A simple, robust flow cytometry-based whole blood assay for investigating sex differential interferon alpha production by plasmacytoid dendritic cells

Abstract

Central to sex differences observed in outcome from infection and vaccination is the innate immune response, and specifically production of type I interferons by plasmacytoid dendtiric cells (pDCs), the main producers of IFN-α. Evaluation of IFN-α production by pDCs is therefore critical for studies of innate immune function. However, reliable measurement of pDC IFN-α is hampered by reduced cell yields and cytokine production after cryopreservation or after even short delays in stimulating freshly isolated cells. We here describe a simple yet robust method for measuring IFN-α production in pDCs that preserves cell activation and cytokine production through immediate stimulation of whole blood and subsequent maintenance at 37 °C.

Keywords: Immune sex differences; Innate immunity; Interferon alpha; Plasmacytoid dendritic cell; Toll-like receptor 7.

Fig. 1

Kinetics of the pDC response to CL097 stimulation in whole blood. Time course of pDC activation and cytokine production in immediately-stimulated whole blood for 6 healthy donors (median age 23 yrs; range 19-24 yrs; male n = 3; female n = 3). 1 ml blood was drawn into vacutainers containing 1 ml R10 and 2 μg/ml CL097 + 10 μg/ml Brefeldin A (1 μg/ml and 5 μg/ml final conc. respectively) and maintained at 37 °C using a dry block-heater during transport to the laboratory (~30mins) before transfer to loose lid tubes and further incubation in a tissue-culture incubator for indicated times. Red blood cells were then lysed and remaining cells stained as detailed in Methods. Time points were done in duplicate per donor. Dashed purple line represents the average of unstimulated controls processed at 4 h. A) raw count of gated pDCs. B), C), E), G) mean fluorescence intensity (MFI) of gated pDCs of: HLA-DR, CD123, IFN-α, and TNF-α respectively. D), F) percentage of gated pDCs expressing IFN-α and TNF-α respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

pDC activation and IFN-α production in whole blood compared to PBMC and cryopreserved PBMC. Comparison of immediately in-tube stimulated cells with isolated PBMC and frozen PBMC. Blood was taken from 4 healthy donors (median age 23 yrs; range 19-24 yrs; male n = 2; female n = 2) and subjected to three conditions: i) immediate stimulation as for Results 2.2; maintained at 37C by dry block heater for 2 h, then incubated for a further 4 h [WB], ii) PBMC isolated after 2 h incubating blood at room temperature then stimulated for 6 h [PBMC], iii) remaining PBMC from ii) cryopreserved and thawed before 6 h stimulation [Frozen PBMC]. Experiments were performed in duplicate per donor. A) raw count of gated pDCs. B), C), E), G) mean fluorescence intensity (MFI) of gated pDCs of: HLA-DR, CD123, IFN-α, and TNF-α respectively. D), F) percentage of gated pDCs expressing IFN-α and TNF-α respectively. Pairwise comparisons performed using Student's paired t-test.

Fig. 3

Effect of simulated transport temperature on pDC activation and cytokine response. Comparison of immediately in-tube stimulated blood incubated at either i) 37C, ii) room temperature (20-25C) [RT], or iii) 4C for 2 h before 4 h further incubation in a tissue culture incubator up to 6 h post-stimulation. Conditions were done in duplicate per donor. Donors were matched for age and sex (median 23 yrs; range 19-24 yrs; male n = 2; female n = 2). A) raw count of gated pDCs. B), C), E), G) mean fluorescence intensity (MFI) of gated pDCs of: HLA-DR, CD123, IFN-α, and TNF-α respectively. D), F) percentage of gated pDCs expressing IFN-α and TNF-α respectively. Pairwise comparisons performed using Student's paired t-test.

Fig. 4

Longitudinal robustness of pDC activation and cytokine production. Assay reproducibility over 4 weeks for 6 healthy donors (median age 23 yrs; range 19-24 yrs; male n = 3; female n = 3) for immediately in-tube stimulated blood maintained at 37C and incubated for 6  as described in Methods 2.2. Blue symbols indicate male donors and red female donors. A) raw count of gated pDCs. B), C), F), H) mean fluorescence intensity (MFI) of gated pDCs of: HLA-DR, CD123, IFN-α, and TNF-α respectively. D), G) percentage of gated pDCs expressing IFN-α and TNF-α respectively. E) average percentage of pDCs expressing IFN-α for each sex.

Fig. 5

Application of immediate whole blood stimulation in a large clinical research cohort. Application of the whole blood assay in a large dataset (n = 109; males n = 44; females n = 65; age range 6-69 yrs; median 36.5 yrs). A) B), D), F) mean fluorescence intensity (MFI) of gated pDCs of: HLA-DR, CD123, IFN-α, and TNF-α respectively. C), E) percentage of gated pDCs expressing IFN-α and TNF-α respectively. Pairwise comparisons performed using Student's unpaired t-test with Welch's correction for unequal variance.