Apc conjugated anti cd38

We assessed T cell proliferation by measurement of Ki-67 (BD Biosciences) (Figure 1). In brief, 1 £ 10 6 PBMCs were incubated with Pac-Blue-conjugated anti-CD3 (BioLegend), AmCyan-conjugated anti-CD8 (BD Biosciences), and APC-conjugated anti-CD38 (eBioscience), washed twice with FACS buffer, and fixed and permeabilized with ice-cold methanol. Samples were then incubated after the addition of FITC-conjugated anti-Ki-67 (BD Biosciences) and analyzed.
We assessed the potential for granule-mediated cytotoxicity by using granzyme B content as a surrogate (Figure 1). In brief, 1 £ 10 6 PBMCs were incubated with Pac-Blue-conjugated anti-CD3 antibody (BioLegend), AmCyan-conjugated anti-CD8 (BD Biosciences), APC-conjugated anti-CD38 (eBioscience), and FITC-conjugated anti-CD57 (BD Biosciences). Cells were then washed, fixed, and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences) in accordance with the manufacturer's instructions. Samples were then incubated after the addition of PE-conjugated granzyme B (BD Biosciences) and analyzed.

Treated PBMCs were harvested, washed three times with PBS, and resuspended with 100 μL PBS. To evaluate the proportion of regulatory T cells (Tregs), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience, San Diego, CA) and PerCP-cyanine-labeled anti-CD25 (eBioscience) were added to single-cell suspensions. After fixation/permeabilization, the cells were stained with phycoerythrin (PE)-conjugated anti-Foxp3 (eBioscience). To measure the ratio of Th1, Th2, Th17 cells, the treated PBMC were incubated with 50 ng/mL phorbol myristate acetate (PMA), 1 μg/mL Ionomycin and 10 μg/mL Brefeldin-A (BFA) for 4 hours. Cell suspensions were stained with surface FITC-conjugated anti-CD3 and PerCP-cyanine-labeled anti-CD4 (eBioscience) for 30 minutes and stained with allophycocyanin (APC)-conjugated anti-IFN-γ, PE-conjugated anti-IL-4, or PE-conjugated anti-IL-17A (eBioscience) for 30 minutes. For surface activation markers, such as CD25, CD38, PD-1, and ICOS, treated PBMCs were stained with APC-conjugated anti-CD25, APC-conjugated anti-CD38, PE-conjugated anti-PD-1, and FITC-conjugated anti-ICOS antibodies (eBioscience). After being washed twice with PBS, samples were processed and analyzed using a flow cytometer.

To further characterize virus specificity, we performed EBV and CMV tetramer staining in patients for whom HLA typing was amenable to available viral-specific tetramer staining. We had samples available for 16 patients in the acute GVHD cohort and 8 patients in the no acute GVHD cohort (Supplementary Table 1). In brief, 2 £ 1 £ 10 6 thawed PBMCs were stained with appropriate PE-conjugated tetramers with peptide sequences and specificities shown in Supplementary Table Biosciences), APC-conjugated anti-CD38 (eBioscience), FITC-conjugated anti-CD45RA, and 7-amino-actinomycin D (BioLegend) to assess for viability.