Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials

Abstract

A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.

Keywords: Cryopreservation; Peripheral blood mononuclear cells; Repository.

Fig. 1

IFN-γ ELISpot responses. The IFN-γ ELISpot responses are reported as spot forming cells per 10⁶ PBMC (y-axis). The stimulation conditions reported on the x-axis were unstimulated (medium only representing the background), the CEF peptide pool and CMV pp65 peptide pool. The mean and the standard error of the mean are indicated by the line and whiskers.

Fig. 2

Correlation between frequency of IFN-γ T cell responses detected by ELISpot and intracellular cytokine staining. The frequency of IFN-γ T cell responses detected by ELISpot is reported as spot forming cells per 10⁶ PBMC (y-axis). The frequency of the total viable CD3+IFN-γ+ T cells is reported on the y-axis. The Spearman correlation r values were determined using Prism software for responses to: A) the CEF peptide pool; and B) the CMV pp65 peptide pool. The p values for significance are reported in each figure.

Fig. 3

Frequency of HIV-1-specific T cell responses by intracellular cytokine assays. The frequency of viable CD3+ T cells against the PTE peptide pools representing the HIV-1 Env (A), Gag (B), Nef (B), Pol (C) gene products are reported on the y-axis. The responses to each peptide pool are grouped according to the gray arrows above each plot. The cell CD4+ and CD8+ CD3+ T cell subsets responding as detectable by the production of IFN-γ and IL-2 are reported on the x-axis.

Fig. 4

Longitudinal quality assessment of functional recall antigens responses. Five samples selected to represent a range of positive responses to the recall antigens represented by the CEF (A) and CMVpp65 (B) peptide pools are reported as spot forming cells per 10⁶ PBMC. Each line represents the longitudinal assessment for each individual sample as reported in the figure legend. The time of testing are reported on the y-axis.

Fig. 5

Functionality of cryopreserved NK cell-mediated ADCC. Cryopre-served human PBMC samples were genetically screened to identify donors with low affinity (158F, white bars) and high affinity (158V, grey bars) allelic variants of FcγR IIIa. The results are reported as the maximum % Granzyme B (%GzB) activity on the y-axis. The target cells were represented by the A1953 HIV-1 chronically infected target cell line. The purified NK populations were tested at an effector to target cell ratio of 5:1. Palivizumab (anti-RSV mAb) was used as negative control along with the plasma collected from a HIV-1 seronegative donor (seronegative plasma). The polyclonal IgG preparation, HIVIG, was tested as a positive control. The A32 and 2G12 mAb are anti-HIV-1 Env gp120 specific mAb tested as expressed with a consensus or AAA variant sequence of the Fc region. The height of each bar represents the average of two independent experiments, and the whiskers represent the standard error mean.

Fig. 6

Functionality of cryopreserved B cells detected by ELISPOT assay. Cryopreserved PBMCs were stimulated in the presence of two co-stimulatory TLR agonists CpG-C and R848 to detect the production of total or HIV-1 BaL gp140-specific IgG-producing B cells. The KLH reagent was used as negative control to determine background IgG detection. The results are reported as average of the IgG spot forming cells per 2 × 10⁵ cells in triplicate wells, and the whiskers represent the standard error mean. Each bar represents a different condition for detection of IgG producing cells, and the corresponding colors are depicted in the figure.