Anti cd4 pacific blue

Spleens were minced using a wire mesh, and splenocytes were collected and counted. For the immunodetection of specific populations of T cells within the spleen, anti-CD4-Pacific Blue, anti-CD8-Alexa647, anti-CD24-PE, anti-CD44-PE-Cy5.5, and anti-CD62L-PE-Cy7 were purchased from BioLegend (San Diego, CA) or BD Biosciences (San Jose, CA). Cell labeling was performed in PBS containing 2% FCS. Flow cytometry studies were performed using a BD LSR II (BD Immunocytometry Systems, San Jose, CA). Data were analyzed using BD FACSDiva software (BD Biosciences). Splenocytes were gated on the live lymphocyte gate and doublet discrimination was performed. Naïve T cells were defined as CD44^loCD62L^hi, central memory (CM) T cells as CD44^hiCD62L^hi, and effector memory (EM) T cells as CD44^hiCD62L^lo.

For in-vitro studies, the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, anti-CD4 PE-Cy7, anti-CD8 APC, and anti-CD44 PE, all at a 1:200 dilution (Biolegend). For in-vivo and ex-vivo studies, the following flow cytometry panel was used: anti-CD3 PE-Cy7, anti-CD4 Pacific Blue, anti-CD8 APC, anti-CD44 PE, anti-CD62L APC-Cy7, and anti-CD25 Alexa 700, all at a 1:200 dilution, except anti-CD25 at a 1:50 dilution (Biolegend). Endogenous Foxp3 expression was detected either via eGFP of eYFP fluorescence, which is compatible with all panels but not when used with BODIPY-FL dye (493/503) or 2-NBDG (465/540). In those assays, Foxp3-based uptake of FA or Glucose was not performed, but all other cellular subsets described were tested. All acquisition was performed using a BD Fortessa flow cytometer (BD, MD, USA) at the Northwestern University Comprehensive Flow Cytometry core.

Leukocytes were extracted from murine spleen, salivary glands and lungs as described previously [13 (link),60 (link)]. For CD4⁺ functional responses, isolated leukocytes were stimulated for 2 hours with 3 μg/ml m09, (GYLYIYPSAGNSFDL), M25 (NHLYETPISATAMVI), m139 (TRPYRYPRVCDASLS), and m142 (RSRYLTAAAVTAVLQ) MCMV MHCII peptides (Genscript). Brefeldin A (Sigma) was then added and cells incubated for a further 4 hours. For CD8⁺ functional responses, leukocytes were stimulated with 2 μg/ml m139, IE3, M38, and M45 MCMV MHCI peptides in the presence of anti-mouse CD107a-FITC (Biolegend) with monensin (BD Biosciences) and Brefeldin A for 6 hours. Cells were subsequently stained with Zombie Aqua fixable viability dye (Biolegend) or LIVE/DEAD-Aqua (Life-Technologies), stained with anti-CD16/CD32 Fc-block (Biolegend) and then with either anti-CD4 Pacific-Blue or PercP, (clone RM4-5, Biolegend) or with anti-CD8 PercP (clone 53–6.7, Biolegend) and anti-CD262 (TRAILR DR5, clone MD5.1, eBioscience). Cells were fixed, saponin permeabilised and stained for anti-IFNγ FITC or Pacific-Blue (clone XMG1.2, Biolegend) and anti-IL-10 APC (clone JES5-16E3, eBioscience). Data was acquired using a BD FACSCantoII or a BD LSR II fortessa flow cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar).