA) is an inflammatory rheumatic disease, associated with a dysfunction of the T cell-mediated tolerance and leading to the disability of working population.  The regulatory CD4+ T cells are play important role in the regulation of autoimmunity and can suppress immune responses. With that, there is no consensus on the content of  these lymphocytes  and their role in the pathogenesis of RA. Objective: The aim of the study was to assess the content of peripheral blood regulatory T cells (Treg) according to the expression of membrane markers CD4, CD25, CD127 and intracellular FOXP3 marker, as well as the expression of two functional molecules (CTLA-4 and CCR4) in Treg cells of patients with RA. Methods: Peripheral blood samples of RA patients (mean age 61,1±10,5) and healthy controls (mean age 52,2±14,0) were analyzed. Cell count and the expression level of molecules were assessed by flow cytometry. Results: Peripheral blood samples of 36 RA patients and 20 healthy donors were analyzed. The number of the cells with Treg-associated phenotypes CD4+CD25hi and CD4+CD25hiCD127low/– was higher in RA patients in comparison with healthy donors. Increased levels of RA CD4+ T cells expressing FOXP3 were also observed. This may be due to increasing in the number of CD4+FOXP3+CD25- lymphocytes, whereas the content of RA CD4+FOXP3+CD25+ Treg cells was at the level of the control. The expression of the functional molecule CTLA-4 in Treg cells of patients with RA was not different from the control, while the expression level of the chemokine receptor CCR4, which provides migration of lymphocytes at sites of inflammation and barrier tissues, was significantly increased in RA patients. Conclusion: Increase in the levels of certain Treg-associated lymphocyte populations were detected in peripheral blood of RA patients. During the natural course of RA, alterations in the level of the chemokine receptor CCR4 might indicate the enhanced lymphocyte migration.

Novel IL-2Ralpha (CD25)-Binding RNA Aptamer to Target T Regulatory Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3697-3697
Author(s):  
Suresh Veeramani ◽  
Sue E Blackwell ◽  
William H Thiel ◽  
Paloma H Giangrande ◽  
George J Weiner
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Rna Aptamers ◽  
Target Antigen ◽  
Mrna Levels ◽  
Rna Aptamer ◽  
Foxp3 Mrna ◽  
Whole Cell

Abstract Background: RNA aptamers are short RNA molecules that bind to antigens and ligands in a manner analogous to antibodies. RNA aptamers are being evaluated as clinical therapeutic agents based on their advantages and flexibility as cell targeting agents. Here, we report development and evaluation of a novel human IL-2Ralpha (CD25)-binding RNA aptamer that can be used to target T regulatory (Treg) cells. Methods: A. RNA aptamers: A whole cell SELEX strategy was designed to enrich for RNA aptamers that specifically bind to CD4+CD25high Tregs cells, but not to lineage-related CD4+CD25-T effector (Teff) cells. Selection began with a library of random RNA aptamers (SEL2-N20) with an expected structural diversity of ~1012. The aptamer pool was pre-cleared of aptamers that bound to common T cell antigens using Teff cells before aptamers were positively selected that bound to Tregs from the same donor. This process was repeated for eight successive enrichment rounds, each round using T cells from different donor. Enriched aptamers were sequenced and the top enriched aptamer (Tr-1) was characterized for its antigen specificity. B. Target antigen identification: The specificity of Tr-1 aptamer and its structural mutants was evaluated by testing its binding to recombinant CD25 by RT-qPCR. C. Targeting Tregs with chimeric Tr-1: Chimeric Treg-targeting reagents were created by linking Tr-1 aptamers with cytotoxic Saporin (Tr-1-Sap) or with Treg-inhibiting Foxp3 silencing RNA (Tr-1-Foxp3 siRNA). Unfractionated CD4+ T cells or Tregs were treated with Tr-1-Sap or Tr-1-Foxp3 siRNA. Reduction in Treg percentage (for Tr-1-Sap treatment) or Foxp3 mRNA levels (for Tr-1-siRNA treatment) was determined by flow cytometry or RT-qPCR, respectively. Results: A. Treg-binding RNA aptamers: A large panel of Treg-binding RNA aptamers was identified using whole cell SELEX and were synthesized. Most of the selected aptamers, particularly Tr-1, bound to CD25high Treg cells to a greater degree than to CD25low Teff cells (Figure 1). B. Target antigen identification: Tr-1 bound to human CD25 (IL-2Ralpha) protein while a control aptamer (C-248) did not. Structural mutants of Tr-1 that had lost Treg binding ability showed significantly reduced binding to CD25 protein (Figure 2). C. Treg targeting with chimeric Tr-1: Treg-targeting agents created with Tr-1 aptamers successfully delivered toxic Saporin and Foxp3 siRNA into Tregs. a. CD4+ T cells treated with Tr-1-Sap had a decrease in the percentage of CD25+ Treg population as determined by flow cytometry. b. Enriched human Tregs treated with Tr-1-Foxp3 siRNA chimera showed reduction in their Foxp3 mRNA levels. Conclusion and significance: RNA aptamers that target human Tregs were identified. The most predominant Treg-binding aptamer, Tr-1, binds to human IL-2Ralpha/CD25, a clinically-targeted molecule expressed by Tregs. Chimeric reagents based on Tr-1 aptamer effectively targeted Tregs signifying their potential use as novel immunomodulators. Ongoing studies are further exploring the significance of Tr-1 aptamer as a diagnostic agent and as a therapeutic modulator of Treg activity. Disclosures No relevant conflicts of interest to declare.

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