Home › Cell Biology › Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade
Cell Biology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/2673-v
What you'll learn
  • ✓ Induce alloantigen-specific anergy in human PBMCs using co-stimulatory blockade
  • ✓ Assess efficacy of anergy induction via mixed lymphocyte reaction (MLR)
  • ✓ Measure alloantigen specificity and proliferation by thymidine incorporation
  • ✓ Generate non-alloreactive donor cells for clinical hematopoietic stem cell transplantation
Protocol

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Difficulty
advanced
Total time
~5–7 days (primary culture, MLR setup, secondary MLR, thymidine incorporation readout)
Model organism
Human peripheral blood mononuclear cells (PBMCs)
Biosafety
BSL-2

Steps

1
Prepare PBMC and establish bulk alloanergizing cultures

Isolate human peripheral blood mononuclear cells and initiate co-stimulatory blockade cultures with alloantigen stimulation to induce anergy in responder T cells.

▶ 02:00
2
Measure co-stimulatory blockade efficacy by primary MLR

Perform mixed lymphocyte reaction on treated PBMCs to quantify T-cell proliferation reduction following co-stimulatory signal blockade during initial alloantigen exposure.

▶ 03:57
3
Assess specificity of alloanergizing across multiple alloantigen sources

Evaluate whether anergy induced against one alloantigen donor transfers or remains specific when cells are re-exposed to different alloantigen donors.

▶ 06:48
4
Quantify proliferation in primary and secondary MLR

Measure thymidine incorporation to determine DNA synthesis and confirm reduced proliferation in secondary MLR of alloanergized cells versus controls.

▶ 09:05
5
Interpret co-stimulatory blockade and anergy results

Analyze data showing reduced primary MLR proliferation and decreased secondary MLR proliferation in alloanergized PBMC populations, validating anergy induction.

▶ 09:43
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