Anti cd62l pe cy7

Flow cytometry of BAL cells was performed by using the following monoclonal antibodies: anti-CD8a-AlexaFlour750 (clone 5H10, Thermo Fisher Scientific; AB_10374588), anti-Ly6c-FITC (clone AL-21, BD Biosciences, San Jose, CA USA; AB_394628), anti-CD4-PerCP-Cy5.5 (clone RM 4–5, BD Biosciences; AB_393977), anti-CD62L-PE-Cy7 (clone MEL-14, eBioscience, affymetrix, Frankfurt am Main, Germany; AB_469632), anti-CD3-eFlour450 (clone 17A2, eBiosciene; AB_1272229), anti-CD25-APC (clone PC61, BD Biosciences; AB_398623), anti-Gr1-PacificOrange (clone Rb6-8C5, Thermo Fisher Scientific; AB_2556571), anti-CD19-PE-Cy7 (clone 1D3, BD Biosciences; AB_10894021), anti-CD11b-APC-Cy7 (clone M1/70; BD Biosciences; AB_396772), anti-CD11c-FITC (clone HL 3, BD Biosciences; AB_395060), anti-CD45-AlexaFlour700 (clone 30-F11, BioLegend, San Diego, CA USA; AB_493714), anti-F4/80-APC (clone BM8, eBioscience; AB_469451) and anti-MHC-II (I-A/I-E)-PerCP-Cy5.5 (clone M5/114.15.2, BioLegend; AB_2191072) [23 (link)]. Data were acquired using a LSRII flow cytometer (BD Bioscience) and further analyzed with FACSDiva software (BD Biosciences) and FlowJo V.7.2.2 (Tree star, Ashland, USA). The gating strategy is displayed in S1 Fig.

Splenocytes were prepared as described above, plated in 96-well round-bottom plates, and stimulated using peptide pools for EBOV GP, SUDV GP, or MARV GP (as described above) at a final concentration of 5 µg/mL or media only. Stimulation and staining was then performed as described previously [33 (link)] except that the following antibodies were used: anti-CD4-Qdot605, anti-CD127-APCef780 (Invitrogen), anti-CD62L-PeCy7, and anti-CD8-PerCP/Cy5.5 antibodies (eBioscience), as well as LIVE/DEAD^® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), anti-TNF-Alexa488, anti-IL-2-PE, and anti-IFN-γ-e450 antibodies (eBioscience). Antigen-specific cells were identified by gating based on doublet negative, size, live cells, and either CD4⁺ or CD8⁺ surface expression. Background responses in unstimulated control samples were subtracted from responses of peptide stimulated T cells.

Peripheral blood was sampled from the retro-orbital sinus of each of 15 mice once a month for 8 time points (a total of 120 samples). Mononuclear cells were isolated by density gradient centrifugation using Ficoll (Ficoll Paque plus, GE Health Care–according to the manufacturer’s instructions). At the termination of the experiment, single-cell suspensions were prepared from the thymus and spleen of each mouse. For cell sorting, the cells were stained with the following fluorescently labeled monoclonal antibodies: anti-CD4 Pacific Blue (BD), anti-CD25 PE (eBioscience), anti-CD44 APC (BD) and anti-CD62L PE-Cy7 (eBioscience); viability was determined using the Fixable Viability stain 450 (BD Horizon). Cell sorting was performed using a FACS ARIA III sorter; CD4⁺CD62L^hiCD44^lo T cells, the most prevalent T-cell population, was used for CDR3 sequencing. After sorting, the cells were pelleted and resuspended with 300μl of RNAprotect cell reagent (Qiagen). The cells were stored at minus 80°C until RNA extraction. RNA was purified from RNAprotect-stabilized cells using the RNeasy Plus Mini Kit. After RNA extraction, samples were run on TapeStation to estimate quality.

Lung T-cell phenotypes were assessed as described [19 (link)]. 2 x 106 lung cells/well were surface-stained with anti-CD3-eFluor450, anti-CD8-APC-Cy7, anti-CD62L-PE-Cy7, anti-CD69-PE, anti-CD127-PerCP-Cy5.5 (eBiosciences, San Diego, CA), and Live/Dead fixable green viability stain Vivid for 488 nm excitation (Invitrogen). The following tetramer was obtained through the NIH Tetramer Facility: NP_147–155-H2-K^d Tetramer-APC (TYQRTRALV) (Atlanta, GA). 50,000 events per sample were acquired on an LSRII flow cytometer.
FACS Diva V6 software (BD Biosciences, San Jose, CA) was used for data acquisition and FlowJo V7.6.5 (TreeStar, Ashland, OR) for data analysis and display. Single color-stained cells were used for compensation, and fluorescence minus one (FMO) controls were used for gate setting.

Splenocytes were isolated for analysis via IFN-γ ELISpot, surface and intracellular cytokine staining (ICS) and flow cytometry as previously described^{69 (link)–71 (link)}. Splenocytes were restimulated with 2 μg/mL of the appropriate antigenic peptide pool (comprised of individual peptides that were 20 amino acids in length with 10 amino acids of overlap) spanning the full-length LASV GPC sequence (Mimotopes). Reference sequences for peptide synthesis are as follows: NP_694870.1 (Josiah), AIT17836.1 (Pinneo), AAF86703.1 (803213), CAA36645.1 (GA391). Cells were restimulated for 18–20 h or 6 h (at 37 °C and 5% CO₂) for IFN-γ ELISpot and ICS, respectively. Surface staining and ICS were carried out using the following antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fischer Scientific); anti-CD8a-PerCP/Cy5.5, anti-CD62L-PeCy7, anti-IFN-γ-eFluor 450, anti-TNF-α-Alexa Fluor 488, anti-IL-2-PE (eBioscience); anti-CD4-Brilliant Violet 650, anti-CD44-Alexa Fluor 700 (BioLegend); and anti-CD127-eFluor 660 (Invitrogen). Antigen-specific T-cell responses were quantified by subtracting the response (SFU for IFN-γ ELISpot and the percentage of cytokine-positive cells for ICS) measured without stimulation from that observed after restimulation.

For H2-K^b Ova_257-264 (SIINFEKL)-specific CD8⁺ T-cell analysis, spleen cells were obtained 12 days after tumor inoculation and stained with AF647-coupled dextramer loaded with Ova_257-264 peptide (Dex^OT–I; Immudex, Copenhagen, Denmark), FITC-anti-CD11b, PerCP-anti-CD43, BV421-anti-CD8 (all from BioLegend, San Diego, CA, USA), Fixable Viability Dye eFluor 780, PE-Cy7-anti-CD62L (eBioscience), and BV510-anti-CD44 (Becton-Dickinson).
The frequency of CD8⁺ T cells specific for the H2-D^b-restricted Leader-Gag-derived epitope GagL_85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetI^GagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL_85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown in Supplementary Figure 2.