Cryopreservation decreases receptor PD-1 and ligand PD-L1 coinhibitory expression on peripheral blood mononuclear cell-derived T cells and monocytes

Abstract

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.

FIG. 1.

Three-color flow cytometric evaluation of PD-1 and PD-L1 expression on unstimulated and PHA-stimulated PBMC-derived CD3⁺/CD8⁻ and CD3⁺/CD8⁺ T cells. Freshly prepared PBMC from an adult control were cultured for 3 days with and without PHA (20-μg/ml final concentration).

FIG. 2.

Flow cytometric evaluation of competitive binding of various concentrations of unlabeled functional anti-PD-1 antibodies with a constant concentration of PE-conjugated anti-PD-1 antibodies for PD-1 receptors on 2 × 10⁵ PHA-stimulated PBMC.

FIG. 3.

TH-1/TH-2 cytokine levels in culture fluids of PHA-stimulated adult control PBMC with and without coinhibitory blockade. Freshly isolated (A) and cryopreserved (B) PBMC were cultured for 24 h with and without monoclonal antibodies specific for the coinhibitory receptor/ligands PD-1, PD-L1, and PD-L2.

FIG. 4.

TH-1/TH-2 cytokine levels in culture fluids of Candida-stimulated adult control PBMC with and without coinhibitory blockade. Freshly isolated (A) and cryopreserved (B) PBMC were cultured for 48 h with and without monoclonal antibodies specific for the coinhibitory receptor/ligands PD-1, PD-L1, and PD-L2.

FIG. 5.

TH-1/TH-2 cytokine levels in culture fluids of PHA-stimulated HIV-positive PBMC with and without coinhibitory blockade. Freshly isolated (A) and cryopreserved (B) PBMC were cultured for 24 h with and without monoclonal antibodies specific for the coinhibitory receptor/ligands PD-1, PD-L1, and PD-L2.

FIG. 6.

Influence of coinhibitory signaling blockade on IL-4 (A) and IL-5 (B) production by PHA-stimulated, freshly isolated control and HIV⁺ PBMC harvested at 48 h.