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- Luo, Zhengkang, et al.
(författare)
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Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry
- 2019
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Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :144
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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.
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- Luo, Zhengkang, 1994-
(författare)
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Immunological strategies for counteracting type 1 diabetes focusing on IL-35 producing regulatory immune cells
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 1 diabetes (T1D) is an autoimmune disease where pancreatic β-cells are attacked by immune cells. Regulatory T (Treg) cells play critical roles in suppressing immune responses and their involvement have been intensively studied in T1D. Low dose IL-2 has been proposed to selectively boost Treg cells in T1D, with only limited success. We thus further decreased the IL-2 dosage and treated multiple low dose streptozotocin (MLDSTZ) mice with an ultra-low dose IL-2, but it did not protect STZ mice from hyperglycemia. Similarly, low dose IL-2 only partially prevented diabetes. Treg cells’ phenotype was not protected by either dose. These data suggest that alternative IL-2 therapies might be considered. Regulatory B (Breg) cells suppress pro-inflammatory immune responses by producing anti-inflammatory cytokines IL-10 and IL-35. Decreased IL-35+ and increased IFN-γ+ Breg cell proportions were found in T1D patients, and in diabetic mice. IL-35 treatment prevented increased IFN-γ+ Breg cell proportions in STZ mice. These data illustrate Breg cells’ involvement in T1D, and IL-35 treatment prevents hyperglycemia by maintaining Breg cells’ phenotype.Treg cells’ involvement in diabetic nephropathy (DN) has not been studied. Lower plasma IL-35 was found in DN patients than in T1D patients without DN and healthy controls, and was strongly correlated with kidney function. Decreased IL-35+ and increased IL-17+ Treg cells were found in DN patients. Moreover, Foxp3+ cell infiltration was found in the kidneys of diabetic mice, but it failed to counteract mononuclear cell infiltration. IL-35 treatment prevented DN and Treg cells’ phenotypic shift in STZ mice by maintaining the transcription factor Eos. These results demonstrate that IL-35 may be used to prevent DN. Given the instability of IL-35, we explored the effect of IL-6 signaling blockade. Anti-IL-6R completely protected STZ mice from diabetes. Proteomics indicated enhanced metabolism and down-regulated pro-inflammatory pathways. It maintained Treg cells’ phenotype by increasing IL-35 and decreasing IFN-γ production. It also reduced the number of macrophages and conventional dendritic cells type 2 and their CD80 expression. STZ mice remained normoglycemic despite the discontinuation of anti-IL-6R treatment. Therefore, our results illustrate the outcomes of several potential T1D immunotherapies and highlight the involvement of IL-35 producing immune cells in controlling the disease.
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- Luo, Zhengkang, et al.
(författare)
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Interleukin-35 Prevents Development of Autoimmune Diabetes Possibly by Maintaining the Phenotype of Regulatory B Cells
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:23
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Tidskriftsartikel (refereegranskat)abstract
- The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35(+) Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35(+) Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35(+) Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35(+) Breg cells. A higher proportion of IFN-gamma(+) cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-gamma(+) cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.
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- Luo, Zhengkang, et al.
(författare)
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Kinetics of immune cell responses in the multiple low dose streptozotocin mouse model of type 1 diabetes
- 2019
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Ingår i: FASEB BioAdvances. - : Wiley. - 2573-9832. ; 1, s. 538-549
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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.
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