CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 +

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1 Supplements Supplemental Materials and Methods Depletion of CD25 + T-cells from PBMC. Fresh or HD precultured PBMC were stained with the conjugate CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 + T-cells using MACS (Miltenyi Biotec). Pentamer staining. Frequencies of PP65_HCMV specific T-cells were detected using Pro5 Pentamer HLA-A21 PP65_HCMV Pro5 Pentamers HLA-A21 was used as a negative control (Proimmune). Phenotyping of CD8 T-cells. The following additional antibodies and chemicals were used for phenotyping of CD8 T-cells by flow cytometry: acd152 (CTLA-4)-PE, acd279 (PD-1)-PerCP/Cy5.5, acd357(gitr)- PE (BioLegend), Annexin V-FITC (ImmunoTools) and 7-AAD (BD Biosciences).

2 Figure S1. Time and density-dependence of PBMC preculture effect on virus-and tumor-directed CD8 T-cells responses. (A) CD8 T-cell responses of a healthy donor to.1 µg/ml of the PepMix TM CEF standard were obtained by IFN-γ ELISPOT assay. PBMC preculture conditions were tested by varying time and cell densities. Unstimulated cells from all preculture conditions tested did not release IFN-γ. Data represent mean ± SD for triplicate samples. (B) IFN-γ responses from PBMC of a representative HLA-A21 positive leukemia patient to titrated WT1_HUMAN LD preculture was performed with 1x1 6 cells/ ml. Experiment was repeated 5 times. 2way ANOVA P <.5; P <.1. (C) The RESTORE effect is independent of cryopreservation. PBMC of a representative leukemia patient were frozen, thawed and tested with or without the HD preculture step. IFN-γ secretion of cryroconserved and non-cryoconserved fresh or HD precultured samples were compared by an unpaired t test: n.s; The RESTORE effect was significantly analyzed by 2way ANOVA. Experiment was repeated 3 times. Figure S2. RESTORE effect is not due to a reduction in Treg activity. (A, Left) Similar frequencies of regulatory T-cells in unstimulated fresh and precultured PBMC of a representative healthy donor. Intracellular staining and flow cytometry analysis of regulatory T-cells; Gating of Foxp3 + CD25 + cells was performed on viable CD4 T-cells. (Right) Regulatory T-cells were depleted from fresh or precultured PBMC by magnetically labeling CD25 positive cells. (B) Similar increase in virus-specific CD8 T-cell responses upon HD preculture of total and CD25-depleted PBMC. Cells from Panel A were stimulated with titrated PepMix TM CEF standard in IFN-γ ELISPOT assays. Responses of Fresh/ CD25 depleted Fresh PBMC and Precultured/ CD25 depleted Precultured PBMC, respectively were compared by an unpaired t test: ; Significantly enhanced IFN-γ secretion of CD8 T-cells upon HD preculture of total or CD25 depleted PBMC relative to fresh or CD25 depleted fresh PBMC were detected after stimulation with.33 (unpaired t test: P <.5),.1 ( P <.5) and.3 µg/ml ( P <.5) of the PepMix TM CEF standard. Data represent mean ± SD for triplicate samples. Experiment was repeated 3 times.

3 Figure S3. HLA-A21-restricted WT1 specific CD8 T-cell responses in fresh and HD preculture PBMC. (A,B) PBMC of HIV sero-negative hematopoietic stem cell transplanted patients that show WT1 dependent IFN-γ releases to WT1_HUMAN (Figure 5A) or WT1_HUMAN (Figure 5B) were also treated in IFN-γ ELISPOT assays with titrated amounts of the irrelevant HLA-A21-restricted peptide HIV-1 pol (C) Nonspecific effects of WT1 peptide stimulation were excluded by treating PBMC of an HLA-A21 negative healthy donor with titrated WT1_HUMAN or No unspecific responses were detected. Unpaired t test: ; The RESTORE effect was demonstrated by TAB8 responses, exclusively in HD precultured PBMC, but not in fresh PBMC of all tested patients (S3A,B) and healthy donors (S3C). Data represent mean ± SD for triplicate samples. Figure S4. HD preculture prepares T-cells to better respond to antigen, but does not increase the frequency of antigen-specific cells. Similar frequencies of PP65_HCMV specific T-cells were detected in unstimulated Fresh, HD and LD precultured PBMC by flow cytometry upon pentamer staining. An HLA-A21-restricted pentamer was used as a negative control. Gating was performed on viable CD8 T-cells. Experiment was repeated with 2 HLA-A21 positive healthy donors. Figure S5. Raw data of the summary Figures 2B,D, 3E and 5C. IFN-γ pos cells / 1 6 fresh (F) or HD precultured (P) PBMC upon stimulation with titrated (A) PepMix TM Influenza A, (B) PP65_HCMV , (C) PepMix TM CEF standard and (D) WT1_HUMAN Wilcoxon matched-paired signed rank test: P <.5, P <.5; P <.5; P <.1;

