For the quantification of epitope-specific T cells, PE-conjugated I-Ed LukE296–311 and I-Ab LukE153–168 and LukE185–200 class II pMHC tetramers were synthesized by the NIH Tetramer Core Facility. Lymphocytes were isolated from spleens and draining lymph nodes and processed into single-cell suspensions and then blocked with anti-CD16/32 antibody (clone 2.4G2, Bio X Cell), followed by incubation for 60 min with tetramers at RT. Tetramer enrichment procedures were performed as described previously (23). Briefly, following tetramer staining, tetramer-bound cells were enriched using anti-PE magnetic beads and columns (Miltenyi Biotec). The enriched fractions were collected and used for flow cytometric analysis. Cells were stained for flow cytometry using AquaFluor LiveDead (Life Technologies) solution to exclude dead cells, and a cocktail of dump antibodies was used to exclude unwanted cells: DX5, CD11b (M1/70, BioLegend), F4/80 (BM8), CD19 (1D3), and TER119. Additional antibodies against CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), and CD44 (IM7) were used to stain T cells.

For quantification of TFH cells, draining lymph nodes were collected 2 weeks following a second vaccination with LukE, and tetramer staining was performed as described above. For surface staining, cells were incubated with anti-CD49b (DX5), anti-TER119, anti-CD19, anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD4, anti-CD8, anti-CD44, and anti–PD-1 (29F.1A12). For transcription factor staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for 30 min in the dark at RT following two subsequent washes with stain buffer [PBS and 2% fetal calf serum (FCS)] after surface staining. Cells were then washed twice with Permeabilization Wash Buffer and incubated with anti-Foxp3 (FJK-16s) and anti–Bcl-6 (7D1) overnight in the dark at 4°C. Antibodies were purchased from eBioscience, BioLegend, or Becton Dickinson. Flow cytometry was performed on an LSRII or Fortessa (BD Biosciences) cytometer and analyzed with FlowJo software.

To quantify OVA-specific responses in the context of S. aureus SSTI, splenocytes were prepared as single-cell suspensions from 2W-OVA transgenic C57BL/6 mice and lysed with ACK (ammonium chloride potassium) buffer. The cells were washed twice in PBS and were subcutaneously injected (10 × 106 cells per mouse) into naïve C57BL/6 mice, along with concomitant S. aureus SSTI (or PBS). To quantify 2W-specific T cells, mice were euthanized 8 days later, and lymphocytes from draining lymph nodes (dLNs) and splenocytes were stained with a fixable LIVE/DEAD stain (Aqua, Invitrogen) and blocked with anti-CD16/32 antibody (2.4G2), followed by staining with 2W-specific I-Ab pMHC tetramers PE-2W(EAWGALANWAVDSA) and allophycocyanin (APC)–2W(EAWGALANWAVDSA) for 1 hour at RT (tetramers were obtained from the NIH tetramer facility). Cells were then stained with anti-CD49b (DX5), anti-TER119, anti-CD19 (1D3), anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), and anti-CD44 (IM7). Surface-stained cells were then fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for 30 min at RT, followed by two washes with staining buffer (PBS and 2% FCS). Cells were then washed twice with Permeabilization Wash Buffer and incubated with anti-Foxp3 (FJK-16s) overnight at 4°C. Flow cytometry was performed on a BD LSRII and analyzed with FlowJo software. 2W-specific T cells were identified as PE-2W:I-Ab tetramer and APC-2W:I-Ab tetramer double-positive cells. Tconv cells were identified as Foxp3 cells.

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