The overall goal of this procedure is to use the trans vivo DTH assay to monitor both pro and anti-inflammatory antigen specific responses in human subjects. First, isolate peripheral blood mononuclear cells from the human subject's peripheral blood. Then mix PBMC preparation with PBS.
Recall antigen test antigen and test antigen plus recall antigen. Proceed with the footpad prem measurements and inject prepared mixtures into skid mice. Footpads repeat measurements 24 hours later.Three.
Definitive patterns of DTH responses are possible regulatory, non-regulatory and sensitized. The trans vivo delayed type hypersensitivity assay can be applied to determine clinical parameters like allo react responses where the source of antigen is the cells of the transplant donor. The main advantage of this technique over existing in vitro methods is that one can easily detect both pro-inflammatory and regulatory responses to allo antigens, auto antigens, or tumor antigens using a single assay system.
Egon, a researcher in my laboratory will now demonstrate the procedure Using fresh human peripheral blood collected in acid citrate dextrose tubes to avoid platelet activation. Isolate PBMC using lymphocyte separation medium according to standard methods. Wash the PBMC preparation with PBS to remove contaminating platelets.
If there is a noticeable red blood cell contamination, perform lysis of the red cells using a CK lysis buffer. Then perform two more washes with PBS Resus. Suspend the PBMC in PBS at a concentration of 10 million PBMC per milliliter for the allo antigen.
Start with P BMCs, isolated from the donor peripheral blood using the procedure shown earlier. Then resus. Suspend the donor cells in PBS at a concentration of 120 million cells per milliliter and add one micromolar of PMSF to prevent protein degradation.
Now sonicate the cell suspension using seven one second pulses with a two millimeter probe sonica. Using a hemo cytometer. Verify the disruption of greater than 90%of the cells.
Next, centrifuge the mixture at 14, 000 RPM at four degrees Celsius for 20 minutes. Carefully transfer the supernatant to a two safe lock tube and determine the protein concentration by standard methods for each injection. Aliquot seven times 10 to the sixth P BMCs into two milliliters.
Safe lock tubes pellet the cells by centrifugation. First, prepare the negative control suspension of PBMC in 35 microliters of PBS. Then for the positive control, add 25 microliters of a recall antigen, tetanus, toxoid, diptheria, toxoid, and adjust the injection volume to 35 microliters with PBS.
As per the experimental design for evaluating donor specific response, resuspend the cell pellet in 10 microliters of donor antigen plus 25 microliters of PBS for a total volume of 35 microliters. For evaluating the experimental donor antigen specific regulation, resus suspend the cell pellet in 35 microliters by adding 10 microliters of donor antigen and 25 microliters of recall TT DT antigen. After anesthetizing, the skid mouse with isof fluorine place a spring loaded caliper at the center of a footpad with one edge touching the last walking pad of the foot to provide a benchmark to keep the measurement site consistent when the gauge reading has stabilized.
Record the baseline footpad thickness for the footpad injection. Place the syringe with the needle pointing towards the toes and the bevel facing up. Now begin subcutaneous injections of each cell suspension into the rear foot pads for mouse.
Number one, slowly inject the right foot pad with negative control prep. Next, inject the left foot pad with the positive control of PBMC plus TT dt. Now return the mouse to its cage for mouse.
Number two, inject the right footpad with PBMC plus allo antigen and the left footpad with PBMC plus allo antigen plus TT dt. Make sure that there is no leakage around 18 to 24 hours after injection. Anesthetize each mouse with isof fluorine and repeat the measurement of footpad swelling.
Subtract the thickness of each foot pad prior to injection from the post-injection value to obtain the footpad swelling value. Then calculate the antigen specific net swelling by subtracting control footpad swelling value from the footpad swelling values obtained from the treatments. Verify the positive control reading a positive control response to recall antigen TT DT of greater than or equal to 25 times 10 to the minus fourth inches over.
Background response to PBS is required for the test to be considered valid. Next, determine the inhibition of recall responses in the presence of donor antigens. This experiment evaluates renal transplant recipients for donor antigen specific response and for regulation.
There are three main patterns of delayed type hypersensitivity in transplant recipients, regulatory, non-regulatory, and sensitized. All patients responded strongly to the positive control of recall antigen tt. Patient number 62 shows the regulatory pattern characterized by a weak response to donor antigen and a marked linked suppression to recall antigen response in the presence of donor antigen.
This is the pattern that has been found to be associated with organ allograft tolerance. Patient number 48 exhibits a non-regulatory pattern, which has the feature of a weak response to donor, but no linked suppression. This pattern has been frequently observed in patients taking immunosuppressive drugs.
Patient number eight exhibits the donor sensitized pattern characterized by a high response to donor antigen and no linked suppression. This response is associated with graft rejection. In clinical case studies, P BMCs from subjects enrolled into an observational trial were assayed for donor specific indirect pathway, T effector and T regulatory responses to evaluate the role of donor specific regulation in clinical tolerance.
The indirect tector responses to donor antigens reveals a distinct spectrum across the enrollment groups. The footpad swelling increases as the patient's clinical status moves from those that are tolerant to those that are chronically rejecting. Here, the linked suppression assay measures the anti donor indirect pathway of T regulatory response.
These data show a decrease in regulatory responses over the range from most tolerant patients through intermediate to least tolerant chronically rejecting patients. Taken together. Immune regulation as measured by the trans vivo DTH assay appears to be an important mechanism for renal allograft acceptance.
A promising area is the application of the TDTH assay in monitoring of autoimmunity. Our study of the role of collagen type five in the pathologic process of bronchiolitis OBL syndrome revealed that the highest relative risk of BOS development was observed in patients with a positive response to collagen. Five pbmc from lung transplant recipients, but not from healthy controls or collagen.
Four reactive good pasture syndrome Patients after renal transplant were frequently collagen five reactive. The TDTH assay can also be used to test the reactivity of pbmc from cancer patients to tumor specific antigens. For example, the presence of PAP specific T regulatory cells in prostate cancer patients may limit the response to vaccination with PAP antigen.
The TRANSVAAL DTH assay is a novel diagnostic test with clinical application in assessing cell-mediated immune responses in transplant cancer and autoimmune patients. It is valuable because it is not only useful in monitoring t effector recall responses, but it can also be used to detect T regulatory responses because it does not rely on a particular cytokine measurement for detecting effect or regulatory T cells. It is very flexible and broadly applicable.
