The combination of T cells and dendritic cells rapidly rearranges MHC II transport
Overview: The CV1000 live cell dynamic observation system integrates microscope, confocal scanning system, live cell culture, etc., changing the traditional confocal complex connection, the synchronous monitoring mode of the personnel, and abandoning all external human disturbance factors, real-time dynamics. It was observed that the combination of T cells and dendritic cells rapidly rearranges the transport process of MHC II. The major histocompatibility complex (MHC) presenting antigens as short peptides to T lymphocytes is in the endoplasmic reticulum. Assembled, once assembled, they will be shipped to their final destination. MHC class II molecules reach the cell surface according to the endocytic pathway to obtain the antigen. The transport of MHC class II molecules in professional antigen presenting cells (APCs) is tightly regulated and responds to inflammatory stimuli such as lipopolysaccharide. To investigate the transport of MHC class II molecules in antigen-presenting cells (APCs), we replaced MHC class II molecules in mice with MHC class II molecules with enhanced green fluorescent protein (EGFP) tagging. The resulting mice were immunologically indistinguishable from wild mice. In bone marrow-derived dendritic cells, we found that MHC class II molecules in late endocytic structures are similar to those in Langerhans cells. We have found that when dendritic cells presenting antigens encounter T cells of suitable characteristics, the tubular endosomes will extend within the cells and polarize toward the T cells in which they act, which promotes subsequent responses of T cells.
Objective: To reveal the transport direction of MHC class II molecules in antigen presentation
Experimental Materials:
Instruments: ZEISS inverted microscope + spinning wheel confocal eyepiece, flow cytometry reagent: anti-EGFP monoclonal antibody, Alexa Fluor 594 labeled transferrin, chicken egg lysozyme (HEL), ovalbumin (Ova), Hoechst 33258 Dye test subject: dendritic cells
Experimental steps:
1. Construction of MHC II-EGFP knock-in mice MHC class II molecules with enhanced green fluorescent protein (EGFP) tag were inserted into embryonic stem cells (ES cells) of 129/Sv mice by homologous recombination, by western blot. MHC II-EGFP protein expression was confirmed by fluorescence activated cell sorting (FACS), and MHC II-EGFP knock-in mice were obtained.
2. To demonstrate the immunological difference between transgenic mice and wild mice. MHC class II molecules were matured in mice by pulse-chase analysis and immunoprecipitation, and cell sorting was performed by flow cytometry. There was no significant difference in immunity between transgenic mice and wild mice.
3. Distribution of MHC II-EGFP molecules in dendritic cells
Figure 1 shows the distribution of MHC II-EGFP protein in the late endosomes of dendritic cells. Observation by time-lapse confocal microscopy
Figure 1 summarizes: MHC II-EGFP is present in dendritic cells, which induces antigen-presenting pathways similar to MHC class II molecules. Lipopolysaccharide can alter the morphology of dendritic cells and the distribution of MHC II-EGFP on the cell membrane.
4. T cells trigger MHC II directed transport
Dendritic cells of MHC II-EGFP mice (cultured for 4 days) were incubated overnight with chicken egg lysozyme (HEL) (panel a) and ovalbumin (Ova) (panel b), respectively, making the source HEL or Ova short The peptides were displayed on the cell membrane by MHC II, and each of them was added with HEL or Ova-specific T hybridoma cells. The time-lapse confocal microscopy was used to randomly select dendritic cell-T cell complexes and observed that 2 hours and MHC II-EGFP pores towards T cells were produced after 6 hours (panel a, panel b). A red staining solution capable of staining α-tubulin was added to the dendritic cell/T hybridoma cell cluster, and microscopic observation showed that the T cell-forming tubule was the MHC II-EGFP pore channel (Fig. c).
Figure 2 summarizes: Confocal microscopy observations demonstrate that T cells trigger directional transport of MHC II towards itself.
5. Quantitative analysis of T cell-priming tubules In the absence and presence of ovalbumin (Ova), native OTII T cells (Ova-specific T cells) stained with Hoechst 33258 were incubated with dendritic cells, respectively. The analysis showed that the number and length of tubules induced by ovalbumin (Ova) toward T cells were much higher than those produced in the absence of ovalbumin (Ova) (Fig. 3).
Figure 3 summarizes: Tubules triggered by T cell-induced directional transport of MHC II are not accidental events and are statistically significant.
in conclusion:
Summarizing the above series of experimental results, MHC class II molecules are distributed in dendritic cells, which present antigens through a series of processing processes such as endocytosis. Lipopolysaccharide can induce changes in the morphology of dendritic cells and increase MHC class II molecules. Distribution on the cell membrane. In the presence of T hybridoma cells, the addition of the corresponding antigen induces dendritic cells to produce tubules directed to T cells, resulting in the transport of MHC class II molecules to T cells. On the other hand, rearrangement of this MHC II molecular trafficking pathway in turn enhances the chance of T cell activation and promotes subsequent T cell responses.
Recommended reading reason:
This is an article published by Nature at Harvard Medical School. It is selected as a cover story because of its dynamic interpretation of the rearrangement of MHC class II molecules in dendritic cells and the capture of high-resolution images; The live cell dynamic observation confocal technique (YOKOGAWA) is used to stand out from many top scientific research articles, and once again verifies the undeniable truth that high-end technology promotes the development of scientific research and leads the trend of cutting-edge research. The era of closed-door manufacturing has been As soon as the scientific research workers pay attention to the scientific research dynamics in their own fields, the progress of experimental technology for scientific research services cannot be ignored. Otherwise, mistakes will be misunderstood.
This article was published in 2002, and its selection of YOKOGAWA live cell dynamic observation confocal technology has also made great progress and has maintained a leading position in the world. In 2010, YOKOGAWA improved and integrated this technology into the model CV1000. In the all-in-one machine, the observation control of living cells has greatly increased by a step, changing the traditional confocal complex connection, the operation mode of personnel synchronous monitoring, and abandoning all external human disturbance factors, which has been unanimously recognized by the industry. The rapidly increasing installed capacity and the amount of articles will be actually rewarded.
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