Application of popliteal lymph node assay to evaluate tolerogenic effect of donor leukocyte infusion

Popliteal lymph node assay (PLNA) has long been proposed to detect immunostimulating potential of chemicals. Here, the PLNA was used to evaluate the effect of donor leukocyte infusion on recipients' reaction to donor-specific antigens.

Donor rats' peripheral blood leukocytes (ranging from 1 x 10(4) to 500 x 10(4) cells) were intravenously (i.v.) infused into recipients. A week later recipients' reaction to donor-specific antigen was evaluated, using the PLNA technique, by subcutaneous injection of donor spleen cells to one hind footpad of recipients and injection of saline to the other. Seven days later all recipients were killed and their PLNs' weight and cellularity indices were determined. While the same process was applied to the positive control (PC) animals, rats without leukocyte infusion, negative control (NC) animals, rats without leukocyte infusion, were injected in both hind footpads with saline.

The PLN weight indices of recipients of: > or =5 x 10(4) leukocytes were significantly lower than PC animals (P < 0.001), whereas the weight indices of recipients of 1 x 10(4) cells were similar to PC group but higher than NC animals (P < 0.0001). However, the PLN cellularity indices of recipients of < or =10 x 10(4) cells were not different from PC animals but the PLN cellularity indices of recipients of: > or =50 x 10(4) cells were significantly lower than PC group (P < 0.05).

Overall, these results suggest that donor leukocytes infusion dose-dependently decrease reaction to donor-specific antigens, but a state of tolerance to donor antigen might be induced at the dose of: > or =50 x 10(4) cells. PLNA appears to represent a simple test model to quantify efficacy of immunotolerance protocols.