determination-regulatory-t-cell-subsets-murine-thymus-pancreatic

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Uppsala University

Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4(+)CD8(-)CD25(+) Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice, and thymus of NOD and C57BL/6 mice. The frequency of Helios(+) cells was higher than that of Nrp1(+) cells in CD4(+)CD8(-)CD25(+) and CD4(+)CD8(-)CD25(+)Foxp3(+) Treg cells in thymus. Interestingly, the proportion of IL-10(+), Ebi3(+)and CTLA-4(+) cells was higher in Helios(+) than Nrp1(+) tTreg cells. The anti-apoptotic activity of Helios(+) tTreg cells was higher in thymus compared to Nrp1(+) tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4(+)CD25(+) T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and -CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1. 

Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice.

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells. 

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis.

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases. 

Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.

Heparanase is an endo-glucuronidase that degrades heparan sulfate chains. The enzyme is expressed at a low level in normal organs; however, elevated expression of heparanase has been detected in several inflammatory conditions, e.g. in the synovial joints of rheumatoid arthritis (RA) patients. Herein, we have applied the model of collagen-induced arthritis (CIA) to transgenic mice overexpressing human heparanase (Hpa-tg) along with wildtype (WT) mice. About 50% of the induced animals developed clinical symptoms, i.e. swelling of joints, and there were no differences between the Hpa-tg and WT mice in the incidence of disease. However, Hpa-tg mice displayed an earlier response and developed more severe symptoms. Examination of cells from thymus, spleen and lymph nodes revealed increased innate and adaptive immune responses of the Hpa-tg mice, reflected by increased proportions of macrophages, antigen presenting cells and plasmacytoid dendritic cells as well as Helios-positive CD4 and CD8 T cells. Furthermore, splenic lymphocytes from Hpa-tg mice showed higher proliferation activity. Our results suggest that elevated expression of heparanase augmented both the innate and adaptive immune system and propagated inflammatory reactions in the murine RA model.

Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis.

 To characterize the plasma levels of vascular endothelial growth factor (VEGF) in type 1 diabetes mellitus (T1D) and its relation to both present and historical metabolic control and microvascular complications.  Plasma levels of VEGF and routine clinical parameters were analyzed in 115 patients with long-standing T1D and 45 healthy controls (HC). All patients were under clinical routine diabetes treatment at Uppsala University Hospital.  The plasma levels of VEGF were increased by 37% in patients with T1D when compared to HC (18.2 ± 0.8 versus 13.2 ± 1.0 pg/ml,  < 0.001). The levels of VEGF correlated to insulin needs and BMI but not to present or historical metabolic control. The levels of VEGF were similar in patients with T1D and microvascular complications (microalbuminuria and retinopathy) when compared with patients without microvascular complications. Historical HbA1c levels were found to be the best predictor for present metabolic control.  Circulating plasma levels of VEGF do not correlate to present or historical metabolic control in long-standing T1D and the levels are not affected by the presence of microvascular complications.

The Increased Circulating Plasma Levels of Vascular Endothelial Growth Factor in Patients with Type 1 Diabetes Do Not Correlate to Metabolic Control.

Many patients with long-standing type 1 diabetes have remaining functional β-cells. This study investigated immunological differences between patients with or without measurable remaining endogenous insulin production after ≥10 years duration of disease.

Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide.

To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or  knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of  did not affect tumor growth although it increased lung metastasis.  knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of  knockout CD11b/CD8 cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4 lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4 cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice.  knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of  further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an  dependent manner.

Article	Year
Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice. Scientific reports\| PubMed ID: 25586548	2015
Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis. The Journal of biological chemistry\| PubMed ID: 25922077	2015
Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells. Scientific reports\| PubMed ID: 26224624	2015
Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis. Scientific reports\| PubMed ID: 28401953	2017
The Increased Circulating Plasma Levels of Vascular Endothelial Growth Factor in Patients with Type 1 Diabetes Do Not Correlate to Metabolic Control. Journal of diabetes research\| PubMed ID: 28421206	2017
Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide. Diabetes care\| PubMed ID: 28620093	2017
Pro-tumoral immune cell alterations in wild type and -deficient mice in response to 4T1 breast carcinomas. Oncotarget\| PubMed ID: 29721156	2018

Kailash Singh

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