In Vitro e In Vivo valutazione della attività soppressiva T, B e cellule mieloidi e risposte umorali dai destinatari del trapianto

Tolerance induction in transplantation is often associated with emergence of regulatory cell population. Absence of immune response to the donor, but preserved immunity against exogenous antigen. We have different protocol to assess in vitro and in vivo, the suppressive capacity of any regulatory cell subset and the status of humoral responses from the host.

Anesthetize a donor Lewis 1W rat using isoflurane O2 inhalation supplemented with N20 1%after five minutes. Place the animal in dorsal decubitus and disinfect the abdomen with betadine to perform a thoracotomy. Clamp the inferior and superior vena cava, ligatures and then cut them.

Then cut the pulmonary artery and the aorta and save the graft in cold NaCl 0.9%Anesthetize 150 grams Lewis 1A recipient rat of eight to 12 weeks using isoflurane O2 inhalation supplemented with N2O 1%Lay the animal in dorsal decubitis and disinfect the abdomen with betadine to perform cephalo-pubic laparotomy. Externalize the intestines, clamp the abdominal blood vessels. Perform a terminal lateral anastomosis between the graphed aorta and the abdominal aorta.

And between the pulmonary artery and the abdominal vena cava and remove clamps. Suture the muscular plane and skin and disinfect with betadine. Inject and meloxicam subcutaneously and terramycin and buprenorphine intramuscularly and place the animal under a heat lamp until the wakening of the animal.

To alleviate allograph rejection, further proceed with the selected drug. Harvest the spleen from a naive Lewis 1W rat, and save it in cold PBS 1x. Transfer the spleen in the dish, remove PBS and perfuse with five mL of Collagenase D.To improve the enzyme action, cut the spleen in small pieces and incubate 15 minutes at 37 degrees Celsius.

Add 500 microliters EDTA 0.1 molar. Transfer the spleen pieces into a sieve and crush the spleen with a sterile pestle to disassociate the cells. Transfer into a tube and wash the cells with 50 milliliters PBS.

Centrifuge 10 minutes at 430 G.Discard the supernatant. Eliminate red blood cells and platelets. Resuspend the splenocytes pellets in 10 mL of hypotonic solution, and incubate five minutes at room temperature.

Then wash in PBS and centrifuge 10 minutes at 190 G.Discard the supernatant. Remove collagen fibers by filtering in a 100 micrometers tissue filter. Count the cells to adjust cell concentration at five times 10 power seven cells per mL in PBS at CS 2%EDTA 0.5 millimolar.

Incubate 15 minutes at four degrees Celsius with anti-TCR alpha/beta and anti-TCR gamma/delta to label T-cells, anti-CD45RA to label B cells and anti-CD161 to label NK cells. Wash with PBS-FCS-EDTA and centrifuge 10 minutes at 430 G.Wash magnetic dynabeads. Use 35 microliter dynabeads per 10 power six splenocytes with 30 mL PBS-FCS-EDTA.

Place one minute on the magnetic and discard the supernatant. Repeat three times. Resuspend dynabeads in 10 volumes of PBS-FCS-EDTA.

Discard the supernatant of centrifuged cells. Remove unwanted cells by mixing splenocytes with dynabeads for 10 minutes at four degrees Celsius and during agitation. Then place the tube on the magnet one minute and transfer the supernatant in a new tube.

Repeat twice. Count the cells and adjust cell concentration at five times 10 power seven cells per mL in PBS-FCS-EDTA. Sort pDCs using labeled antibodies.

Add an anti-CD45RA antibody to label B-cells, an anti-TCR alpha/beta to label T-cells, an anti-CD4 and anti-CD45R to label pDCs. Sort CD4-positive, CD45R-positive, CD45RA-negative, TCR alpha/beta-negative cells. Label beads with each antibody to establish a compensation matrix.

Incubate 15 minutes at four degrees Celsius and wash the cells with PBS-FCS-EDTA. Resuspend the cells at a concentration of five times 10 power seven cells per mL. Filter on a 60-micrometers tissue filter and label dead cells by adding DAPI at a final concentration of 0.1 microgram per mL.

Sort cells with a 70-micrometers nozzle in FACS Aria by gating on DAPI-TCR-negative, CD45RA-negative, CD4-positive, CD45R-positive. Harvest the spleen of a naive Lewis 1A rat and save it in cold PBS. Transfer the spleen in a sieve placed into a dish and add 10 mLs PBS.

Crush the spleen with a syringe piston to dissociate the cells. Transfer the cells into a tube and complete to 50 mL with PBS and wash them with 10 minutes 430 g centrifugation. This cause a supernatant.

To eliminate red blood cells and platelets, resuspend the splenocytes pellets in 10 mL hypotonic solution five minutes at room temperature, then wash with PBS with 10 minutes, 190 G centrifugation. Discard supernatant. Remove collagen fibers by filtering in 100 micrometers tissue filter.

Count the cells to adjust cell concentration at five times ten to the seven cells per mL in PBS at CSEDT8. Cells of interest should be enriched before sorting. Incubate 15 minutes at four degrees Celsius in NTCD8 alpha to label CD8 cells and TCD45R8 to label B cells and TCD161 to label NK cells and TCD11B/C to label myeloid cells and TT0 gamma delta to label gamma delta T cells then wash with PBS-FCS-EDTA with 10 minutes 430 G centrifugation, discard supernatant.

Wash magnetic dynobeads, use 35 microliters dynobeads for 10 power six splenocytes. With 30 mL PBS PBS-FCS-EDTA, place one minute on the magnet and discard supernatant. Repeat three types.

Resuspend dynobeads in 10 volumes of PBS FCSEDT8. Remove unwanted cells by mixing splenocytes with dynobeads for 10 minutes at four degrees Celsius under agitation. Then place the tube on the magnet one minute.