4 cells / ml Figure S1 A 3x1 7 1x x x1 6 3x1 7 1x x x1 6 3x1 7 3 days 2 days B IFN-γ pos cells / 1 6 PBMC HD preculture 2 days LD preculture 2 days HD preculture 1 day US x1 7 WT1_HUMAN (μg/ml) 3.3x x1 6 1 day US IFN-γ pos cells / 1 6 PBMC C IFN-γ pos cells / 1 6 PBMC Fresh cryoconserved Precultured Precultured cryoconserved US WT1_HUMAN (μg/ml)

5 Figure S2 A before after B Precultured CD25 depletion CD25 depletion IFN-γ pos cells / 1 6 PBMC CD25 depleted Fresh Precultured CD25 depleted Precultured Foxp3 CD25 US PepMix TM CEF standard (μg/ml)

6 Figure S3 A B IFN-γ pos cells / 1 6 PBMC Fresh (TAB8 1 μg/ml: 5 ± 2.4) Precultured (TAB8 1 μg/ml: 19 ± 14) US HIV-1 pol (μg/ml) IFN-γ pos cells / 1 6 PBMC Fresh (TAB8 1 μg/ml: ± ) Precultured (TAB8 1 μg/ml: 527 ± 38.3) US HIV-1 pol (μg/ml) IFN-γ pos cells / 1 6 PBMC C Fresh (TAB8 1 μg/ml: 3.3 ± 2.4) Precultured (TAB8 1 μg/ml: 293 ± 69) US WT1_HUMAN (μg/ml) IFN-γ pos cells / 1 6 PBMC US WT1_HUMAN (μg/ml)

7 Figure S4 Negative HLA-A21 pentamer.3 PP65_HCMV HLA-A21 pentamer HD precultured LD precultured pentamer CD8

8 IFN-γ pos cells / 1 6 PBMC Figure S5 A PepMix TM Influenza A (.12 μg/ml) (.37 μg/ml) (.11 μg/ml) (.33 μg/ml) F (1 μg/ml) P IFN-γ pos cells / 1 6 PBMC B PP65_HCMV (.1 μg/ml) (.1 μg/ml) (.1 μg/ml) (.1 μg/ml) F (1 μg/ml) P IFN-γ pos cells / 1 6 PBMC C PepMix TM CEF standard (.1 μg/ml) (.1 μg/ml) IFN-γ pos cells / 1 6 PBMC D F P F P F P WT1_HUMAN (2.5 μg/ml) (5 μg/ml) (1 μg/ml)

9 Supplementary Table Supplementary Table. Phenotyping of CD8 T-cells and monocytes from fresh or HD precultured PBMC. Expression of the memory (CD45R), co-stimulation or co-inhibition (CD28, GITR and CTLA-4), exhaustion (PD-1) or cell death (7-AAD, Annexin V) markers on CD8 T-cells as well as the co-stimulation (CD8, CD86) and HLA markers on CD14 positive monocytes are given as frequencies (%) and ΔMFI (MFI of the specific marker) MFI (unstained control). % ΔMFI within marker Fresh Precultured Fresh Precultured lymphocytes CD3 CD CD45R CD GITR CD8 T- cells PD CTLA AAD Annexin V CD monocytes CD HLA- A,- B,- C HLA- DR, - DP, - DQ no distinct population