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The Host Response to Grafts and Transplantation Immunology
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
The mixed lymphocyte culture has been used as an in vitro model of the allograft reaction, but it is now thought to represent primarily the direct recognition pathway. Still, it is used by a number of laboratories to determine tissue compatibility for bone marrow transplantation. When lymphocytes from two different individuals are mixed in tissue culture, the cells of each will stimulate the lymphocytes of the other to form blasts and divide. To differentiate between the ability of a lymphocyte to respond and stimulate, a one-way MLC is used, in which the stimulating cells are treated with either mitomycin-C or x-irradiation. Proliferation in the mixed lymphocyte reaction is controlled by the Class II antigens. T cell proliferation is detected by analysis of incorporation of tritiated thymidine (Figure 11.8). Tritiated thymidine is a radioactive DNA precursor that is incorporated into DNA by dividing cells. In the MLC, the lymphocytes identify each other as “nonself” or foreign through recognition of the cell surface histocompatibility antigens, e.g., HLA in man.
Definition of HLA-Dw Determinants Using Homozygous Typing Cells and the Mixed Lymphocyte Culture
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
After the recognition that the strong mixed lymphocyte reaction (MLR) obtained between cells from HLA class II incompatible individuals was genetically controlled by genes within the HLA-D region (distinct from those determining the serologically defined HLA-A, -B, and -C antigens), a number of cellular techniques that identify these determinants have been described. These include the use of homozygous typing cells (HTCs) in the primary MLC26 and the use of primed lymphocyte typing (PLT) reagents in the secondary MLC.27 This chapter concentrates on the description of the former and on the advantages and limitations of this approach.
Self-Recognition and Symmetry in the Immune System
Published in Irun R. Cohen, Perspectives on Autoimmunity, 2020
Yaakov Naparstek, Robert S. Schwartz
Some T-cells can proliferate in response to MHC autoantigens in the absence of exogenous antigens. They are called autoreactive T-cells. They were first demonstrated in mixed lymphocyte reactions, in a phenomenon termed the autologous or syngeneic mixed lymphocyte reaction.63 However, they also occur during the immune response to exogenous antigens.64-66 In normal mice, precursors of autoreactive T-cells outnumber precursors of carrier-specific T-cells.66 The initial in vitro selection of autoreactive T-cells requires the presence of antigen, but the establishment of a line of autoreactive T-cells ultimately depends only on repeated stimulation with class II MHC-compatible cells. In the lupus-prone MRL-lpr/lpr mouse, by contrast, no exogenous antigen is needed to establish lines of autoreactive T-cells, only the presence of cells with autologous MHC determinants.67
Development of an In Vitro Assay to Assess Pharmacological Compounds and Reversion of Tumor-Derived Immunosuppression of Dendritic Cells
Published in Immunological Investigations, 2021
Mikkel Møller Andersen, Jesper Larsen, Morten Hansen, Anders Elm Pedersen, Monika Gad
Buffy coats were acquired from healthy donors at Rigshospitalet (Copenhagen, Denmark), and CD4+ T cells were purified by positive selection using the Dynabeads CD4 positive selection kit (Invitrogen) according to the manufacturer’s instructions. The mixed lymphocyte reaction was carried out in 200 µL DC culture medium in sterile round-bottom 96-well plates by the addition of 1 × 105 freshly purified CD4+ T cells and 5 × 103 freshly harvested mDCs, yielding a 1:20 DC:T cell ratio. The plates were incubated at 37°C in humidified incubators with 5% CO2 for 5 days. The supernatant of the MLR was harvested at day 5 of coculture and stored at −20°C until its concentration of cytokines was measured by ELISA. To measure the proliferation of CD4+ T cells in the MLR, 1 μCi 3H-thymidine (Perkin Elmer) was added to each well 18 hours prior to harvesting the cells. The cells were harvested on day 5 of coculture using a Unifilter-96 cell harvester (Perkin Elmer) and the incorporation of 3H-thymidine was determined by liquid scintillation counting using a TopCount NXT (Packard) after the addition of 20 μL MICROSCINT-O (Perkin Elmer) per well. To examine the expression of transcription factors by CD4+ T cells, the cells were harvested and lysed by the addition of 200 μL RLT lysis buffer from the RNeasy kit (QIAGEN) supplemented with 1% 2-mercaptoethanol. The lysed cells were immediately stored at −80°C, until RNA purification would be performed.
The biological effects of electromagnetic exposure on immune cells and potential mechanisms
Published in Electromagnetic Biology and Medicine, 2022
Chuanfu Yao, Li Zhao, Ruiyun Peng
In addition to PBMCs and NK cells, electromagnetic fields also could produce biological effects on other immune cells. Zhou et al. (2008) exposed human peripheral blood derived DC cells to GSM radio with frequency of 1800 MHz (SAR 4 W/kg, opened for 5 min, stopped for 10 min) for 24 h. They found that microwave significantly decreased several phenotype molecules, such as HLA-DR, CD80, CD86 and CD40. Moreover, according to the results of homologous mixed lymphocyte reaction, the antigen presenting of DCs were also reduced after exposure. These results suggested that GSM radio with frequency of 1800 MHz inhibited the activation of DCs.
In vitro impact of bisphenol A on maturation and function of monocyte-derived dendritic cells in patients with primary Sjögren’s syndrome
Published in Immunopharmacology and Immunotoxicology, 2020
Jing Wang, Chunhui She, Zhiyuan Li, Ning Tang, Lishan Xu, Zhaoyang Liu, Bin Liu
Mixed lymphocyte reaction. BPA-exposed moDCs of pSS patients and HCs were co-cultured with T cells from HCs, respectively. After co-culture, the proliferation of CD4+ T cells was detected by CFSE (A) (n = 3). ELISA analysis (n = 10) of protein levels of IFN-γ, IL17, IL4, and IL10 (B). RT-PCR analysis (n = 10) of mRNA levels of IFN-γ, IL17, IL4, T-bet, RoR-γt, and Gata3 (C, D). Flow cytometry analysis (n = 3) levels of T-bet, RoR-γt, Gata3, and Foxp3 (E and F). *p<.05, **p<.01.