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. 2013:2013:186420.
doi: 10.1155/2013/186420. Epub 2013 Aug 26.

Ex vivo restimulation of human PBMC expands a CD3+CD4-CD8- γδ+ T cell population that can confound the evaluation of CD4 and CD8 T cell responses to vaccination

B J Sedgmen  1 , L Papalia, L Wang, A R Dyson, H A McCallum, C M Simson, M J Pearse, E Maraskovsky, D Hung, P P Eomois, G Hartel, M J Barnden, S P Rockman
Affiliations

Ex vivo restimulation of human PBMC expands a CD3+CD4-CD8- γδ+ T cell population that can confound the evaluation of CD4 and CD8 T cell responses to vaccination

B J Sedgmen et al. Clin Dev Immunol. 2013.

Abstract

The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.

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Figures

Figure 1
Figure 1
Characterisation of an IFN-γ-producing CD3+CD4−CD8− γδ + T cell-positive subject at day 7 after vaccination. PBMCs were expanded in vitro in the absence of exogenous influenza virus antigen, and the expression of CD4, CD8, αβ TCR, CD161, Vα24, and γδ TCR surface antigens was assessed by flow cytometry.
Figure 2
Figure 2
Alternative gating strategy applied to influenza vaccine trial data to eliminate confounding CMI responses from the IFN-γ-producing γδ + T cells. PBMCs from an IFN-γ-producing CD3+CD4−CD8− γδ + T cell-positive subject at day 7 after vaccination were restimulated in (a) the absence of antigen or (b) the presence of exogenous influenza virus antigen (30 HA units per strain/mL), and antigen-specific CD4+ and CD8+ T-cell responses were assessed. For each of the restimulation conditions, the top panels represent the standard gating strategy, and the bottom panels represent the alternative gating strategy, whereby CD3+ γδ + T cells were selectively excluded during post acquisition analysis to avoid reporting misleading cytokine responses that could confound the antigen-specific CD4+ and CD8+ T cell responses.
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