Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years

Abstract

Background: Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation.

Methods: PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced.

Results: All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence.

Conclusions: This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Keywords: HIV reactivation; T cell function; T cell subsets; biobank; cryopreservation; immunophenotyping; memory T cells; viability.

Figure 1

Representative flowplots showing immunophenotype gating for major subpopulations of PBMC.

Figure 2

Major subpopulations of PBMC in recently cryopreserved healthy controls compared with long-term cryopreserved healthy control and viraemic HIV+ patient PBMC.

Figure 3

Representative flowplots showing immunophenotype gating for major subsets of CD4+ and CD8+ T cells in thawed PBMC samples.

Figure 4

Major CD4+ and CD8+ T-cell subsets in recently cryopreserved healthy control PBMC, compared with fresh PBMC, long-term cryopreserved healthy control PBMC, and long-term cryopreserved viraemic HIV+ patient PBMC.

Figure 5

Representative flow plots from original four-colour flow cytometry performed on whole blood samples during 1995–1997. (A) Immunophenotype gating for CD45RA+ and CD45RO+ CD4 T cells. (B) Expansion of highly activated CD38+HLA-DR+ CD8 T cells during early untreated HIV-1 infection compared with an uninfected control.

Figure 6

Correlations between original immunophenotyping performed in 1995–1997 in fresh blood and long-term cryopreserved PBMC separated from the same blood draw (Pearson correlation). Results for PBMC from HIV-uninfected controls are shown as solid circles, and results for viraemic HIV+ donors are shown as white circles for CD45RO+ and CD45RO negative subsets of CD4+ and CD8+ T cells. Results for CD38+HLA-DR+ activated CD4+ and CD8+ T cells are shown only for viraemic HIV+ donors. Results for CD28 negative versus terminally differentiated CD8+ T cells are shown separately for both HIV-uninfected controls and viraemic HIV+ donors.

Figure 7

Antigen-specific CD4 T-cell function assays in long-term cryopreserved PBMC. (A) Representative flow plots of mitogen- and antigen-specific CD4 T cells, gated on day 2 cultured CD3+CD4+ T cells, detected by activation induced marker (AIM) assay (defined as CD25+CD134+ cells). (B) Representative flow plots of proliferating mitogen- and antigen-specific CD4 T cells, gated on day 7 cultured CD3+CD4+ T cells (defined as CD25 high Forward Scatter high blast cells).

Figure 8

Antigen-and mitogen-specific CD4 T-cell function in >20-year cryopreserved PBMC from viraemic HIV+ patients (old HIV) and healthy controls (old), measured by day 2 AIM assay (A), and day 7 proliferating CD4 T-cell blasts (B).

Figure 9

Cytokine response to antigenic and polyclonal stimulation, measured in 48-h culture supernatants from long-term cryopreserved control donor PBMC (Friedman Test p values).

Figure 10

Recovery of HIV RNA after >20-year storage: activated HIV+ donor PBMC stimulated with anti-CD3/CD28/CD2. (A) HIV RNA transcripts in the R and pol regions. (B) Correlation between levels of HIV RNA “Double R” transcripts from activated PBMC and original plasma viral loads from 1995 to 1996. (C) Amplification of long HIV transcripts. (D) Presence of sequence mutations in amplicons from long HIV transcripts.