Figure 1. T Cells Stimulated with ImmunoCult™ Human T Cell Activating Protein Are Compatible with Regulatory T Cell Suppression Assays

Human regulatory T cells (Tregs) and CD4+CD25- responder T cells (Tresps) were isolated from a fresh leukopak (Catalog #70500) using EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Catalog #18063), which allows for isolation of both T cell subsets into separate fractions. The CD4+CD25- Tresps were labeled with a cell proliferation dye, stimulated with ImmunoCult™ Human T Cell Activating Protein, and cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) at various ratios of Tregs:Tresps. Flow cytometric analysis was performed after 4 days of culture. The suppression response at the 1:1 Treg:Tresp ratio was calculated to be 45.4 ± 9.5% (data represent mean ± SD, n = 12 donors).

Figure 2. Morphology of Human T Cells Stimulated with ImmunoCult™ Human T Cell Activating Protein Demonstrates Rapid Activation

Human T cells were isolated from a fresh leukopak (Catalog #70500) using EasySep™ Human T Cell Isolation Kit (Catalog #17951), stimulated with ImmunoCult™ Human T Cell Activating Protein, and cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) supplemented with Human Recombinant IL-2 (CHO-expressed; Catalog #78036). A representative culture image is shown for the T cells demonstrating successful activation after only 1 day of culture.

Figure 3. Human T Cells Stimulated with ImmunoCult™ Human T Cell Activating Protein Display Rapid Expression of the Activation Marker CD25

Human T cells were isolated from a fresh leukopak (Catalog #70500) using EasySep™ Human T Cell Isolation Kit (Catalog #17951), stimulated with ImmunoCult™ Human T Cell Activating Protein, and cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) supplemented with Human Recombinant IL-2 (CHO-expressed; Catalog #78036). Activation of viable CD3+CD4+ and CD3+CD8+ T cells was assessed by detecting CD25 expression via flow cytometry. Following 1 day of culture, the frequency of CD25+ cells was (A) 93.1% for CD3+CD4+ and (B) 95.0% for CD3+CD8+ T cells.

Figure 4. CD8+ T Cells Are Preferentially Expanded in Human T Cell Cultures Stimulated with ImmunoCult™ Human T Cell Activating Protein

Human T cells were isolated from a fresh leukopak (Catalog #70500) using EasySep™ Human T Cell Isolation Kit (Catalog #17951) and expanded over 10 days with ImmunoCult™ Human T Cell Activating Protein in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) supplemented with Human Recombinant IL-2 (CHO-expressed; Catalog #78036). No additional ImmunoCult™ Human T Cell Activating Protein was added during the 10-day culture period. After 10 days in culture, the human T cells (A) expanded 360 ± 248 fold and (B) skewed the CD4:CD8 T cell ratio in favor of CD8+ T cells (data represent mean ± SD, n = 4 donors).

Table 1. Comparison of ImmunoCult™ Human T Cell Activation Reagents