Loss of T cell responses following long-term cryopreservation

Abstract

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-gamma responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4(+) T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8(+) T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8(+) T cell responses to peptides and peptide pools were well preserved. Loss of both CD4(+) and CD8(+) T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4(+) T cell responses to peptide stimulation and partially restored T cell IFN-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4(+) T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4(+) and CD8(+) T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4(+) T cell responses and mild skewing of T cell phenotypic marker expression.

Fig 1

Loss of T cell IFN-γ responses in HIV⁺ individuals after long-term cryopreservation. PBMC from the same phlebotomy were frozen and tested for (a) CD4⁺ IFN-γ and (b) CD8⁺ IFN-γ responses to SEB, HIV peptides, p55 protein, MN and CMV lysate after short-term (<170 days) and long-term (>300 days) storage by 3 or 8-color intracellular cytokine staining. Individual subjects are represented by distinct symbols, with dashed lines representing PBMC from acute HIV infection and solid lines representing PBMC from chronic HIV infection. Subject symbols are consistent throughout the panels and figures.

Fig 2

Confirmation of loss of CD4⁺ IFN-γ responses in HIV⁺ individuals after long-term cryopreservation. Fresh, whole blood was stimulated with CMV pp65 and Gag p55 peptide pools and (a) CD4⁺ T cell and (b) CD8⁺ T cell IFN-γ responses were measured by four-color intracellular cytokine staining. PBMC from the same phlebotomy were cryopreserved for long periods of time (345−1907 days) and stained with six-color intracellular cytokine staining at UCSF CIL. Individual subjects are represented by different symbols, with grey lines representing PBMC from HIV⁻ individuals, dotted and dashed lines representing Towne CMV vaccine recipients, dotted lines representing acute HIV infection, solid lines representing PBMC from chronic HIV infection and solid lines with grey symbols representing individuals from a HIV STI cohort. Subject symbols are consistent throughout the panels.

Fig 3

Correlation of responses in PBMC frozen at two independent laboratories. Long-term cryopreserved PBMC (621–917 days frozen) from six individuals from the chronic HIV cohort (SCOPE) from the same phlebotomy processed at BSRI or at UCSF ASB were stimulated with SEB, HIV peptides, p55 protein or MN. CD4⁺ IFN-γ (a) and CD8⁺ IFN-γ (b) T cell responses were measured by three-color flow cytometry. Two-tailed, paired t tests were performed, and Spearman’s rank correlation coefficient (Rho) was calculated for BSRI processed versus ASB processed PBMC.

Fig 4

Bland–Altman analysis of T cell IFN-γ responses. Bland–Altman plots of fresh or short-term cryopreserved responses minus long-term cryopreserved responses vs. the average response are shown for (a) CD4⁺ T cell IFN-γ and (b) CD8⁺ T cell IFN-γ responses to HIV peptides, p55 protein, Gag p55 peptide pool and CMV pp65 peptide pool in HIV⁺ and HIV⁻ individuals. The mean bias was plotted with a solid line and 95% limits of agreement with dashed lines.

Fig 5

Addition of autologous APC to long-term cryopreserved PBMC. Autologous B-LCL pre-pulsed with SEB, p55 protein, MN or individual HIV peptides were added at a 1:1 ratio to long-term cryopreserved PBMC (634–939 days frozen; hatched bars) and (a) CD4⁺ or (b) CD8⁺ T cell IFN--γ responses were compared to those measured in short-term cryopreserved PBMC (black bars) or in long-term cryopreserved PBMC stimulated without autologous B-LCL (grey bars). The data shown are from four individuals tested from a cohort of individuals with chronic HIV infection.

Fig 6

T cell IFN-γ responses not restored by overnight resting. Long-term cryopreserved PBMC (546–923 days frozen) were rested overnight (hatched bars) or stimulated directly after thawing (grey bars) with SEB, p55 protein, MN or HIV peptide 7874 (within Gag p17) and stained for intracellular IFN-γ expression in (a) CD4⁺ and (b) CD8⁺ T cells using the 3-color panel. Responses measured in the short-term (unrested) cryopreserved PBMC are shown by the black bars. The data shown are representative of 1 out of 10 individuals tested from a cohort of individuals with chronic HIV infection.

Fig 7

Decreased naïve cells after long-term cryopreservation. (a) Naïve cells are defined as CD45RA⁺ CD28⁺; central memory cells as CD45RA⁻ CD28⁺ and effector memory cells as CD45RA^+/− CD28⁻. Summary results are gated on viable (b) CD3⁺CD4⁺ and (d) CD3⁺CD4⁻" unstimulated PBMC from fresh and long-term cryopreserved (>300 days) samples from the same phlebotomy. Two-tailed, paired t tests were performed with significant p values highlighted in bold. Bland−Altman plots showing the difference of fresh PBMC (naïve, central memory or effector memory) minus long-term cryopreserved PBMC (naïve, central memory or effector memory) vs. the average are shown for (c) CD4⁺ and (e) CD8⁺ T cell phenotypes. The bias is plotted with a solid line and 95% limits of agreement with dotted lines.

Fig 8

Increased apoptosis after long-term cryopreservation. Intracellular caspase-3 staining of (a) CD4⁺ IFN-γ⁻ and (b) CD8⁺ IFN-γ⁻T cells from freshly isolated and long-term cryopreserved (>300 days) PBMC following no stimulation or overnight stimulation with SEB, p55 protein, or CMV lysate is shown for individuals from the acute HIV infection cohort. Two-tailed, paired t tests were performed, with significant p values highlighted in bold.