American Society of Hematology

Figure 1

$Figure 1. Tumor-specific CD4+ T cells transferred after Cy-treatment acquire a polyfunctional effector signature. Following the timeline depicted in the schema, mice were treated or not treated with Cy before receiving CFSE-labeled CD4+ T cells derived from HA-TCR Tg mice (Thy1.1 background). Seven days after T-cell transfer, spleen cells were harvested for analysis. (A) The frequency and cell division of the transferred CD4+ T cells are shown in representative dot plots. Results are summarized in bar graphs. (B) The absolute numbers of divided donor CD4+ T cells. The calculation formula is: total spleen cell number × frequency of transferred CD4+ T cells × fraction of divided cells (***P < .001). (C) Phenotypic and functional analyses of the transferred CD4+ T cells. PD1, Foxp3, CD127, and CD40L expression profiles relative to cell division of the transferred CD4+ T cells are shown. Cytokine expression in transferred CD4+ T cells was assayed by intracellular cytokine staining (ICS) after 4 hours of antigenic stimulation in vitro, and the plots shown are gated on the divided CD4+ T cells. Numbers in plots indicate the percentage of cells in the corresponding quadrant. The bar graphs summarize the percentages of divided donor CD4+ T cells positive for the indicated markers. Data shown in each bar graph are results from 2 independent experiments and represented as mean ± SD of at least 6 mice per group. (D) Similar experiments were conducted in CT26HA tumor model. The schema outlines the timeline of the procedures. Seven days after T-cell transfer, spleens and lung draining lymph nodes (DLNs) were recovered and analyzed. Dot plots are representative of the transferred CD4+ T cells in DLNs and spleen. Numbers represent the frequency of the gated donor CD4+ T cells. (E) Expression of CD40L in transferred CD4+ T cells in DLNs and ICS measurement of cytokines in divided donor CD4+ T cells. Numbers in the left panel indicate the percentages of CD40Lhigh cells in divided donor CD4+ T cells. Numbers in the right panel represent the percentages of IFNγ and TNFα double-positive cells in divided donor CD4+ T cells. Data shown are representative of 3 independent experiments with similar results. (F) CT26HA tumor cells remain MHC II negative even in the presence of IFNγ. CT26HA were cultured in the absence of presence of 100 ng/mL murine IFNγ for 2 days and then stained for MHC II. A20HA cells were used as a positive control for constitutive and IFNγ-inducible MHC II expressions.$

Tumor-specific CD4⁺ T cells transferred after Cy-treatment acquire a polyfunctional effector signature. Following the timeline depicted in the schema, mice were treated or not treated with Cy before receiving CFSE-labeled CD4⁺ T cells derived from HA-TCR Tg mice (Thy1.1 background). Seven days after T-cell transfer, spleen cells were harvested for analysis. (A) The frequency and cell division of the transferred CD4⁺ T cells are shown in representative dot plots. Results are summarized in bar graphs. (B) The absolute numbers of divided donor CD4⁺ T cells. The calculation formula is: total spleen cell number × frequency of transferred CD4⁺ T cells × fraction of divided cells (***P < .001). (C) Phenotypic and functional analyses of the transferred CD4⁺ T cells. PD1, Foxp3, CD127, and CD40L expression profiles relative to cell division of the transferred CD4⁺ T cells are shown. Cytokine expression in transferred CD4⁺ T cells was assayed by intracellular cytokine staining (ICS) after 4 hours of antigenic stimulation in vitro, and the plots shown are gated on the divided CD4⁺ T cells. Numbers in plots indicate the percentage of cells in the corresponding quadrant. The bar graphs summarize the percentages of divided donor CD4⁺ T cells positive for the indicated markers. Data shown in each bar graph are results from 2 independent experiments and represented as mean ± SD of at least 6 mice per group. (D) Similar experiments were conducted in CT26HA tumor model. The schema outlines the timeline of the procedures. Seven days after T-cell transfer, spleens and lung draining lymph nodes (DLNs) were recovered and analyzed. Dot plots are representative of the transferred CD4⁺ T cells in DLNs and spleen. Numbers represent the frequency of the gated donor CD4⁺ T cells. (E) Expression of CD40L in transferred CD4⁺ T cells in DLNs and ICS measurement of cytokines in divided donor CD4⁺ T cells. Numbers in the left panel indicate the percentages of CD40L^high cells in divided donor CD4⁺ T cells. Numbers in the right panel represent the percentages of IFNγ and TNFα double-positive cells in divided donor CD4⁺ T cells. Data shown are representative of 3 independent experiments with similar results. (F) CT26HA tumor cells remain MHC II negative even in the presence of IFNγ. CT26HA were cultured in the absence of presence of 100 ng/mL murine IFNγ for 2 days and then stained for MHC II. A20HA cells were used as a positive control for constitutive and IFNγ-inducible MHC II expressions.

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