In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

This protocol describes a potentially novel therapeutic strategy that can be readily modified for human applications. This technique directly enhances the induction of gut-homing regulatory T cells in peripheral lymphoid tissues and can be stably implemented even in highly pro-inflammatory environments. These techniques can also be used to investigate the role of active vitamin D and vitamin A within the peripheral immune system.

This visual demonstration will provide step by step instructions for the generation of high quality engineered dendritic cells that are critical for the success of this technique. Begin by seeding one times 10 to the seven 293T cells in 20 milliliters of CM-10-D cell culture medium in 150 by 25 millimeter culture plates for a 24 hour incubation at 37 degrees Celsius and 5%carbon dioxide. The next day, add 1.62 milliliters of freshly prepared two-fold concentrated HBS solution, 9.5 micrograms of envelope plasmid, 17.5 micrograms of packaging plasmid, and 27 micrograms of the LENTI-CYP-ALDH plasmid to a 50 milliliter conical tube.

Bring the final volume of the tube contents up to 3.0375 milliliters with water followed by the addition of 202.5 microliters of two molar calcium chloride dropwise. Leave the DNA precipitation mixture at room temperature for 20 minutes with occasional mixing before adding 3.24 milliliters of the solution dropwise per 293T culture while gently swirling the dish. Place the plates in the incubator for 24 hours before gently washing each plate with PBS and feeding the cells with CM-4-D culture medium.

After another 24 hours of culture, harvest the supernatants into sterile bottles for storage at four to eight degrees Celsius and feed the cells with fresh CM-4-D medium for another 24 hours. On day four, harvest the supernatants again and strain the supernatants from both days three and four to a 0.45 micrometer filter. Then, concentrate the VSV-G pseudotyped virus by centrifugation for 24 hours.

The next day decant the supernatants. Allow the tubes to drain on a paper towel in a sterile bio-safety cabinet for several minutes, then resuspend the pellets in 33.3 microliters of sterile PBS containing 5%glycerol per culture dish. For bone marrow derived dendritic cell generation, place the tibias and femurs, harvested from BALB/c mice, into a 100 by 20 millimeter culture dish containing fresh culture medium.

Use dissecting scissors and forceps to remove any tissues from the bones. When all of the bones have been cleaned, use the scissors to cut both ends of each bone to expose the bone marrow cavities. Use a 10 milliliter syringe equipped with a 30 gauge needle to flush each bone with 10 milliliters of fresh culture medium, collecting the bone marrow in a 15 milliliter tube.

When all of the bone marrow has been collected, sediment the bone marrow by centrifugation and resuspend the pellet in two milliliters of red blood cell lysis buffer. After four to five minutes at room temperature with occasional shaking, stop the reaction with 10 milliliters of cell culture medium and collect the cells by centrifugation. Resuspend the pellet in 10 milliliters of CM-10-R culture medium for counting and adjust the cells for one times 10 to the six cells per milliliter of CM-10-R culture medium concentration.

Next, add 100 units per milliliter of recombinant murine GM-CSF and 10 units per milliliter of IL-4 to the cells. Plate four milliliters of cells into individual wells of a six well culture plate for their incubation at 37 degrees Celsius and 5%carbon dioxide for 48 hours. At the end of the incubation, gently aspirate the non-adherent cells.

Add fresh medium supplemented with GM-CSF and IL-4 to the cultures before returning the cells to the cell culture incubator for an addition 48 hours. At the end of the second incubation, harvest the non-adherent bone marrow derived dendritic cells into 50 milliliter conical tube for their centrifugation. To generate BMDC-CYP-ALDH cells, resuspend the cultures at a one times ten to the sixth dendritic cells per 500 microliters of CM-10-R medium per well concentration, and plate 500 microliters of cells into each well of a new six well culture plate.

Add 50 microliters of LENTI-CYP-ALDH virus and 8 micrograms per milliliter of protamine to each well, then place the plate in the incubator for 24 hours. The next day, replace the supernatants with fresh culture medium and return the plates to the cell culture incubator for another 24 hours. At the end of the second incubation, the cells can be activated with 100 nanograms per milliliter of lipopolysaccharide per well for 24 hours before harvest for functional analyses.

Compared to parental bone marrow derived dendritic cells, BMDC-CYP-ALDH cells display an enhanced expression of 1-alpha-hydroxylase. The concentrations of the active form of vitamin D in the culture supernatants of BMDC-CYP and BMDC-CYP-ALDH cells are approximately 20 times higher than those observed in parental bone marrow derived dendritic cell and the BMDC-ALDH cell cultures. The mean fluorescent intensities of BMDC-CYP-ALDH cells treated with the substrate are approximately six times higher than those of parental bone marrow derived dendritic cells treated with the substrate.

This suggests that the BMDC-CYP-ALDH cells express significantly enhanced retinal dehydehydrogenase-2 enzymatic activity. DC2.4 cells treated with 25-hydroxy vitamin D and retinal do not induce a significant change in the abundance of foxp3 positive CCR9 positive CD4 positive T cells. In contrast, DC2.4 CYP-ALDH cells treated with 25-hydroxy vitamin D and retinal induce significantly higher numbers of gut-homing regulatory T cells in a concentration dependent manner.

Further, compared to intraperitoneally injected control cells, DC CYP-ALDH cells also induce a significant increase in the number of foxp3 positive CCR9 positive CD4 positive T cells after intraperitoneal injection into BALB/c recipient animals. The success of the procedure depends on the preparation of healthy T cells and a correctly prepared DNA mixture.