Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

The overall goal of the following experiment is to energize recipient allo specific donor T cells by ex vivo stimulation with recipient allo antigens in the presence of antibodies that block antigen presenting cell co-stimulation of T cells via their CD 28 receptor. This is accomplished by first isolating PBMC from two different healthy donors. Next, a bulk allo energizing co-culture is set up by incubating the HLA mismatched PBMC populations in the presence of monoclonal antibodies to the ligands B 7.1 and B 7.2 to block CD 28 stimulation.

Then the efficacy of the allo energization is measured by primary and secondary mixed lymphocyte reactions or MLR. Finally, the allo specificity of the allo energization is measured by determining third party and CMV specific responses. Results are obtained that show that donor T cells are rendered hypo responsive to subsequent allo antigenic stimulation, but that non allo react donor T cells are left intact and functional.

We've successfully employed this strategy in previous and ongoing pilot clinical trials of infusion of allo energized donor T cells during and after HLA mismatch stem cell transplantation and observed improved immune reconstitution fewer infections and less severe acute and chronic graft versus host disease than seen in historical control of recipients of unmanipulated HLA mismatch transplants. A major advantage of the strategy of allo anodization is that off-target effects are minimized as the technique does not reduce pathogen specific or tumor associated antigen specific T-cell responses or eliminate CD four regulatory T cells from the donor T-cell pool demonstrating the procedure will be Christine Vin, a staff scientist in our laboratory. The first step is to draw blood from two healthy volunteer donors.

Cells from one donor will serve as the responder and the cells from another donor will serve as the stimulator. Next, isolate PBMC from the blood by density gradient centrifugation using fial pac. Stay in a small amount of cells with trian blue and count viable PBMC using the hemo cytometer reus.

Spend PBMC from each donor in 10 milliliters. Complete culture media at a concentration of 1 million viable cells per milliliter in separate 15 milliliter conical tubes. Designating one tube of donor PBMC as stimulators the other as responders to set up the bulk allo energizing co-culture.

Add 100 micrograms of anti B 7.1 and anti B 7.2 antibodies to the 15 milliliter tube designated stimulator. Gently agitate the tube and incubate for 30 minutes after the incubation period. Irradiate the stimulator PBMC and add them to a T 25 50 cubic centimeter cell culture flask with a gas permeable cap.

Label the flask bulk allo coal culture. Next, add 10 milliliters of responder PBMC from the other donor at 1 million per milliliter in complete media to the flask. Add a further 100 micrograms of each of the antib 7.1 and Antib 7.2 antibodies to the flask and mixed gently prior to placing the flask upright in an incubator for 72 hours.

Next, irradiate another 10 million of the stimulator PBMC for a bulk control co-culture without blocking antibodies and then add them to a T 25 50 cubic centimeter cell culture flask. Label the flask bulk control coal culture. Add 10 milliliters of the same responder PBMC as used for the bulk allo energizing coal culture at 1 million per milliliter in complete media to the flask and mix gently prior to placing the flask upright in an incubator for 72 hours.

The next step is to measure the function of the allo energized donor T-cell pool on the same day as the bulk coal cultures label one U bottom 96. Well plate primary MLR for each of the three time points to be measured. Then decant five milliliters from each of the bulk control and all energizing co cultures into 15 milliliter tubes.

Next, pipette 200 microliters of cell suspension from each tube into triplicate wells on each of the three plates. Label these wells no co-stimulatory blockade or no CSB for the cells from the bulk control cold cultures and CSB for the cells from the bulk allo energizing cold cultures. Then add 200 microliters of complete media to six wells on each plate as negative controls.

Finally, add 200 microliters of PBS to all the empty wells to reduce evaporation and place the plates in the incubator 72 hours after setting up the bulk co cultures label one UBO 96, well plate secondary MLR for each of the three time points to be measured. To prepare the responder cells for the secondary MLR transfer five milliliters from each of the bulk control and allo energizing co cultures to 15 milliliter tubes and centrifuge the tubes for five to 10 minutes at 2000 RPM at room temperature. Then wash the cells two more times by carefully aspirating the SNAs, disrupting the pellets, adding 10 milliliters of complete media to each tube, and then centrifuge in the cells again after the second wash.

Resus suspend the cells in one milliliter of complete media. Use trian blue staining to enumerate the viable cells and adjust the cell concentration to 1 million viable responder cells per milliliter. Pipette 100 microliters of cell suspension from each tube into triplicate wells of each plate.

