This study investigates the role of iPSC-derived regulatory T cells (Tregs) in modulating arthritis in a mouse model. By injecting Tregs and methylated bovine serum albumin (mBSA), the research explores the contrasting effects of Tregs and activated T cells on joint inflammation.
Take iPSC-derived regulatory T cells, or Tregs, expressing ovalbumin-specific receptors. Inject them into the tail vein of a restrained mouse.
The cells reach the synovial membrane lining the inner surface of the knee joint capsule.
Next, administer methylated bovine serum albumin or mBSA, emulsified with an adjuvant.
Adjuvants enhance the presentation of mBSA to antigen-presenting cells, or APCs, which process and present the fragments via MHC molecules.
The APCs migrate to the lymph nodes, inducing T cell activation. Activated T cells migrate to the knee joint.
Inject mBSA alone and mBSA with ovalbumin in the left and right knee joints, respectively.
In the left knee, APCs present the mBSA fragments to activated T cells, triggering pro-inflammatory cytokine release and leading to tissue damage and joint swelling, termed arthritis.
In the right knee, Tregs interact with ovalbumin-presenting APCs and release anti-inflammatory cytokines, reducing tissue damage and joint swelling, thereby ameliorating arthritis.
Begin by detaching the cell cultures of interest with trypsin, and resuspending each cell type in 10 milliliters of fresh medium. Plate each cell culture in a new 10-centimeter dish, and allow the feeder cells to adhere in the cell culture incubator. After 30 minutes, collect the floating iPSCs, and pass the cultures through individual 70-micron cell strainers to remove the cell clusters for counting.
Resuspend each culture at a 1.5 x 107 cells per milliliter concentration in cold PBS, filtering again as necessary. Then, place 4-to-6-week-old female C57BL6 mice one at a time in a small animal restrainer, and adoptively transfer 200 microliters of one type of cell per mouse into the tail vein of each animal.
Use a dial gauge caliper to measure the swelling of both knees to establish the baseline measurement. 10 days after the cell transfer, use a 1-milliliter syringe to inject the mice with 100 micrograms of methylated BSA emulsified in complete Freund's adjuvant at the base of the tail of each experimental animal.
17 days after the cell transfer, induce arthritis by intra-articular injection of 20 micrograms of methylated BSA in 10 microliters of PBS into the left knee joint and 20 micrograms of methylated BSA, and 100 micrograms of whole ovalbumin in 10 microliters of PBS into the right knee joint of each anesthetized adoptively transferred animal.
Seven days after inducing arthritis, measure the knees again for calculation of the percent increase in the knee diameter and surgically excise the patellas. Fix the joints in formalin. Then, after decalcification in EDTA, embed the knees in paraffin, and obtain 4-micron sections for histochemical staining and analysis.
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