| 1. |
- Luo, Zhengkang, et al.
(författare)
-
Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
- 2019
-
Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :144
-
Tidskriftsartikel (refereegranskat)abstract
- 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, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.
|
|
| 2. |
- Luo, Zhengkang, et al.
(författare)
-
Kinetics of immune cell responses in the multiple low dose streptozotocin mouse model of type 1 diabetes
- 2019
-
Ingår i: FASEB BioAdvances. - : Wiley. - 2573-9832. ; 1, s. 538-549
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- In type 1 diabetes (T1D), the insulin-producing β cells are destructed by immune mechanisms. It has been hypothesized that the very first immune response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the islets. Despite intensive research on characterization of islet-infiltrating immune cells, the kinetics of different immune cells in multiple low-dose streptozotocin (MLDSTZ)-induced T1D mouse model is still much unclear. Therefore, we investigated the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, natural killer (NK) cells, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic-draining lymph nodes, and spleens of MLDSTZ mice on days 3, 7, 10, and 21 after the first injection of STZ by flow cytometry. The proportions of DCs and B cells were increased from day 3, while the proportions of B-1a lymphocytes and interferon-γ+ cells among NK cells were increased, but NK cells were decreased on day 10 in MLDSTZ-treated mice, illustrating that the initial immune response is induced by DCs and B cells. Later, the proportions of T helper 1 and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede adaptive immune cell response in MLDSTZ mice. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, NK cells, B-1a lymphocytes, B, and T cells at the early stage of T1D development.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Singh, Kailash, et al.
(författare)
-
Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naive mice
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5, s. 7767-
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 6. |
- Singh, Kailash, et al.
(författare)
-
Interleukin-35 administration counteracts established murine type 1 diabetes - possible involvement of regulatory T cells
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- 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-beta) and increased pro-inflammatory (IFN-gamma, 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.
|
|
| 7. |
|
|
