Journal: International Journal of Molecular Sciences
Article Title: Reduced Expression of PD-1 in Circulating CD4+ and CD8+ Tregs Is an Early Feature of RRMS
doi: 10.3390/ijms23063185
Figure Lengend Snippet: Gating strategy for CD4+ and CD8+ Treg populations. Gating strategy for flow cytometric analysis of lymphocyte population to identify CD4+CD25+FoxP3+, CD8+CD25+FoxP3+ and CD8+CD122+Helios+ Tregs. A time gate was initially applied to exclude any electronic noise and artifacts (not shown here). Next, based on size and granularity, lymphocytes were gated in a forward scatter area (FSC-A) versus side scatter area (SSC-A) plot ( A ). Then, doublet cells were excluded using FSC-A/FSC-height (FSC-H), FSC-A/FSC-Width (FSC-W) and SSC-A/SSC-height (SSC-H) parameters ( B ). Next, viable lymphocytes were gated and cells that expressed monocyte, NK cell and B cell lineage markers CD14, CD16 and CD19, respectively were excluded (“dump channel”, dump negative gate) ( C ), followed by expression of CD3 ( D ). CD8+CD122+Helios+ Tregs were identified as CD8+CD122+ cells ( E ) with FoxP3 +/− and Helios co-expression ( F ). CD8+CD25+FoxP3+ Tregs were identified by CD8 and CD25 ( G ) and Foxp3 ( H ) expression. CD4+CD25+FoxP3+ Tregs were identified as CD4 + CD25 + FoxP3 + cells ( I , J ). Representative plots are presented.
Article Snippet: The following fluorochrome-conjugated monoclonal antibodies were used according to the manufacturers’ protocols: CD3-eFluor506 (clone UCHT1; eBioscience, San Diego, CA, USA), CD4-BUV496 (clone SK3; BD Biosciences, San Diego, CA, USA), CD8-APC-eFluor780 (clone RPA-T8; eBioscience), CD122-PerCP-eFluor710 (clone TU27; eBioscience), CD25-BV650 (clone M-A251, BD Biosciences), CD14-AF700 (clone: 61D3; eBioscience), CD16-AF700 (clone: eBioCB16; eBioscience), CD19-AF700 (clone: Hib19; eBioscience), CD152(CTLA-4)-PE (clone 14D3; eBioscience), CD357(GITR)-BV610 (clone 621; BioLegend, San Diego, CA, USA) or PE-eFluor610 (clone eBioAITR; eBioscience), CD279(PD-1)-FITC (clone MIH4; eBioscience), CD62L-PE-eFluor610 (clone MEL-14; eBioscience), CD103-SB780 (clone B-LY7; eBioscience), CD28-eFluor506 (clone CD28.2, eBioscience).
Techniques: Expressing