Transfer the supernatant in a new tube, repeat twice. Count the cells and adjust cell concentrations to five times 10 power seven cells per mL in PBS PBS-FCS-EDTA. Add labeled antibodies to sort CD4 positive CD25 positive T cells, and TTCR alpha beta, and TCD4, and TCD25, and incubate 50 minutes at four degrees Celsius.

To simultaneously sort CD8 positive CD45RClow Tregs, add NTCD45RC antibody. Beads, com beads, should be labeled with each antibody to set up a compensation metrics for further processing of flow cytometry. Wash the cells with PBS SCSDT8 and discard the supernatant.

Suspend cells at five times 10 power seven cells per mL. Filter in a 60 micrometer tissue, and label the cells by adding DAPI to find the concentration of 0.1 microgram per mL. Sort cells with a 70 micrometers nozzle facts area by gating on a DAPI TCR positive CD4 positive CD25 negative cells as responder cells.

So that PTCR positive CD4 positive CD45RClow CD4 positive Tregs and DAPI TCR positive CD4 negative CD45RClow CD8 positive Tregs. After respond to cell sorting, wash twice the cells with PBS. Resuspend the cells at a concentration of 10 power seven cells per mL in PBS and incubate with 0.5 micromolar of CSFE for five minutes at room temperature in the dark.

Stop the reaction by adding 1.5 volume of FCS and centrifuge 10 minutes at 430 G at four degrees Celsius. Discard the supernatant. Wash twice the cells with complete medium and counter cells.

Prepare culture medium with RPMI 1640 medium supplemented with beta-mercaptoethanol, five times 10 power minus five molar. Penicillin, 100 unit per mL. Streptomycin, 0.1 milligram per mL.

Sodium pyruvate, one millimolar. Glutamine, two millimolar. Hepes buffer, one millimolar.

Nonessential amino acids, one X.FCS, 10%Culture cells in 96 well U bottom plates. Add cells in the following order. Suppressor cells, responder cells, and allogeneic cells.

Keep proportions of five times 10 power seven responder cells for 200 micro light to medium per well. Transfer the cells from 96 U bottom plates to 96 V bottom plates. And centrifuge one minute at 1200 G at four degrees Celsius.

Save the supernatant in a separate plate and freeze at minus 20 degrees Celsius for cytokine dosage if needed. Wash the cells with 200 microliter of PBS-FCS-EDTA per well and centrifuge at 1200 G one minute at four degrees Celsius. Add antibodies on the cells to further gate on the responder T cells, NTTCR, and NTCD4.

Incubate 15 minutes at four degrees Celsius, and wash twice with 200 microliter of PBS-FCS-EDTA very well. Add 100 microliter of DAPI diluted at 0.1 microgram per mL in PBS, and read the plate on a FACS contour or any equivalent. Analyze the CSFE profile by gating in DAPI negative, leaving cells CPD negative, non CD4 positive Tregs, TCR positive, CD4 positive cells.

Unstimulated responder cells should have maximal CSFE brightness. The day before the graft, anesthetize the rats with intramuscular injection of rampun, eight milligram per kilogram, ketamine, 80 milligram per kilogram, and irradiate them at a dose of 4.5 G rays with x-rays. 12 hours after irradiation, anesthetize rats by inhalation of isoflurane 024 percents.

Infuse the cells, T cells or B cells or myeloid cells or splenocytes as positive control of adaptive transfer of tolerance, intravenously in the penile vein. Next day, perform the allograft procedure and follow graft evolution by palpations through the abdomen. Collect blood in a tube by retro-orbital bleeding and centrifuge at 1200 G for five minutes at four degrees Celsius.

Save the serum, serum can be stored at minus 20 degrees Celsius, or used immediately. Harvest the spleen from a donor Lewis IW rat, and save it in cold PBS. Extract the cells as previously described.

Dilute the serum serially from one portion to one per 270 in PBS, four dilutions per sample. Inactivate complement by incubating the plate with the sera for 30 minutes at 56 degrees Celsius. At two times 10.5 splenocytes per well in a 96 well V bottom plate.

Incubate the cells for one hour at four degrees Celsius with 25 microliter of diluted serum per well. Wash the cells with PBS-FCS-EDTA and discard the supernatant. Incubate the cells with each mouse anti-rat IjJ subype ABS, labeled with free orochromes, for 30 minutes at four degrees Celsius.

Wash the cells with PBS-FCS-EDTA twice and discard the supernatant. Add 100 microliter of DAPI, diluted at 0.1 microgram per mL, and PBS, and read the plates on the FACS contour or any equivalence. Check the image I obtain, with the lowest one W serum from the NFI obtained for each reception of the corresponding dilution.

In this cardiac allograft model in rats, acute rejections occurs rapidly, in about seven days. And these models, tolerance can be induced by the blocking of CD40, CD40 ligand interactions with an add in of various coding CD40 EG.Cells from treated rats can be compared directly to cells from naive rats, and non treated grafted rats for suppressive activity or to any other regulatory cells perplasion. In the range of suppressor effector ratios.

In vivo, while B cells, myeloid cells, or T cells from non triturated rats are not able to any bits acute rejection and prolonged graft survival upon adaptive transfer, cells respond to chated suppressive activity from treated rejection scan. Tolerance can be characterized by the absence of humoral responses to its donor. The conservation of the kappa CT for the receptions to the humoral responses to new antigens and preservation of memory responses.

This method can help answer a key question in turn arounds, in transplantation, before translation to human. For men at all ages of a protocol that we have described here is that they can be used to asses with super specific capacity of any cell subsets, to identify a new biomarker or immunoregulatory molecules, and are adaptible to any setting, such as autoimmune diseases.