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. 2010 Feb 23;107(8):3669-74.
doi: 10.1073/pnas.0915168107. Epub 2010 Feb 4.

Dynamic regulation of functionally distinct virus-specific T cells

Zaza M Ndhlovu  1 , Mathias Oelke, Jonathan P Schneck, Diane E Griffin
Affiliations

Dynamic regulation of functionally distinct virus-specific T cells

Zaza M Ndhlovu et al. Proc Natl Acad Sci U S A. .

Abstract

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expansion of virus-specific CD8+T cells by in vitro stimulation with M1- and H576-loaded aAPCs. Enriched CD8+ T cells were ex vivo-stimulated with aAPCs loaded with either M1 peptide (A) or H576 peptide (B) and analyzed weekly by tetramer staining. Plots are gated on CD8+ tetramer-positive cells and show representative results for three healthy donors. (C) Antigen-specific CD8+ T cell function was analyzed after 3 weeks of expansion with H576aAPC. Frequencies of tetramer-positive, IFN-γ producing cells in response to stimulation with unloaded (Left) or peptide-loaded (Right) aAPC are indicated in the Upper Right quadrants.
Fig. 2.
Fig. 2.
Comparison of virus-specific CD8+ T cells expanded by aAPCs and moDCs. Enriched CD8+ T cells from three donors were cultured with (A) M1 peptide and (B) H576 peptide-loaded aAPCs (open bars) or moDCs (filled bars) for 3 weeks. Tetramer and intracellular cytokine staining was performed at the end of each 7-day stimulation period. Bars represent percentage of CD8+ T cells that were tetramer-positive minus background ± SEM averaged from three donors, each analyzed in triplicate. On day 21 expanded (C) M1-specific CD8+ T cells and (D) H576-specific CD8+ T cells were incubated with either unloaded or peptide-loaded aAPCs for 6 h, followed by intracellular cytokine staining for IFN-γ, TNF-α, and IL-2. Bars represent percentage of total CD8+ T cells expressing the cytokine minus background averaged from three donors ± SEM. Student’s unpaired t test was used for statistical analysis. * P < 0.05, *** P < 0.0001. Cytotoxicity was evaluated by staining for granule membrane protein CD107a (E).
Fig. 3.
Fig. 3.
Cytokine production by CD8+ T cells is determined by the type of APC used for stimulation. (A) To assess the effect and reversibility of APC stimulation on cytokine production, CD8+ T cells stimulated with M1aAPCs for 7 days were restimulated with M1moDCs for a further 7 days or CD8+ T cells initially stimulated with M1moDCs were restimulated with M1aAPCs in week 2. The proportion of cells producing two cytokines (IFN-γ and TNF-α) was assessed by intracellular cytokine staining on day 7 and day 14 (before and after switching APCs). The upper right quadrant represents the proportion of cells expressing IFN-γ and TNF-α. (B) To assess the effect of switching APCs on the cytokines produced, enriched CD8+ T cells were cultured for 3 weeks changing the type of APCs (aAPCs or moDCs) at the end of each 7-day stimulation cycle. The gated cells represent the proportion of cells expressing IFN-γ out of total CD3/CD8+ T cells. (C–G) The effect of switching the type of APC on cytokine, chemokine, and cytotoxicity is shown by a trend line that shows the proportion of cytokine-producing cells at the end of each 7-day stimulation cycle.
Fig. 4.
Fig. 4.
Generation of virus-specific multifunctional CD8+ T cells by aAPCs and moDCs. The multifunctional capacities of individual cells expanded for 3 weeks by M1aAPCs and M1moDCs were analyzed. (A) Every possible combination of cytokine production by M1-specific cells is shown on the x axis. Bars show the relative proportion of the total response contributed by CD8+ T cells with a given functional response. Blue bars show aAPC-stimulated cells. Green bars show moDC-stimulated cells. Red bars show cells stimulated with unloaded aAPCs. Pie charts (Left, aAPC; Center, no stimulation; Right, moDC) show the proportion of cells positive for different numbers of effectors: lavender, 0; yellow, 1; light blue, 2; green, 3; dark blue, 4; and red, 5. (B) Compiled IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a MFI of CD8+ T cells producing cytokines. Dots identify sample means.
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References

    1. Topham DJ, Tripp RA, Doherty PC. CD8+ T cells clear influenza virus by perforin or Fas-dependent processes. J Immunol. 1997;159:5197–5200. - PubMed
    1. Moskophidis D, Cobbold SP, Waldmann H, Lehmann-Grube F. Mechanism of recovery from acute virus infection: Treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response. J Virol. 1987;61:1867–1874. - PMC - PubMed
    1. Rutebemberwa A, et al. High-programmed death-1 levels on hepatitis C virus-specific T cells during acute infection are associated with viral persistence and require preservation of cognate antigen during chronic infection. J Immunol. 2008;181:8215–8225. - PMC - PubMed
    1. Zajac AJ, et al. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med. 1998;188:2205–2213. - PMC - PubMed
    1. Wölfl M, et al. Hepatitis C virus immune escape via exploitation of a hole in the T cell repertoire. J Immunol. 2008;181:6435–6446. - PMC - PubMed

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