Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Summary

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Abstract

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.

Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed ^1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a naïve mouse and DTH-like swelling is measured after 18-24 hr ³. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.

The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts ^2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.

A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity^15,16 and also in tumor immunology ¹⁷.

Protocol

1. Preparation of Lymphocytes

1. Collect blood into ACD (Acid Citrate Dextrose) tubes.
2. Isolate PBMC from fresh human peripheral blood using Lymphocyte Separation Medium according to standard methods.
3. Wash the PBMC three times with PBS to remove contaminating platelets. Platelets were found to interfere with trans-vivo DTH assay. Maximal allowable platelet contamination of PBMC preparation is ≤1x10⁷/injection.
4. If there is a noticeable red blood cell contamination, perform lysis of red cell using ACK lysis buffer after first wash. Remove ACK buffer by washing 2 times with PBS.

2. Preparation of Alloantigen

1. Isolate PBMC from the donor peripheral blood using the procedure shown above.
2. Resuspend donor PBMC in PBS at a concentration of 120x10⁶ cells/ml (4x10⁶ cells / 30 μl).
3. Add 1 μM PMSF to the mixture to prevent protein degradation.
4. Sonicate the cell suspension using seven 1-second pulses with a 2 mm-probe sonicator. (Note: Keep the material cold and avoid excessive bubbles. If foaming occurs, let the cell suspension sit on ice for a 2-3 min.)
5. Verify the disruption of >90% of the cells using a hemocytometer.
6. Centrifuge the mixture at 14,000 rpm at 4 °C for 20 min in refrigerated microfuge.
7. Transfer supernatant to a new 2.0 ml safe-lock tube and determine protein concentration.

3. Cell Preparation for Injections

1. For each injection aliquot 7x10⁶ PBMC into 2.0 ml safe-lock tubes.
2. Centrifuge at 6,000 rpm for a minute in microfuge and remove the supernatant.
3. Resuspend the cells in PBS with or without antigens. Adjust the injection volume to 35 μl with PBS. The following scheme is used for injections:
   Negative Control: PBMC + PBS
   Positive Control: PBMC + TT/DT (25 μg/injection)
   Experimental Antigen Specific Response: PBMC + test Ag (4-8 μg/injection)
   Experimental Antigen Specific Regulation: PBMC + test Ag + TT/DT

4. Pre-measurement, Injection, and Post-measurement

1. Anesthetize CB17 SCID mouse with isoflurane. Measure rear footpad thickness using a spring-loaded caliper. Put caliper at the center of the footpad, with one edge touching the last walking pad of the foot, to provide a benchmark to keep the measurement site consistent. Footpad thickness is recorded when gauge reading has stabilized.
2. Slowly inject cell suspensions subcutaneously into the footpads of the mice using ½ cc insulin syringes with 28-gauge needle. Perform injection with the needle pointing toward the toes, and the bevel facing up. (Note: make sure there is no leakage).
3. 18-24 hr after injection, anesthetize mouse with isoflurane and repeat measurement of footpad swelling.
4. Subtract the thickness of each footpad prior to injection from the post injection value to obtain the footpad swelling value, expresses the data in units of 10^-4 inches. Calculate the antigen-specific net swelling by subtracting control footpad swelling (PBMC+PBS) from the footpad swelling values obtained from the treatments (PBMC + donor alloantigen, TT/DT, or donor alloantigen + TT/DT). A positive control response to recall antigen TT/DT of ≥ 25x10^-4 inches over background response to PBS is required for the test to be considered valid.
5. Determine the inhibition of recall responses in the presence of donor antigens by comparing the net swelling of each injection using the following formula:
   

Results

1. Evaluation of renal transplant recipients for donor antigen-specific response using Tv-DTH assay

To test the donor-reactive cellular immunity in renal transplant recipients we injected PBMC from these patients with donor antigens alone or with a recall antigen tetanus toxoid (TT). As a positive control cells were injected with TT alone. We observe three main patterns of delayed type hypersensitivity in transplant recipients (Figure 1). All patients responded strongly to Recall...

Discussion

The trans-vivo DTH assay is a novel diagnostic test with a potential clinical application in assessing cell-mediated responses in transplant, cancer and autoimmune patients. It is valuable because it is not only useful in monitoring recall T effector responses, but also it can detect T regulatory responses. A reliable way to detect human DTH regulation might predict safety of immunosuppression withdrawal in patients who are candidates for monotherapy or tolerance trials.

The Tv-DTH is very sen...

Materials

Name	Company	Catalog Number	Comments
ACD tube for blood collection	BD	02-684-26
Lymphocyte Separation Medium	Cellgro	25-072-CV
Dulbecco's Phosphate-Buffered Saline	Cellgro	21-031-CM	Without calcium & magnesium
ACK Lysis Buffer	BioWhittaker	10-548E
TT/DT or EBV	Sanofi Pasteur Inc./ Meridian Life Science, Inc.		TT/DT 25 μg/injection EBV 8 μg/injection
Protease inhibitor PMSF	Sigma-Aldrich	78830
Eosine for cell count	Sigma-Aldrich	E-6003
Alloantigen			Purified HLA antigens, synthetic allopeptides can be used instead of donor cell-free lysates
50 ml sterile centrifuge tubes	Fisher Scientific	06-443-18
10 ml pipettes and pipettor	BD Falcon	13-675-20
2 ml safe-lock tubes	Costar	3213
1000 μl, 100 μl , 10 μl pipettes with sterile tips
Hemocytometer	Fisher	02-671-10
Full size centrifuge and microfuge	Beckman Coulter/Eppendorf
1/2cc or 1cc insulin syringes	Becton Dickinson	14-826-79	28 gauge
Vibracell sonicator	Divtech Equipment Co. Sonocs Materials Inc		2 mm probe
Dial thickness gauge	Mitutoyo, Japan
SCID mice	Harlan
Isoflurane	Piramal Healthcare		Inhalant anesthesia