Cd19 6d5

Cells were incubated with αCD16/32 (clone 2.4G2; hybridoma supernatant) and plated on U-bottom 96-well plates at ≤ 3 × 10⁶ cells/well in complete RPMI. Where indicated, cells were stained with Kb-SIINFEKL tetramer APC (NIH tetramer core) at 37°C for 30 min in the presence of αCD8α (53–6.7; Biolegend). For live-dead and surface staining, cells were washed with media, and stained in media for 20 min at RT with Fixable Viability Dye 780 (eBioscience) and surface antibodies for CD19 (6D5, Biolegend), CD8α (53–6.7, Biolegend), CD44 (IM-7; Tonbo), CD127 (A7R34, Tonbo), KLRG1 (2F1/KLRG1, Biolegend), CD45.1 (A20, Biolegend), CD45.2 (104, Biolegend), CD122 (TM-b1, Biolegend). After wash with RPMI, cells were fixed and permeabilized for 45 min at RT in Foxp3/Transcription Factor 1× Fix/Perm solution (Tonbo), followed by wash with 1× Flow Cytometry Perm Buffer (Tonbo) and intracellular staining for 45 min at RT with intracellular antibodies, including TCF1 (C63D9; Cell Signaling Technology), FOXO1 (C29H4, Cell Signaling Technology), T-bet (4B10, Biolegend), EOMES (Dan11mag; eBioscience). The cells were washed twice and resuspended in 1× Flow Cytometry Perm Buffer (Tonbo). Flow cytometry data was acquired on a four-laser (405, 488, 561, 638 nm) CytoFlex S (Beckman Coulter) and analyzed using FlowJo software (BD Biosciences).

Bronchoalveolar lavage (BAL) was collected using HBSS + 30μM EDTA; the cellular component was isolated and resuspended in HBSS for enumeration. The superior/upper right lobe (URL) was harvested and processed into a single cell suspension, as previously described^{26 (link)}. Each BAL and URL sample represents a single adult animal; infant BAL samples (2-4 infant mice) were pooled to acquire sufficient cell numbers for flow cytometry. To maintain consistency with the BAL, the same infant URL samples were pooled prior to enumeration. Cells (0.5 – 1 × 10⁶) were surface stained with antibodies against TCR_β-H57-597, CD62L-MEL-14, and CD19-6D5 (Biolegend, San Diego, CA), CD4-GK1.5, CD8a-53-6.7, CD44-IM7 (BD Biosciences, San Jose, CA). Cells were fixed and permeabilized for transcription factor staining using the BD Pharmingen Transcription Factor Buffer Set, according to manufacturer recommendations (BD Biosciences). Intracellular staining of GATA3-16E10A23 and Tbet-4B10 (Biolegend) was performed following overnight incubation in BD Fix/Perm solution. Where indicated, BAL samples were incubated with RSV F-protein MHC I pentamer (H-2kd KYKNAVTEL, ProImmune, Sarasota, FL). Samples were run on a BD LSRFortessa (BD Biosciences) managed by the University of Pittsburgh United Flow Core and analyzed using FlowJo V10 software (FLOWJO, Ashland, OR).

Dissociated cells were incubated with the following antibodies (1:500) in Flow Cytometry Staining Buffer on ice for 30 minutes. Allophycocyanin (APC)-conjugated CD45 (30F-11, eBioscience [San Diego, CA, USA] 17–0451-82), phycoerythrin (PE)-conjugated CD48 (HM48-1, BioLegend [San Diego, CA, USA] 103405), CD41 (MWReg30, BioLegend 133905), CD79b (HM79-12, BioLegend 132803), F4/80 (BM8, BioLegend 123109), Gr-1 (RB6-8C5, BioLegend 108407), brilliant violet 650 (BV650)-conjugated CD150 (TC15-12F12.2, BioLegend 115931), CD19 (6D5, BioLegend 115541), CD11b (M1/70, BioLegend 101239). Flow cytometry analysis was performed using a four-laser BD LSR Fortessa (Ex. 405/488/561/640 nm) and FACSDiva software. Acquired raw data were further analyzed on FlowJo software (TreeStar, Ashland, OR, USA). Representative plots of at least three independent biological samples are shown in the figures. ImageStream analysis was performed using Amnis ImageStream^X Mark II (MilliporeSigma, Burlington, MA, USA). Acquired raw data were further analyzed on Amnis IDEAS software.

Cell suspensions prepared as described above were blocked with CD16/CD32 (Mouse BD Fc Block, 2.4G2, BD Biosciences). For B cell compartment analysis, suspensions were stained with antibodies against CD45 (30-F11, Tonbo), CD4 (GK1.5, eBioscience), CD19 (6D5, Biolegend), IgD (11–26c.2a, Biolegend), IgG (polyclonal, Jackson ImmunoResearch), IgM (II/41, Thermofisher), CD95 (DX2, Biolegend), and GL7 antigen (GL7, Biolegend). Dead cells were excluded with eBioscience Fixable Viability Dye eFluor 455UV (Thermofisher). In phagocyte depletion experiments, suspensions were stained with antibodies against CD45 (30-F11, Tonbo), I-A/I-E a (M5/114.15.2, Biolegend), CD11c (N418, Biolegend), CD11b (M1/70, Thermofisher), CX3CR1 (SA011F11, Biolegend), and CD103 (2E7, Thermofisher). Dead cells were excluded with eBioscience Fixable Viability Dye eFluor 506 (Thermofisher). Bacterial FISH staining was performed with Cy5-labeled EUB338 5’-GCTGCCTCCCGTAGGAGT-3’ probe (Integrated DNA Technologies; Table S1). Images were acquired under an inverted Nikon Eclipse Ti microscope (Nikon). Flow cytometry was performed using a LSRFortessa (BD Biosciences) and data were analyzed with FlowJo software (TreeStar).