Depletion of human NK and CD8 cells prior to in vitro H1N1 flu vaccine stimulation increases the number of gamma interferon-secreting cells compared to the initial undepleted population in an ELISPOT assay

Abstract

In order to study the respective roles of CD4, CD8, and CD56 (NK) cells in gamma interferon (IFN-gamma) production after in vitro stimulation with flu vaccine in a healthy adult human population, we depleted these cellular subtypes before stimulation with antigen (inactivated split vaccine, A/Texas H1N1, or A/Sydney H3N2). We observed that while CD4 cells were required for IFN-gamma secretion in both conditions in vitro, CD56 (NK) cells and, to a lesser extent, CD8 cells had a negative effect on such synthesis upon H1N1 stimulation, as judged by an increased number of spots compared to the initial undepleted population. This regulation of IFN-gamma secretion was associated with an increase in ICAM-1 expression, in particular on T and B cells. This study points out the importance of evaluating in vitro immune responses on a whole-cell population in addition to isolated subtypes if one needs to address potential cellular interactions occurring in vivo in some situations (H1N1 stimulation in the present case). Such cross-regulations occur even in vitro during the antigenic stimulation step.

FIG. 1.

Influence of cell depletion on IFN-γ synthesis after H1N1 stimulation. Depletion was performed before stimulation with flu vaccine. Spot numbers (for each donor) correspond to values obtained with PBMC before depletion or after CD4, CD8, or CD56 depletion. Stimulation was done in ELISPOT plates during 24 h by using A/Texas-H1N1 vaccine at a final concentration of 10 μg/ml; 10⁵ cells were used per well, and experiments were performed in triplicate for each condition. The results are representative of at least two independent experiments for each donor.

FIG. 2.

Influence of cell depletion on IFN-γ synthesis after H1N1 stimulation in three different experiments on the same donor. Spot numbers correspond to values obtained with PBMC before depletion or after CD4, CD8, or CD56 depletion. Stimulation was done in ELISPOT plates during 24 h by using A/Texas-H1N1 vaccine at a final concentration of 10 μg/ml; 10⁵ cells were used per well, and experiments were performed in triplicate for each condition.

FIG. 3.

Influence of cell double depletion on IFN-γ synthesis after H1N1 stimulation. Depletion was performed before stimulation with flu vaccine. Spot numbers (for each donor) correspond to values obtained with PBMC before depletion or after CD4-CD8, CD4-CD56, or CD8-CD56 depletion. Stimulation was done in ELISPOT plates during 24 h by using A/Texas-H1N1 vaccine at a final concentration of 10 μg/ml; 10⁵ cells were used per well, and experiments were performed in triplicate for each condition. The results are representative of three independent experiments.

FIG. 4.

Influence of cell depletion on IFN-γ synthesis after H3N2 stimulation. Depletion was performed before stimulation with flu vaccine. Spot numbers (for each donor) correspond to values obtained with PBMC before depletion or after CD4, CD8 or CD56 depletion. Stimulation was done in ELISPOT plates during 24 h by using A/Sydney-H3N2 vaccine at a final concentration of 10 μg/ml; 10⁵ cells were used per well, experiments were performed in triplicate for each condition. The results are representative of at least two independent experiments for each donor.

FIG. 5.

Percentage of ICAM-1 (CD54)-positive cells for each considered population before and after H1N1 or H3N2 stimulation. Stimulation was done in culture plates during 24 h (10 μg of the corresponding vaccine/ml). The results are representative of three independent experiments.

FIG. 6.

Potential regulatory mechanisms involved in IFN-γ secretion after stimulation with H1N1 or H3N2 flu vaccine antigens. Both H1N1 and H3N2 antigens captured and processed by APCs stimulated a CD4 T-cell response, but only H1N1 stimulation triggered NK and CD8-negative roles, directly or indirectly, through CD4 cells. This negative regulation could result from an effect on APCs and/or IFN-γ-producing CD4 cells through direct cell lysis or inhibition of activation.