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Clinical and Vaccine Immunology, November 2009, p. 1648-1653, Vol. 16, No. 11
1071-412X/09/$08.00+0     doi:10.1128/CVI.00259-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cryopreservation Decreases Receptor PD-1 and Ligand PD-L1 Coinhibitory Expression on Peripheral Blood Mononuclear Cell-Derived T Cells and Monocytes{triangledown}

D. E. Campbell,* N. B. Tustin, E. Riedel, R. Tustin III, J. Taylor, J. Murray, and S. D. Douglas

The Joseph Stokes Jr. Research Institute, the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Received 26 June 2009/ Returned for modification 29 July 2009/ Accepted 27 August 2009

The B7-CD28 immunoglobulin superfamily of costimulatory and coinhibitory ligands and their cell receptors play a critical role in modulating immune responses. Imbalances in these immune regulatory signals occur in pathological conditions characterized by chronic antigenic stimulation. Clinical studies often rely on the use of cryopreserved peripheral blood mononuclear cells (PBMC) to evaluate cellular immune responses. The impact of cryopreservation on these coinhibitory ligands and their cell receptors is unknown. In our studies, cryopreservation significantly reduced the expression of both PD-1 and PD-L1 on PBMC-derived CD3+/CD8+ T cells and CD45+/CD14+ monocytes obtained from adult control subjects. Blockade of PD-1, PD-L1, and PD-L2 using both freshly isolated and cryopreserved PBMC led to higher levels of phytohemagglutinin (PHA) and Candida-induced gamma interferon (IFN-{gamma}), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-{alpha}) with no effect on IL-10 production. Coinhibitory signaling blockade of freshly isolated, PHA-stimulated PBMC from normal adult controls and human immunodeficiency virus (HIV)-infected subjects led to increased production of IL-4 and IL-5. Candida-stimulated PBMC preferentially induced IFN-{gamma} and TNF-{alpha} production, with reduced production of IL-2 and IL-10. This is in contrast to high levels of IFN-{gamma}, IL-2, and TNF-{alpha} production with PHA-stimulated cells. The effects of coinhibitory blockade on PHA and Candida-induced lymphoproliferation were varied, with freshly isolated PBMC from adult control subjects and HIV-infected patients yielding higher levels of lymphoproliferation in response to PD-1/PD-L1 blockade. Immune function studies employing cryopreserved cells may lead to increased T-cell effector cytolytic and regulatory immune responses.

* Corresponding author. Mailing address: Division of Allergy and Immunology, The Children's Hospital of Philadelphia, 34th St. & Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 590-2353. Fax: (215) 590-3044. E-mail: campbelld{at}email.chop.edu

{triangledown} Published ahead of print on 2 September 2009.

Clinical and Vaccine Immunology, November 2009, p. 1648-1653, Vol. 16, No. 11
1071-412X/09/$08.00+0     doi:10.1128/CVI.00259-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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