This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.
Our protocol describes a method to build an RA model. Its success rate is over 93 percent and its pathogenesis is depends on T cells, B cells, and innate immunity, which allows researchers to better study the pathogenesis of RA from a global immune perspective. Then we use INKT cells with immunomodulatory effects for their treatment.
INKT cells have good effect on the treatment by in vivo introduction which provide basic data support for the clinical application of INKT cells and truly explodes the clinical application value of INKT cells. The greatest advantage of this technology is that it is simple to operate and easy to copy. Demonstrating the procedure will be Chen Shengde, Gao Xiang, and Zhang Jingnan, graduate student from my laboratory.
To begin, weigh 1.75 milligrams of both HGPI325 to 339 and HGPI469 to 483 fragments and dissolve them in 5.25 milliliters of 4 degrees Celsius triple distilled water. Dissolve Complete Freund's Adjuvant in a 50 degree Celsius water bath. Draw 5.25 milliliters into another 10 milliliter centrifuge tube and cool it for use.
Put the mixture of HG6PI solution and Complete Freund's Adjuvant solution in an artificial emulsification unit with two glass syringes, connected. In an ice bath, push the syringe at a constant speed and frequency of ten to twenty times per minute to completely emulsify the mixture peptide solution and complete Freund's Adjuvant solution. Then, keep the emulsion droplets in the water for ten minutes.
Next, inject 150 microliters of the emulsified HG6PIs into the mouse's tail root subcutaneously. Immediately and after 48 hours, inject 200 milligrams of pertussis toxin into the mouse intraperineally. Now, inject normal DBA1 mouse intraperineally without the gausser at the dosage of 0.1 milligrams per kilogram of body weight.
Three days after modeling, after anesthetization, isolate the spleen of the mouse. Prepare a single cell suspension by cutting and grinding the spleen in a 200 mesh sieve. Wash the cell suspension with PBS.
Centrifuge at 200 times g for five minutes and discard the supernatant. Re suspend the cells with one milliliter of whole blood tissue solution. Add three milliliters of mouse lymphocyte separation medium.
And then centrifuge the cells for 20 minutes at 300 times g at room temperature. To purify the INKT cells, resuspend 10 to the seventh cells with 100 microliters of four degree Celsius PBS, add 10 microliter of alpha gausser loaded CD1 tetramer PE and incubate them at 4 degrees Celsius for fifteen minutes in the dark. Wash the cells twice with PBS and resuspend them in 80 microliters of PBS.
Add 20 microliters of anti-PE microbeads and incubate them at four degrees Celsius for twenty minutes in the dark. Wash them twice with PBS and resuspend the cells with 500 microliters of PBS. Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 microliters of PBS.
Add the prepared cell suspension to the sorting column, collect the flow through, and rinse three times with PBS buffer. Remove the magnetic field and add one milliliter of PBS buffer to the sorting column. Quickly push the plunger at a constant pressure to drive the labeled cells into the collection tube.
And obtain the purified INKT cells. Count with an automated cell counter. To identify the INKT cell phenotype, first, take one times ten to the sixth cells from the purified INKT cells and resuspend them in 50 microliters of PBS.
Add 0.5 microliters of alpha gausser PE CD1 tetramer, or 10 microliters of FITC TCR beta in the single positive control tube. Add 0.5 microliters of alpha gausser PE CD1 tetramer, Add 10 microliters of FITC TCR beta in the sample tube. Incubate them at four degrees Celsius for thirty minutes in the dark.
After that, wash the cells in PBS and then centrifuge at 200 times g for five minutes. Discard the supernatant and add one milliliter of FoxP3 fixation permeabilization working solution. And incubate the cells for 45 minutes at four degrees Celsius in the dark.
Then, add one milliliter of 1x permeabilization working solution and centrifuge the cells at 500 times g at room temperature for five minutes. Discard the supernatant. Add one microliter of AlexaFluor 647 mouse anti-PLZF and one microliter of PerCP Cy5.5 mouse anti-Tbet and incubate for thirty minutes at room temperature in the dark.
Next, add two microliters of permeabilization buffer working solution and centrifuge at 200 times g for five minutes for cleaning. Discard the supernatant. Resuspend the cells in 500 microliters of PBS and measure by flow cytometry.
In this study, compared with the control group, the toes of the RA model group began to show red swelling after modeling with gradual aggravation. At 14 days, the red swelling in the ankle joint peaked followed by gradual relief. The pathological results show that the infiltration degree of inflammatory cells in the ankle synovial tissue of the RA model mice was different at different stages.
Peak inflammation occurred on day 14 post modeling. In the RA model group, the serum levels of pro-inflammatory cytokines significantly increased 11 days and 14 days after modeling. While anti-inflammatory cytokines significantly decreased.
This figure shows the rate of INKT2 in normal mice was around five percent. The rate of INK2 was about 82 percent after in vivo induction. The rate of INK2 was more than 92 percent after MACS purification.
Here we show a method to emulsify the major polypeptide we see in RA.The emulsified droplets are kept in the water for 10 minutes. Results is dispersing. This protocol is a new add-on for the treatment of RA raised special NKT cells.
Which can be applied towards cell immunotherapy or other autoimmune disease and tumors. This model can stimulate the overall proliferation of CD4 T cells and NKT cells deficits in RA patients, which lays the foundation for in depth study of RA immune pathways. In this protocol, pertussis toxin is injected the interperitoneally during modeling.
Please wear experimental clothes and disposable gloves. And carry all the standards of operation so as to prevent stab wound an infections.
