Cd4 ecd

Two panels were designed; Immune Activation Panel and Immune Exhaustion Panel. The Immune Activation Panel consisted of the following fluorochrome-coupled monoclonal antibodies: CD3-FITC; CD4-ECD; CD8-PC-7; CD45-KO; CD38-PE and; HLA-DR-APC (Beckman Coulter, MI). The Immune Exhaustion Panel was composed of CD3-FITC; CD4-ECD; CD8-PC7; CD45-KO; CTLA-4-PE and; PD-1-APC. Flow cytometry data acquisition was performed using a Navios flow cytometer (Beckman Coulter). Optimal volatges for acquisition were determined, compensation and fluorescence minus one analyses were performed before testing of study samples. The gating strategy is shown in Additional file 1: Figure S1. Data analysis was performed using Beckman Coulter Kaluza software.

The available number of PBMC allowed further analysis of eight samples by flow cytometry. In detail, 0.5 × 10⁶ cells were transferred to a 96-well round-bottom plate in 100 µL culture medium with fetal calf serum in the presence of the corresponding peptide pool or SEB or culture medium alone. Following 1-h incubation at 37 °C in a humidified 5% CO2, brefeldin A (Sigma-Aldrich) at a final concentration of 10 ug/mL was added. After overnight incubation, cells were washed in phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid (EDTA) 2mM and incubated with the Live/Dead Fixable Far Red Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA, USA) for 30 min at 4 °C. Cells were then washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm (BDBiosciences, San Jose, CA, USA) according to the manufacturer’s instructions for intracellular staining with the following monoclonal antibodies: IFNγ-FITC, CD4-ECD, CD8-PC7, CD3-Pe-Cy5 (all from BeckmanCoulter, Brea, CA, USA). Finally, the cells were resuspended in 4% paraformaldehyde and analyzed with a Navios flow cytometer (Beckman Coulter, Brea, CA, USA), obtaining the percentages of IFN-γ secreting lymphocyte cell responses and allowing phenotypical discrimination of individual cytokine-producing cells

The following monoclonal antibodies (mAbs) were used in different combinations. CD8-PB, CD3-APC-H7, PD-1-PECy7, PD-1-PB, IFN-γ-AF700, IL-2-PE, CD4-PB, granzyme B-AF700, perforin-APC were purchased from Becton Dickinson (BD, San Diego, CA), CD45RA-ECD, CD4-ECD from Beckman Coulter (Fullerton, CA, USA), CCR7-FITC from R&D Systems (Minneapolis, MN, USA), 2B4-PECY5.5, CD160-APC, CD160-PE from BioLegend (San Diego, CA, USA), CD4-eFluor650NC, CD8-eFluor625NC from eBioscience.

The prostate single cell suspension was centrifuged, and the supernatant was removed, and 50uL normal saline was added to resuspend. Add CD45-KO, CD3-FITC, CD4-ECD, CD8-PC5, CD25-PC7, CD127-APC flow antibodies (both 2μL, Beckman Coulter) to the cell suspension and incubate at room temperature for 20 minutes. After incubation, centrifuge to remove the supernatant, add 50uL saline to resuspend, and use a Beckman Navios flow cytometer. The results were analyzed using Kaluza 3.13 flow cytometry analysis software.

The ability of expanded Tregs to generate new Tregs infectiously from naïve responder cells was measured using the “Treg-MLR” as described previously^{24 (link)}. Briefly, MLR cultures were set up with “recipient” CFSE-labelled responders stimulated with irradiated (3,000 rads) and PKH26-labelled donor-specific or allo-irrelevant PBMC. PKH26-labelled modulator cells composed of either Tregs or R-PBMC treated in an equivalent manner to serve as controls were then added at modulator: responder ratios of 1:10, 1:50, and 1:250. On day 7, flow cytometry was performed on the cultured cells after labelling with CD127-PE, CD4-ECD, CD25-PC7 (all from Beckman-Coulter), and FOXP3-PC5 (eBioscience). PKH26-labelled modulators and any surviving stimulators as well as CD127-PE⁺ responder cells were gated out, and CD4⁺ cells that proliferated (as determined by reduced CFSE expression) were analyzed for CD25 and FOXP3 expressions. Thus, the percentage of CD4⁺CD127⁻CD25^HighFOXP3⁺ cells that were newly generated in the proliferating responder cells was determined.

As previously described [1 (link), 8 (link), 9 (link)], after Ficoll-Hypaque gradient centrifugation, PBMC were labeled with CFSE following the manufacturer’s instructions (labeling efficiency of >99%). 5x10⁵ CFSE labeled responding PBMC from healthy volunteer (A) were cultured with 5x10⁵ PKH26 labeled stimulator cells from HLA-2DR-matched (Bx) or HLA-mismatched (Ix; indifferent 3^rd party) laboratory volunteers in 48-well culture plates. On days 0 (baseline), 5, 7 and 9 flow cytometric analyses were performed for surface markers using CD4-ECD, CD25-PC7, CD127-PE (all from Beckman-Coulter, Miami, FL) and for intracellular FOXP3-PC5 (eBioscience, San Diego, CA) as previously described [7 (link), 8 (link)]. Gated viable lymphocytes were further gated followed for CFSE bright and dim cells which were negative for both CD127-PE and PKH26 (thus gating out CD127⁺ responders and any residual stimulators). This was followed by gating for CD4⁺ cells. CFSE dilution in these CD4⁺ responders assessed the extent of proliferation, i.e., non-proliferating (CFSE high) or proliferating (CFSE low) cells. The expression of CD25 and FOXP3 were analyzed in the non-proliferating and proliferating populations. Data were calculated as percentage of CD127^-CD4⁺ cells that were CD25⁺FOXP3⁺ (total Tregs) or CD25^HighFOXP3⁺ (natural Tregs; nTregs) [10 (link)].

Laboratory analyses included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), total serum IgE, total serum immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 subclasses. Flow cytometry was performed using a Navios cytometer (Beckman Coulter, Brea, CA, USA) on fresh peripheral blood collected in ethylenediaminetetraacetic acid tubes using a lyse-no-wash technique (ammonium chloride) and the following panel of directly conjugated antibodies: CD3-fluorescein isothiocyanate, CD4-ECD, CD8-Pacific Blue, CD19-A700, CD20-allophycocyanin, CD27-phycoerythrin-cyanine 7, CD38-A750, CD45-Krome Orange, CD56-phycoerythrin, CD138-PC5.5 (Beckman Coulter). Naive B cells, memory B cells, plasmablasts, and plasma cells were identified within the CD19⁺ gate as CD19⁺CD20⁺CD27⁻CD38⁺ cells, CD19⁺CD20⁺CD27⁺CD38⁻ cells, CD19⁺CD20⁻CD27⁺CD38^+bright cells, and CD19⁺CD20⁻CD38⁺CD138⁺ cells, respectively. Total B cells were identified both as CD19⁺ cells (CD19⁺/side scatter [SSC] within the leukogate) and CD20⁺ cells (CD20⁺/SSC within the leukogate).