Anti hla dr pe cy7

Flow cytometry was used for phenotyping of engrafted human cells. Probes of blood or re-suspended cells retrieved from solid organs were incubated with inactivated mouse serum to block unspecific Fc receptors. Then, mixtures of monoclonal antibodies against human antigens were added and cells were incubated for 30 min at room temperature. The following antibodies were used: anti-CD45 AmCyan, anti-CD14 PE, anti-CD3 Pacific Blue, anti-CD4 APC, anti-CD8 PE, anti-CD33 PeCy5.5, anti-HLA-DR PE.CY7, (BD Bioscience), anti-CD80 Alexa Fluor 488 (Biolegend, San Diego, USA). After staining, erythrocytes were lysed with BD Pharm Lyse solution (BD Biosciences) for 10 min, washed with PBS with 2% Newborn Calf Serum (NCS), and re-suspended in 0.5% paraformaldehyde in PBS. Cells were acquired using the FACS Canto II flow cytometer and Diva software (BD Bioscience, San Jose, CA). Analysis of immunophenotype was carried out applying the FlowJo 10 software (TreeStar, Inc., now part of FlowJo LLC, Ashland, OR, USA). The applied anti-human antibodies were verified for cross-reactivity with cells of non-transplanted mice and no staining was present.

Colorectal tissue was digested with an enzyme cocktail including dispase I and DNase I, as previously described [23 (link)]. The resulting cellular suspension was washed in PBS, counted and stained for flow cytometry analysis at a concentration of 10⁶ cells/mL. Mononuclear cells (MNCs) were fixed in a PBS solution containing 2% paraformaldehyde, 60 mM sucrose at pH 7.4, for 15 min at room temperature. Fixation is necessary for MAb 45523 to bind to cell surface CCR5 on primary CD4⁺ T-cells. The fixed cells were washed twice with PBS containing 20 mM glycine (buffer A) and incubated for 15 min at room temperature in buffer A supplemented with 1% BSA and 0.05% NaN₃ (buffer B). Cells were then stained with mixtures of MAbs in buffer B, anti-CD4-APC-Cy7, anti-CD13-PE, anti-CCR5-2D7-FITC, anti-CXCR4-12G5-FITC, anti-HLA-DR-PE-Cy7 (BD Pharmingen, San Diego, CA), anti-CCR5-45531-FITC, anti-CCR5-45523-FITC, anti-DC-SIGN-PE (R&D Systems), anti-CD14-ECD, anti-CD64-PE, or anti-89-PE (Beckman Coulter), for 1 h at room temperature. After three washes in buffer B, cells were resuspended for analysis using a BD LSR II flow cytometer (BD Biosciences; San Jose, CA, USA). The parameters used to select cell populations for analysis were forward and side-light scatter, with a total of 10,000 events being collected for analysis as described previously [24 (link)].

Single cell suspensions of leukocytes from peripheral blood were stained using the following monoclonal antibodies: anti-CD3 APC-H7 (BD, 641397), anti-CD4 Alexa Fluor 700 (BD, 557922), anti-CD8 Alexa Fluor 700 (BD, 557945), anti-CD25 APC (Miltenyi 130-092-858), anti-CD28 BV421(BD, 562613), anti-CD38 BV605 (Biolegend 303532), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-CD39 BV421 (BD 563679), anti-CD45 RO BV510 (BD, 563215), anti-CD56 APC (Biolegend 318310), anti-CCR7 PE (BD 561008), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-Ki-67 FITC (BD 556026), anti-HELIOS (Biolegend, 137214), anti-CTLA-4 APC (BD 560938), anti-OX40 FITC (Biolegend 35006), and anti-PD1 BV510 (Biolegend 329932). Cells were stained in FACs buffer (1 % FBS in PBS with 0.01 % NaN₃) and fixed according to the eBioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were analyzed on a BD X20 Fortessa Flow cytometer. We used Fluorescence Minus One to discriminate between positive and negative cells for each antibody [33 (link)].

B-cell subsets were measured in freshly thawed cryopreserved PBMCs. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD19-PE-Cy5, anti-CD21-PE, anti-CD20-PE-Cy7, anti-CD27-APC, anti-IgM-FITC, anti-CD38-FITC, anti-HLA-DR-PE-Cy7 (BD Bioscience); anti-CD10-APC-Cy7, anti-BAFFr-APC-Cy7 (Biolegend); anti-TACI-PE, anti-CxCr5-APC (R & D Systems) and analyzed with Guava easyCyte 8HT (Millipore) and FlowJo (Treestar). Subsets were expressed as percentages of the parent CD19+ B-cell population.

Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).