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Human lymphocyte activation assay: an in vitro method for predictive immunotoxicity testing.

Mark Collinge1, Susan H Cole, Patricia A Schneider

  • 1Drug Safety Research and Development, Pfizer Global Research and Development, Groton, CT 06340, USA. mark.collinge@pfizer.com

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Summary

A new human lymphocyte activation (HuLA) assay uses human cells to detect drug-induced immune suppression early in development. This in vitro method is sensitive to immunosuppressive compounds and suitable for high-throughput screening.

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Area of Science:

  • Immunology
  • Pharmacology
  • Drug Development

Background:

  • Preclinical immunotoxicity assessment is crucial in pharmaceutical development.
  • Current in vivo T-dependent antibody response (TDAR) assays are sensitive but impractical for early drug development due to limited test article quantities.
  • Existing in vitro assays like the Mishell-Dutton assay have limitations including inter-laboratory variability and use of rodent cells, hindering human translation.

Purpose of the Study:

  • To develop a novel in vitro human cell-based assay for assessing drug-induced immunosuppression.
  • The assay should be sensitive to immunosuppression, require minimal test article, and be amenable to moderate to high-throughput screening.
  • The assay should mimic the multi-compartmental cellular cooperation seen in in vivo T-dependent antibody response (TDAR) assays.

Main Methods:

  • Developed the human lymphocyte activation (HuLA) assay using frozen-stored human peripheral blood mononuclear cells.
  • Measured influenza antigen-specific responses.
  • Assessed sensitivity to known immunosuppressive compounds (cyclosporine, dexamethasone, rapamycin, mycophenolic acid, methotrexate).
  • Characterized proliferating lymphocyte subsets using flow cytometry.
  • Measured flu-specific antibody-secreting cells via ELISPOT.

Main Results:

  • The HuLA assay demonstrated sensitivity to immunosuppressive compounds within their therapeutic ranges.
  • Influenza antigen-specific proliferation involved CD4+ and CD8+ T-lymphocytes and B-lymphocytes.
  • ELISPOT analysis confirmed the detection of immunosuppression through antibody-secreting cells.
  • The assay showed robustness and reproducibility.

Conclusions:

  • The human lymphocyte activation (HuLA) assay is a straightforward, sensitive in vitro method for detecting immune suppression in human cells.
  • This assay is suitable for early-stage pharmaceutical development, compound ranking, and immunotoxicity assessment.
  • The HuLA assay offers a valuable alternative to in vivo methods, improving human relevance and efficiency.