Label these wells first party or FP alloy stimulation. Then pipette 100 microliters of cell suspension from each tube into another three wells on each plate and labeled these wells CD three slash 28 stimulation PBMC from the same donor used to supply first party stimulator PBMC for the allo. Energized and control co cultures are used for stimulator cells for secondary MLR suspend 10 million of the first party donor PBMC at a concentration of 1 million per milliliter and a radiate add.

100 microliters of irradiated stimulator cells to the wells labeled fp allo restimulation on each secondary MLR plate. For positive controls, add one microliter each of CD three and CD 28 monoclonal antibodies and 98 microliters of complete media to the wells labeled CD 3 28 stimulation on each secondary MLR plate. In addition, add 200 microliters of complete media to six empty wells on each plate to serve as negative controls and add 200 microliters of PBS to all empty wells to reduce evaporation.

Place the plates in the incubator. The next step is to measure the specificity of the allo PBMC isolated from a new healthy donor termed the third party donor are used to test the specificity of the allo process in a secondary MLR. Suspend the third party PBMC at 1 million cells per milliliter and irradiate label 3 96 while plates secondary MLR specificity day three, five and seven.

To prepare the responder cells for the secondary MLR to measure specificity, transfer five milliliters from each of the bulk control and allo energizing co cultures to 15 milliliter tubes and centrifuge the tubes for five to 10 minutes at 2000 RPM. Then wash the cells twice by carefully aspirating the supra names, disrupting the pellets, adding 10 milliliters of complete media to each tube and centrifuging the cells again after the second wash. Re suspend the cells in one milliliter of complete media.

Use trian blue staining to count the cells and adjust the cell concentration to 1 million viable responder cells per milliliter and then pipette 100 microliters of cell suspension from each tube into triplicate wells on each plate. Label these wells TP or third party allo stimulation. Then add 100 microliters of irradiated TP stimulator cells to the wells labeled TP allo stimulation.

To further test the specificity of allo responses to pathogens are measured after energization of PBMC from donors with previously established proliferative responses to CMV label 3 96. Well plates secondary M-L-R-C-M-V day three, five and seven, and transfer five milliliters from each of the bulk control and allo energizing coal cultures to 15 milliliter tubes. Then wash count and resuspend cells at 1 million per milliliter in complete media pipette 100 microliters of cell suspension from each tube into three wells on each plate, and label these wells CMV stimulation.

Then add 0.1 microliters of CMV lysate in 100 microliters complete media to wells labeled CMV stimulation. Then add 200 microliters of complete media to six wells on each plate as negative controls. Finally, add 200 microliters of PBS to all empty wells to reduce evaporation and place the plates in the incubator to measure proliferation in primary and secondary MLR.

Add one micro curry of tritiated thymidine to the negative control and cell containing wells 16 hours prior to harvesting the plates. Collect the thymidine on one filter mat for each plate using a TomTec harvester or similar air. Dry the filter mats for one hour.

Then place them in individual sample bags. Add five milliliters scintillation fluid to each and seal. Measure the tri thymidine incorporation in the last 16 hours of the experiment by reading the filter mats with a wallach micro beta scintillation counter, or similar using HLA mismatch stimulator and responder PBMC.

It can be seen in this figure that the presence of co-stimulatory blockade in the primary MLR reduces mean allo proliferation of responder PBMC at day five to around 30%of that seen in control primary MLR in the absence of co-stimulatory blockade. This is equivalent to a mean inhibition of primary allo proliferation of around 70%at day five. As seen in this figure.

It can be seen in this figure that in the secondary MLR first party allo proliferation of allo energized PBMC at day five is typically 10 to 15%of that seen with control PBMC. This is equivalent to a mean inhibition of proliferation of 85 to 90%demonstrating that allo energized PBMC are hypo responsive to first party allo stimulators. As seen in this figure, Once mastered bulk co cultures to allo energized donor peripheral blood mononuclear cells can be set up in two to three hours.

The approach can be easily standardized for clinical use to generate donor T cells with reduced allo reactivity, but retained pathogen specific immunity. Such cells can be adoptively transferred in the setting of allogeneic hematopoietic stem cell transplantation to improve immune reconstitution without excessive graft-versus-host disease. While Attempting this procedure, it's important to remember to work quickly.

This helps preserve the viability of cells and avoid non-specific activation or loss of antigen presenting cells. If using cryo-preserved PBMC for stimulators or responders, a validated protocol for resuscitation of frozen cells that gives consistently high yields of viable cells should be used. After watching this video, you should have a good understanding of how to allo anize human donor, peripheral blood mononuclear cells, and how to perform assays to assess the efficacy and specificity of this procedure, the strategy can be used to generate human donor T cells with reduced allo responses, which can be used clinically to improve immune reconstitution after allogeneic hematopoietic stem cell transplantation.