The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming

Abstract

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

Figure 1

Relationship between ex vivo and cultured ELISPOT responses to recall antigens in unvaccinated controls. (a) Correlation of ex vivo with cultured interferon-γ (IFN-γ) ELISPOT responses to purified protein derivative (PPD; n = 13) but not cytotoxic T lymphocyte (CTL; n = 12). (b) No correlation between interleukin-2 (IL-2) and ex vivo or cultured ELISPOT against CTL but (c) correlations for PPD. For PPD (d) weak or no correlations between CFSE proliferative index and ELISPOTs (n = 6).

Figure 3

Correlation of ex vivo and cultured interferon-γ (IFN-γ) ELISPOT responses following prime-boost ME-TRAP vaccination. Relationships are investigated between (a) ex vivo day 104 against cultured day 104 (NS); (b) ex vivo day 7 against ex vivo day 104 (NS); (c) ex vivo day 7 against cultured day 104 (NS); (d) cultured day 104 against cultured day 7 (P = 0·011) (n = 12).

Figure 2

ELISPOT responses following prime-boost ME-TRAP vaccination. (a) Time–course for ex vivo interferon-γ (IFN-γ) responses. Mean (+ SEM) IFN-γ SFC/M is shown. (b) Significant correlation between ex vivo and cultured responses on day 7 following vaccination (n = 12).

Figure 5

Time–course for memory marker expression on tetramer⁺ T cells during cultured ELISPOT. Tetramer⁺ T cells, as in Fig. 4, were examined for expression of CCR7 and CD62L. (a) Percentage of tetramer⁺ CCR7⁺ cells; (b) percentage of tetramer⁺ CD62L⁺ cells; (c) percentage of tetramer⁺ CCR7⁺ CD62L⁺ cells. Data are representative of at least two experiments for the full time–course and at least three experiments for key time-points (days 0 and 10).

Figure 4

Time–course for ELISPOT and tetramer positivity during cultured ELISPOT. (a) Interferon-γ (IFN-γ) ELISPOT was monitored during culture at days 0, 3, 7 and 10. Mean (+ SEM) IFN-γ SFC/M is shown. (b) Tetramer positivity was also monitored during culture. Gating was performed on lymphocytes, CD3⁺, CD4⁺ or CD8⁺ T cells, followed by tetramer positivity. Separate individuals were examined for each peptide. Data are representative of at least two experiments for the full time–course and at least three experiments for key time-points (days 0 and 10). (c) An example of tetramer⁺ cells against flu8 following culture is shown.

Figure 6

Effect of depletion of memory T-cell subsets on ex vivo and on cultured ELISPOT responses to recall antigens, and to vaccine antigens following prime-boost ME-TRAP vaccination. Ex vivo responses (mean SFC/M + SEM) to (a) flu8; (b) purified protein derivative (PPD); cultured responses to (c) flu8 and PPD; (d) circumsporozoite (CS) peptides; (e) TRAP peptides are shown in conjunction with depletion of CCR7⁺ or CD62L⁺ cells. Results are for one individual for each recall antigen (Flu8 and PPD) and two individuals (#1 and #2) for vaccine antigens (CS and TRAP). Data are representative of at least three experiments.