Anti cd45ra fitc

Specific antibodies against CD3-PE-Cy7 (Biolegend) or anti-CD3-FITC (Biolegend), anti-CD25-PE (BD Pharmingen™), anti-CD127-APC (Biolegend), anti-CD4-FITC (Biolegend), anti-CD8-APC (Biolegend), anti-CD45RA-FITC (Biolegend), anti-CCR7-PE-Cy7 (Biolegend), anti-CD4-PE (BD Biosciences), anti-PD-1-FITC (Biolegend), anti-CD4-PErCP-Cy5.5 (BD Biosciences), anti-Tim-3-PE (BD Pharmingen™), anti-FoxP3-PE (Biolegend), and anti-IFN-γ-APC (eBioscience) were used. 7AAD viability staining (Biolegend) was used to assess the viability of the cells. Tils were examined at day 0, 5, 10, 15, 20 and 25 by flow cytometry. Briefly, 1 × 10⁶ T cells was mixed with 5 μl of each antibody and was incubated on ice for 20 min in the dark. After incubation the samples were washed with FACS buffer (5% BSA in PBS, 0.09% sodium azide). The pellets were suspended in 300 μl of FACS buffer and acquired on a BD FACS Conto II flow cytometer, and then the data was analyzed with FlowJo software (TreeStar Inc). The data is displayed as background-corrected values with control sample or by fluorescence minus one (FMO).

Cell migration assays were performed with CD4+ T lymphocytes isolated from eight patients using a transwell system with a pore size of 3 µm (Costar, Kennebunk, ME, USA). Briefly, between 2 × 10⁵ and 5 × 10⁵ CD4+ T lymphocytes in 100 µL of medium were placed in the upper chamber of a transwell system suspended over a larger well containing 600 µL of culture medium or in the presence of IL-15 (50 ng/mL). Cells were allowed to migrate through the pores to the other side of the membrane. After 18 h, migratory cells (MCs) and non-migratory cells (NMCs) were collected. Then, they were surface stained with anti-CD45RA (FITC) (Biolegend), anti-CD28 (PE), and anti-CD4 (PerCP) (BD Bioscience) monoclonal antibodies. Flow cytometric analysis was made using the Accuri C6 (BD Biosciences). For the cell count, 20 µL of the sample was acquired at medium speed. We have tested different concentration of IL-15 (from 0.5 to 100 ng/mL) to perform these transwell assays and we have observed a significant increment in the cell migration capacity with all the IL-15 concentrations tested comparing to unstimulated conditions. The maximum peak of migration was already reached for both T CD4+ lymphocyte subsets studied with 50ng/mL (data not shown). Based on this, we decided to use this IL-15 concentration for the rest of experiments.

CMV-specific T cells (CMV-CTL) and WT1-specific T cells (WT1-CTL) frequencies were quantified and phenotyped in patients by staining with PE-A^*0201 CMV_NLVPMVATV Dextramer, PE-A^*0201 WT1_RMFPNAPYL Dextramer and APC-A^*0201 WT1_SLGEQQYSV Dextramer (Immudex, Copenhagen, Denmark) (Supplementary Figure 1). Briefly, PBMCs were stained with Dextramers for 30 min at room temperature. Anti-CD3-Brilliant Violet 570, anti-CD4-Brilliant Violet 510, anti-CCR7-Brilliant Violet 421, anti-CD45RA–FITC, anti-PD-1-PE Cy7 (from Biolegend, San Diego, CA, USA), anti-CTLA-4–PE Cy5, anti-CD8-AF700 (from BD Biosciences, San Jose, California, USA) and anti-TIM-3–PerCP eFlour 710 (from eBioscience, USA) were added for the final 20 minutes of incubation. Surface expression of CD45RA and CCR7 was used to characterize naïve (T_Naive, CD45RA+CCR7+), central memory (T_CM, CD45RA-CCR7+), effector memory (T_EM, CD45RA-CCR7-), and terminally differentiated (T_EMRA, CD45RA+CCR7-) phenotypes (Supplementary Figure 2). Appropriate isotype controls or fluorescence minus one control for each fluorochrome were used to assess for nonspecific staining and determine gating strategy, respectively. FACS Aria flow cytometer (BD Biosciences, San Jose, California, USA) and FlowJo software were used for acquisition and data analysis.

LN-derived cells were thawed and stained for viability using a fixable viability dye e506 (Thermo Fisher Scientific, 65-0866-14) and for different surface markers depending on the experimental set-up. The following surface antibodies were used: anti-CD3-PerCP-Cy5.5, anti-CD4-PE-Dazzle, anti-CD8-APC-Cy7, anti-CD45RA-FITC, anti-CD25-BV421, anti-CD31-BV605, anti-CXCR5-BV711, anti-TIM3-BV711, anti-CD278-BV605, anti-PD1-PE-Cy7, anti-CD69-AF700 and anti-CD244-BV421 (all BioLegend, Supplementary Table 4). For subsequent intracellular staining, cells were fixed and permeabilized with the intracellular fixation/permeabilization buffer set (Thermo Fisher Scientific, 88-8824-00) and stained with anti-Ki67-BV785, anti-FOXP3-AF647, anti-IKZF3-PE or adequate isotype controls (Thermo Fisher Scientific, BD Biosciences, Supplementary Table 4). Then, cells were analysed using an LSR Fortessa (BD Biosciences) and FACSDiva (BD Biosciences, version 8). For analysis and gating of flow cytometry data, FlowJo (v10.8.0) was used.

The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A^*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65_341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).

Daily peripheral blood from warm ischemia model and blood before transplant and on days 7 and 21 for the transplant model was collected in heparin tubes and separated in cytometry tubes to add the antibodies and lysis buffer needed to continue with the cytometry protocol.
Splenocytes were preserved at −80 °C after its isolation using the Ficoll (GE Healthcare) density gradient. Standard methods were used to thaw, wash, and recover the cells. An incubation of 25 min in the dark at room temperature with different monoclonal antibodies was performed for the cytometry technique using FACS Canto II Cytometer and the subsequent analysis using FACS DIVA software (BD Biosciences, San Jose, CA, USA). The antibodies were titrated, mixed, and formulated for optimal staining performance.
Cocktail T/B/NK (BD 558495) containing anti-CD3 APC, anti-CD45RA FITC, and anti-CD161 PE for T, B, and NK cell detection; anti-CD43 PE (Biolegend 202812), anti-CD161 AF647 (AbD Serotec MCA1427A647), and anti-ED9 FITC (AbD Serotec MCA620F) for monocytes detection; and anti-CD3 PerCP efluor (eBioscience 46-0030-80), anti-CD4 FITC (eBioscience 11-0040-85), anti-CD25 APC (eBioscience 17-0390-82), and anti-Fox P3 PE (eBioscience 12-4774-42) for Tregs detection. Cell membrane and nucleus permeabilization was needed for Tregs cytometric detection.