Cervical T Cell Immunophenotyping

Cervical cells were obtained from Digene cytobrushes, processed within 4 h, and stained immediately to measure T cell frequencies and activation by flow cytometry, as previously described (21 (link)). The gating strategy is shown in Fig. S1 in the supplemental material. The following panel of antibodies was included: APC-H7-CD3, BV605-CCR6, and APC-CCR5 (BD Biosciences); BV711-CD8, PE-CCR10, and Alexa-700-human leukocyte antigen (HLA) DR isotope (BioLegend); PE-Cy5.5-CD4 (Invitrogen); and PE-Cy7-cluster of differentiation 38 (CD38) (eBioscience, Inc.). LIVE-DEAD cell, Pacific-blue-CD14, and Pacific-blue-CD19 (Invitrogen) staining were performed to exclude dead cells, monocytes, and B-cells, respectively. Cells were acquired using an LSRFFortessa (BD Immunocytometry Systems). FlowJo v9.9.3 (FlowJo, LLC) was used for the data analysis.

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Happel A.U., Gasper M., Balle C., Konstantinus I., Gamieldien H., Dabee S., Gill K., Bekker L.G., Passmore J.A, & Jaspan H.B. (2022). Persistent, Asymptomatic Colonization with Candida is Associated with Elevated Frequencies of Highly Activated Cervical Th17-Like Cells and Related Cytokines in the Reproductive Tract of South African Adolescents. Microbiology Spectrum, 10(2), e01626-21.

Publication 2022

APC-H7-CD3 BV605-CCR6 APC-CCR5 BV711-CD8 PE-CCR10 Alexa-700-human leukocyte antigen (HLA) DR isotope PE-Cy5.5-CD4 PE-Cy7-cluster of differentiation 38 (CD38)

Antibodies B cells Ccr5 Ccr6 Cells Cervical Cy5 5 Flow cytometry Human leukocyte antigen dr Isotope Monocytes T cell

Corresponding Organization : University of Cape Town

Other organizations : Namibia Institute of Pathology, Desmond Tutu HIV Foundation, Centre for the AIDS Programme of Research in South Africa, National Health Laboratory Service, University of Washington

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Variable analysis

independent variables

Cervical cells obtained from Digene cytobrushes

dependent variables

T cell frequencies
T cell activation

control variables

Cells processed within 4 h
Cells stained immediately
Gating strategy shown in Fig. S1
Panel of antibodies used: APC-H7-CD3, BV605-CCR6, APC-CCR5, BV711-CD8, PE-CCR10, Alexa-700-HLA DR, PE-Cy5.5-CD4, PE-Cy7-CD38
LIVE-DEAD cell, Pacific-blue-CD14, and Pacific-blue-CD19 staining to exclude dead cells, monocytes, and B-cells
Cells acquired using an LSRFFortessa (BD Immunocytometry Systems)
Data analysis using FlowJo v9.9.3 (FlowJo, LLC)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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