Computational protocol: A Human Trypanosome Suppresses CD8+ T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4+ Foxp3+ T Cells

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Protocol publication

[…] Synthetic peptides SIINFEKL, VNHRFTLV and ANYKFTLV were purchased from Genscript (Piscataway, New Jersey). The biotinylated tetramer H-2Kb-SIINFEKL was purchased from ProImmune Inc. (Oxford, UK).Tetramer staining was performed before other FACS staining per manufacturer’s instructions. The intracellular cytokine staining of spleen cells was performed after 6 h of ex vivo restimulation with SIINFEKL 10 μM as described earlier []. CTLA-4 staining was performed following ICS procedures. The intranuclear staining of transcription factors was performed following the manufacturer’s instructions using Foxp3 Fixation/Permeabilization Concentrate and Diluent and Permeabilization Buffer (eBioscience). BrdU staining was performed with BD FITC BrdU Flow Kit according to the manufacturer’s instructions. EdU staining was performed with Click it Plus EdU Alexa 488 kit (Molecular Probes). Samples were acquired immediately after staining in a BD FACSCanto II flow cytometer and analyzed in FlowJo 8.7 (Tree Star).For flow cytometry stainings the following antibodies were used: CD11c APCCy7 (HL3, BD), CD40 APC (2/23, BD), CD80 PerCP (16-10A1, BD), CD86 PECy7 (GL1, BD), H-2Kb FITC (AF6-88.5, BD), I-Ab PE (AF6-120.1, BD), CD8 PerCP or PB or FITC (53–6.7, BD), CD4 PECy7 (RM4-5, BD), Vβ5.1,5.2 FITC (MR9-4, BD), Vα2 APCCy7 (B20.1, BD), CD44 PE (IM7, BD), CD62L APC (MEL-14, BD), IL-2 PerCP (JES6-5H4), TNF PE (MP6-XT22, BD), IFN-γ APC (XMG1.2, BD), IL-4 PE (11B11, BD), IL-10 PE (JES5-16E3, BD), IL-17 PE (TC11-18H10, BD), CD11a FITC (2D7, BD), CD49d FITC (R1-2, BD), CD38 PE (90, BD), CD27 FITC (LG3A10, BD), CD69 PerCP (H1.2F3, BD), KLRG-1 FITC (2F1, eBioscience), CD25 FITC or PE (7D4 or PC61, BD), CD122 FITC (TM-β1, BD), CD43 PECy7 (1B11, BioLegend), CD45 FITC (30-F11, BD), CD70 PE (FR70, BioLegend), CD71 FITC (C2, BD), CD134 PE (OX-86, BioLegend), CD127 PE (SB/199, BD), CTLA-4 PE (UC10-4B9, eBioscience), CD272 PE (8F4, eBioscience), CD279 FITC (J43, eBioscience), CD274 PE (MIH5, BD), Tim-3 Alexa Fluor 647 (B8.2C12, BioLegend), CD223 PE (C9B7W, BD), CD262 PE (MD5-1, BioLegend), CD254 PE (IK22/5, BioLegend), CD95 PECy7 (Jo2, BD), CD178 PE (MFL3, BD), CD195 FITC (C34-3448, BD), CD197 Alexa Fluor 647 (4B12, BD), CD199 Alexa Fluor 647 (9B1, BioLegend), CD183 PerCP/Cy5.5 (CXCR3-173, BioLegend), CD184 PE (2B11/CXCR4, BD), CXCR7 PE (8F11-M16, BioLegend), β7 PerCP (FIB27, BioLegend), T-bet PE (4B10, BD), Eomes FITC (Dan11mag, eBioscience), GATA-3 PE (L50-823, BD), RORγ-t PE (Q31-378, BD), FOXP3 PE (R16-715, BD), H-2KbSIINFEKL PE (25-D1.16, eBioscience), Ki67 e450 (SolA15, eBioscience). To stain biotinylated H-2Kb-SIINFEKL tetramer was used streptavidin APC or PE (BD). As isotype controls were used IgG1kappa FITC or PE or PECy7 or APC, IgG2a kappa FITC or PE or APC, IgG2b kappa FITC or PE, IgG2 kappa FITC, and IgG1 lambda PE, all from BD.The transcription of cytokine mRNA in BMDC after T. cruzi exposure was assessed by RT-PCR. To this end, 10×106 BMDC from each condition were used. Total RNA was extracted with Trizol (Invitrogen) and purified with Quick RNA Miniprep columns (Zymo Research) according to the manufacturer instructions. RNA was quantified in Nanodrop and cDNA was synthesized with SuperScript III kit (Invitrogen), following manufacturer instructions. The absence of genomic DNA was confirmed by using controls to which no reverse transcriptase was added. The resulting cDNA was amplified in a StepOne Plus equipment (Applied Biosystems) with SYBR Green (Thermo Scientific) using specific primers. Relative expression of target genes was normalized using gapdh as endogenous control and calculated by the Δ Δ Ct method. Primer sequences are given in .To perform in vitro antigen presentation assays, spleen cells from OTI naïve animals were harvested and the CD8+ T cell population was isolated through negative selection with CD8+ T cell MACS beads isolation kit (Miltenyi) according to the manufacturer specification. The isolated lymphocytes were co-cultured for 5 days with BMDC previously exposed or not to T. cruzi, stimulated with LPS and loaded with SIINFEKL peptide. The ratio of 1 BMDC to 5 CD8+ T cells was used (with 1 x 105 T cells/well) and the number of IFN-γ secreting cells was determined by Elispot as described elsewhere [,]. [...] Groups were compared using Two-Way ANOVA followed by Tukey’s HSD test (http://faculty.vassar.edu/lowry/VassarStats.html). Differences were considered significant at a P value of <0.05. […]

Pipeline specifications

Software tools FlowJo, VassarStats
Applications Miscellaneous, Flow cytometry
Organisms Trypanosoma cruzi, Mus musculus, Homo sapiens
Diseases Infection