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D. Negative fraction after isolation of

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1 D. Negative fraction after isolation of
Qesari et al., SDC, Figure 1 IFN-ϒ CD3 CD4 CD8 A. Unstimulated control B. pp65 stimulated PBMCs before magnetic enrichment C. pp65 stimulated PBMCs after magnetic D. Negative fraction after isolation of pp65 specific T-cells

2 SDC, Figure 1 Representative FACS dot plots for the evaluation of the purity after isolation of CMV-CTLs by gamma-catch.

3 Qesari et al., SDC, Figure 2 CD3 CD4 CD8 IFN-ϒ Unstimulated control pp65 stimulated cells

4 SDC, Figure 2 Representative FACS dot plots for the detection of intracellular IFN-ϒ staining in expanded CTLs.

5 Unloaded Target only Target + CTLs Unloaded
Aqua CFSE CD4 CD8 SSC-A FSC-A Viable target cells Target cells Unloaded Target only Target + CTLs Unloaded Qesari et al., SDC, Figure 3 Pp65-loaded Target only Pp65-loaded Target + CTLs

6 SDC, Figure 3 Representative FACS dot blots and gating strategy for the calculation of the percentage of specific lyses by CMV-CTLs, as described in the Material and Methods section. For each experimental condition, cells were gated with a consecutive order on lymphocytes, CD4+ T cell subset, CD4+ / CFSE+ T-cell subset (target cells), aqua- / CFSE+ T-cell subset (viable target cells). An additional gating on count beads was applied, on each experimental condition, for the calculation of the absolute number of target cells.

7 MLR, Autologous: Recipient + Recipient Qesari et al., SDC, Figure 4
MLR, Allogeneic: Unselected-PBMCs + Recipient FSC-A CD45 CD45 CD45 CD3 CD4 CD4 CD8 CD45 Aqua CD14 CD3 CD25 CD8 CD25 CD25 MLR, Allogeneic: CMV-CTLs + Recipient CD25 FSC-A CD45 Aqua CD14 CD3 CD4 CD8 CD45 Aqua CD14 CD3 CD25 CD8 FSC-A CD4

8 SDC, Figure 4 Representative FACS dot plots for the evaluation of the CD25 activation of T-cells in the MLR.

9 A. HLA-virus restrictions matches * GvHR crosstabulation
Qesari et al., SDC, Figure 5 A. HLA-virus restrictions matches * GvHR crosstabulation CMV-CTLs Donor-Recipient GvHR Total GvHR + GvHR - HLA-matches Unmatched 3 2 5 60.0% 40.0% 100.0% Matched 1 66.7% 33.3% 8 62.5% 37.5% B. Chi-Square Test Value df Asymp. Sig. (2-sided) Exact Sig. (2-sided) Exact Sig. (1-sided) Point Probability Pearson Chi-Square 0.036 1 .850 1.000 .714 Continuity Correction 0.000 Likelihood Ratio .036 Fisher's Exact Test Linear-by-Linear Association 0.031 .860 .536 N of Valid Cases 8 SDC, Figure 5 A: Chi-Square test reporting the incidence in percentage between the HLA donor-recipient pairs matched or unmatched for the possible virus restrictions and the presence or absence of GvHR (n=8). B: Fisher’s Exact Test: P=1

10 Correlation Coefficient
Qesari et al., SDC, Figure 6 Correlations GvHR % of CD4+ CTLs Spearman's rho Correlation Coefficient 1.000 .894 Sig. (2-tailed) .106 N 4 % of CD4+ CMV-CTLs SDC, Figure 6: Spearman’s rank correlation between the frequency of CD4+ CTL population and the incidence of GvHR. Sperman’s correlation coefficient for n=4 is

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