Figure 2.
Characterization of panfungal T-cell product. Donor HPCs were stimulated with lysates of A terreus and C krusei and expanded as described in “Methods.” Average composition or percentage of (A) T cells (CD3+), natural killer (NK) cells (CD3−CD56+), natural killer T cells (NKT) (CD3+CD56+), B cells (CD19+), and monocytes (CD14+) of live cells; (B) CD4+ T cells and CD8+ T cells of CD3+ T cells; (C) T central memory (CD45RA−62L+), T terminal effector (CD45RA+62L−), T naive (CD45RA+62L+), and T effector memory (CD45RA−62L−); (D-E) exhaustion marker expression profile of CD4+ T cells (PD-1, Tim-3 and CTLA-4, and CD57 and Lag-3) and (F) the CD4 T helper subtypes TH1 (CCR4−CCR6−CCR10−CXCR3+), TH2 (CCR4+CCR6−CXCR3−), TH9 (CCR4-CCR6+), TH17 (CCR4+CCR6+CCR10−CXCR3−), TH22 (CCR4+CCR6+CCR10+), and TH1/TH17 (CCR4−CCR6+CXCR3+) was determined at the end of culture by flow cytometry (A-D) or mass cytometry by time of fight (E-F). Results are means of 3 to 11 experiments (±SEM) and shown as percentage of live cells, CD3+ T cells, or CD4+ T cells. (G) Quantification of the TCR-β sequences expression in CD4+CD154+ sorted cells from T-cell products expanded for 11 days or 19 days after 16-hour activation with A fumigatus–pulsed DCs. Results are from 5 experiments and give the proportion of the product made up of the top N clones, where N is the number of clones shown. (H) Immunoblot analysis of cytokines in supernatants of panfungal T cells cultured for 24 hours with noncontrol or fungus-pulsed DCs, shown as representative immunoblot image from 2 experiments (cytokines measured in the blot are described in the tables; upper table refers to top 2 blots, and lower table refers to bottom 2 blots). Relative density measurement corresponds to the relative cytokine expression levels from panfungal T cells cultured with pulsed DCs and control DCs and calculated as follows: X(Ny) = X(y) × P1/P(y), where P1 is the mean signal density of positive control spots on reference array, P(y) is the mean signal density of positive control spots on array “y,” X(y) is the mean signal density for spot “X” on array for sample “y,” and X(Ny) is the normalized signal intensity for spot “X” on array “y” spots on reference array “y.”

Characterization of panfungal T-cell product. Donor HPCs were stimulated with lysates of A terreus and C krusei and expanded as described in “Methods.” Average composition or percentage of (A) T cells (CD3+), natural killer (NK) cells (CD3−CD56+), natural killer T cells (NKT) (CD3+CD56+), B cells (CD19+), and monocytes (CD14+) of live cells; (B) CD4+ T cells and CD8+ T cells of CD3+ T cells; (C) T central memory (CD45RA−62L+), T terminal effector (CD45RA+62L−), T naive (CD45RA+62L+), and T effector memory (CD45RA−62L−); (D-E) exhaustion marker expression profile of CD4+ T cells (PD-1, Tim-3 and CTLA-4, and CD57 and Lag-3) and (F) the CD4 T helper subtypes TH1 (CCR4−CCR6−CCR10−CXCR3+), TH2 (CCR4+CCR6−CXCR3−), TH9 (CCR4-CCR6+), TH17 (CCR4+CCR6+CCR10−CXCR3−), TH22 (CCR4+CCR6+CCR10+), and TH1/TH17 (CCR4−CCR6+CXCR3+) was determined at the end of culture by flow cytometry (A-D) or mass cytometry by time of fight (E-F). Results are means of 3 to 11 experiments (±SEM) and shown as percentage of live cells, CD3+ T cells, or CD4+ T cells. (G) Quantification of the TCR-β sequences expression in CD4+CD154+ sorted cells from T-cell products expanded for 11 days or 19 days after 16-hour activation with A fumigatus–pulsed DCs. Results are from 5 experiments and give the proportion of the product made up of the top N clones, where N is the number of clones shown. (H) Immunoblot analysis of cytokines in supernatants of panfungal T cells cultured for 24 hours with noncontrol or fungus-pulsed DCs, shown as representative immunoblot image from 2 experiments (cytokines measured in the blot are described in the tables; upper table refers to top 2 blots, and lower table refers to bottom 2 blots). Relative density measurement corresponds to the relative cytokine expression levels from panfungal T cells cultured with pulsed DCs and control DCs and calculated as follows: X(Ny) = X(y) × P1/P(y), where P1 is the mean signal density of positive control spots on reference array, P(y) is the mean signal density of positive control spots on array “y,” X(y) is the mean signal density for spot “X” on array for sample “y,” and X(Ny) is the normalized signal intensity for spot “X” on array “y” spots on reference array “y.”

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