Fig 1.
Optimal concentrations of ConA and PMA/I are limited by impact on viability.
(A–D) Mitogens were used to activate PBMCs (n = 3) at different concentrations to determine optimal dose. CD5+ T cell activation was determined by cell surface expression of CD25 and viability was evaluated as an indicator for activation-induced cell death. (A) Frequency of activated T cells after ConA stimulation. (B) Impact of ConA dose increase of on cell viability. Arrows indicate the difference in cell mortality at each dose compared to the lowest dose. Green arrows indicate less than 10% loss in viability, which was deemed acceptable. Red arrows indicate a greater than 10% loss in viability which was disqualifying. (C) Frequency of activated T cells after PMA/I stimulation. (D) Impact of PMA/I dose increase of on cell viability. For (A, C) horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; ** p < 0.01, **** p < 0.0001. For (B, D), viability was defined as the frequency of viable singlets as determined by flow cytometry. Horizontal lines indicate mean values, and error bars represent standard error of the mean.
Fig 2.
Bead-bound antibody utilizing 5B8 co-stimulation demonstrates superior T cell activation.
(A) Schematic representation of experimental design. Bead-bound stimulatory antibodies (6F9=CA17.6F9; 2A12=CA17.2A12) were used to activate PBMCs (n = 3) and frequency of activated CD5+ T cells was determined by cell surface expression of CD25. (B-G) Different antibody doses were utilized to determine optimal dose for superior T cell activation. The stimulatory effects of four different αCD3 + αCD28 bead-bound antibody combinations (1:1 ratio) and two different αCD3 + isotype bead-bound antibodies (1:1 ratio) were evaluated: (B) 6F9 + 5B8 beads, (C) 2A12 + 5B8 beads, (D) 6F9 + 1C6 beads, (E) 2A12 + 1C6 beads, (F) 6F9 beads, and (G) 2A12 beads. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 3.
5B8 co-stimulation demonstrates superior T cell activation for plate-bound antibody.
(A) Schematic representation of experimental design. Plate-bound stimulatory antibodies (6F9=CA17.6F9; 2A12=CA17.2A12) were used to activate PBMCs (n = 3) and frequency of activated CD5+ T cells was determined by cell surface expression of CD25. (B-G) Different antibody doses were utilized to determine optimal dose for superior T cell activation. The stimulatory effects of four plate-bound αCD3 + αCD28 antibody combinations (1:1 ratio) and two solitary plate-bound αCD3 antibodies were evaluated: (B) 6F9 + 5B8, (C) 2A12 + 5B8, (D) 6F9 + 1C6, (E) 2A12 + 1C6, (F) 6F9 alone, and (G) 2A12 alone. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 4.
Mitogen and plate-bound antibody with 5B8 co-stimulation outperform other T cell activation strategies.
At optimal doses of mitogens, plate-, and bead-bound antibodies (6F9=CA17.6F9; 2A12=CA17.2A12), this is a summary of all activation assays performed and directly compares the impact of these T cell stimulation strategies on canine T cell activation after three days of stimulation (n = 3). Because ConA and isotype antibodies were used as positive and negative controls respectively for the activation assays utilizing antibody stimulation, ConA and isotype values for Donors #1-3 represent the average activation efficiency for each donor. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; **** p < 0.0001, ns not significant.
Fig 5.
Plate-bound antibody utilizing 5B8 co-stimulation results in a superior short term T cell expansion profile.
(A-B) CFSE proliferation assay (n = 3) over four days using mitogen, plate-, or bead-bound antibody (6F9=CA17.6F9; 2A12=CA17.2A12). CFSE intensity in CD5+ T cells was evaluated on day 4. (A) Representative histogram (Donor #1) of proliferation profiles in CD5+ T cells after utilizing different stimulation methods. (B) Graphical representation of CFSE proliferation assay illustrating the number of cellular divisions over four days in CD5+ T cells for individual donors. (C-E) Degree of proliferation in PBMCs from stimulation was determined by cell counts over four days. All experimental samples started with 106 PBMCs on day 0. Each symbol represents the mean cell count on the corresponding day. (C) is an evaluation of antibody-coated plate stimulation only, (D) is an evaluation of antibody-conjugated bead stimulation only, and (E) evaluates all stimulation strategies concurrently. (F) Evaluates the fold-change between day 0 and day 4 in all donors. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; p < 0.05 is considered significant.
Fig 6.
PMA/I stimulation results in superior T cell transduction efficiency.
PBMCs from three canine donors were transduced after three days of stimulation using mitogen, plate-, or bead-bound antibody (6F9=CA17.6F9; 2A12=CA17.2A12). PBMCs were transduced with GFP gamma retrovirus at an MOI of 13 and the frequency of GFP expressing CD5+ T cells was evaluated three days after transduction. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; *** p < 0.001, **** p < 0.0001, ns no significance.
Fig 7.
PMA/I and plate-bound 6F9 + 5B8 stimulated T cells demonstrate better expansion over 14 days after removal from stimulation.
(A) Schematic representation of experimental design and timeline. After three days of stimulation with mitogen or plate-bound antibody (6F9=CA17.6F9; 2A12=CA17.2A12), PBMCs (n = 5) were removed from stimulation and 106 cells from each sample were expanded in culture. (B) Cell count was performed and the mean count was recorded over 14 days and (C) fold change was evaluated on day 14. (D) Mean viability was recorded over 14 days with (E) statistical analysis of viability performed on day 14. For (C, E), Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; p < 0.05 is considered significant.
Fig 8.
Plate-bound 6F9 + 5B8 and ConA stimulation reduce proportion of double negative T cells.
After three days of stimulation with mitogen or plate-bound antibody (6F9=CA17.6F9; 2A12=CA17.2A12), PBMCs (n = 5) were removed from stimulation. Cell surface expression of CD4 and CD8 was evaluated before stimulation (naive), and at 7 and 14 days after removal from stimulation. Horizontal lines indicate mean values, and error bars represent standard error of the mean.
Fig 9.
PMA/I and 6F9 + 5B8 stimulation significantly reduces the proportion of Tregs.
After three days of stimulation with mitogen or plate-bound antibody (6F9=CA17.6F9; 2A12=CA17.2A12), PBMCs (n = 5) were removed from stimulation. Identification of Tregs was determined by surface expression of CD4 and CD25 and intracellular expression of Foxp3. Frequency of Tregs was evaluated before stimulation (naive), and at 7 and 14 days after removal from stimulation. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed by One-way ANOVA with Tukey’s multiple means comparison; p < 0.05 is considered significant.
Fig 10.
Culturing at 38.8 °C improves canine T cell transduction and expansion.
After three days of stimulation with PMA/I or plate-bound antibody (2A12=CA17.2A12), PBMCs (n = 3) were removed from stimulation and 106 cells were transduced with GFP gamma retrovirus. Cells were then expanded in IL-2 enriched media. (A) Transduction efficiency was determined by the frequency of CD5+ T cells expressing GFP three days after transduction. (B) Cell count was performed and the mean count was recorded over seven days and (C) fold change was evaluated on day 7. Horizontal lines indicate mean values, and error bars represent standard error of the mean. Pairwise statistical analysis was performed using paired two-tailed t-test; * p < 0.05.