Profiling Antigen-Specific Lymphocyte Responses

At day 7 (peak of T cell responses; (Moon et al., 2009 (link))) and 14 post-injections (peak of B cell responses; (Pape et al., 2011 (link))), the mice were sacrificed and the skin draining lymph nodes (axillary, brachial and inguinal) harvested. Single-cell suspensions were generated using mechanical disruption through cell strainers. The mRNA-LNP platform does not require the use of T cell tetramers or fluorochrome-labeled antigen for B cells to study antigen-specific responses. There is only one antigen that the T and B cells react to, and the response is so robust that no need for magnetic enrichment either. Therefore, the cells were stained with either Tfh cell or B cell panels. The Tfh cell panel contained: fixable viability dye (Thermo Fisher), CD4 (GK1.5), CD44 (IM7), CD45 (OX-7), CD62L (H1.2F3), CD69 (H1.2F3), CXCR5 (L138D7), PD-1 (29F.1A12) and Bcl-6 (K112–91) from BioLegend and BD Biosciences. The gating strategy can be found in Suppl. Figure 3A. The B cell panel consist of dump (fixable viability dye, F4/80, CD11b), CD38 (90), B220 (RA3–6B2), CD138 (281–2), GL-7 (GL-7), Sca1 (D7), IgD (11–26c.2a) and IgM (RMM-1). The gating strategy is presented in Suppl. Figure 3B. The stained samples were run on LSRFortessa^™ (BD Biosciences) and the resulting data analyzed with FlowJo 10.

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Ndeupen S., Bouteau A., Herbst C., Qin Z., Hutchins Z., Kurup D., Diba L.Z, & Igyártó B.Z. (2021). Langerhans cells and cDC1s play redundant roles in mRNA-LNP induced protective anti-influenza and anti-SARS-CoV-2 responses. bioRxiv.

Publication Preprint 2021

fixable viability dye LSRFortessa™

Antigen Axillary B cell Bcl 6 Cd11b Cd138 Cd44 Cd62l Cells Cxcr5 Dump Fluorochrome Inguinal Lymph nodes Mice Mrna Sca1 Skin T cell Tetramers Tfh cell

Corresponding Organization : Thomas Jefferson University

Other organizations : Baylor University

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Variable analysis

independent variables

Day 7 post-injections (peak of T cell responses)
Day 14 post-injections (peak of B cell responses)

dependent variables

Frequency of Tfh cells
Frequency of B cells (including GC B cells, plasma cells, and memory B cells)

control variables

Skin draining lymph nodes (axillary, brachial and inguinal) harvested
Single-cell suspensions generated using mechanical disruption through cell strainers
Staining with Tfh cell or B cell panels
Samples run on LSRFortessa™ and analyzed with FlowJo 10

positive controls

No positive controls explicitly mentioned

negative controls

No negative controls explicitly mentioned

Annotations

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