The emergence of novel immunomodulatory therapeutics has brought more attention to the evaluation of potential adverse effects on the immune system, including the developing immune system. The T-dependent antibody response (TDAR) is considered as the main functional test to address immunotoxicity in non-clinical setting; however, assay designs need optimization for use in younger animals.

In this study, Keyhole Limpet Hemocyanin (KLH), selected as the T-cell-dependent antigen, was used to immunize juvenile rats. Cyclophosphamide (CPA) served as a positive control for immunotoxicity evaluation and demonstration of assay design appropriateness. Twenty-two days old rats were injected for 42 days with CPA at 0, 2, 4 or 6 mg/kg/day. Primary and secondary KLH immunizations were given on Days 7 and 35. Anti-KLH antibody response was evaluated by ELISA from serum samples collected prior and following each immunization. Complementarily, immunophenotyping was performed on terminal blood samples to evaluate immunotoxic effects on lymphocyte subsets.

An anti-KLH IgG response was detected following both immunizations, although the secondary challenge produced a more robust response. CPA treatment at 6 mg/kg/day significantly inhibited the anti-KLH response for both sexes on all occasions. Inhibition was also achieved at 4 mg/kg/day only for the secondary response, suggesting that the secondary immunization provides a more sensitive assessment of immunosuppression by CPA. Reduction in T lymphocytes was achieved with CPA at 2 mg/kg/day, and at 4 mg/kg/day for B lymphocytes and NK cells.

The identification of a well-tolerated dose of CPA which induced significant immunosuppression will support future immunotoxicology studies in juvenile rats.