Fig 1.
A novel T cell:PBMC assay to detect CD4+ T cell response to BPs in healthy donors.
The figure shows a schematic representation of the T cell:PBMC assay format: PBMCs were obtained from naive healthy donors. CD4+ T cells were enriched and plated in 24-well or 96-well plates at respectively 5x105 or 1x105 cells/well in multiple wells containing irradiated allogeneic PBMC at a concentration of either 2x105 or 1x106 cells/well. The co-cultured cells were challenged with KLH (30 μg/ml), CMV (2 μl/ml), infliximab, rituximab, adalimumab and natalizumab (all 45 μg/ml). To the 24-wells for ELISpot analysis 5 μl/ml anti-CD28/CD49d mAbs were added. At day six, the 24-well cell cultures were washed thoroughly, re-stimulated with the corresponding compounds and plated in triplicates in 96-well plates for 18 hours prior to spot detection. At day six and eight, proliferation (n = 6) was measured after 18-h pulse with 3[H]-thymidine. Reprint from Servier Medical Art by Servier under a CC BY license with permission from Servier Medical Art, original copyright Creative Commons Attribution 3.0 Unported License.
Fig 2.
PBMCs from a healthy donor were isolated and used for both (A) CD8+ T cell-depleted PBMC assay and (B) our optimized T cell:PBMC assay. The cultures were challenged with keyhole limpet hemocyanin (KLH, 30 μg/ml), Protective antigen (PA, 3 μg/ml) and cytomegalovirus (CMV, 2μl/ml). Proliferation was measured by 3[H]-thymidine incorporation at day six and IL-2 secretion was measured by ELISpot analysis at day seven. The proliferative response in counts per minute (cpm) and ELISpot IL-2 secretion (spw) was converted to stimulation index (SI). Shown are graphs of SI of proliferation and IL-2 secretion, which is also visualized by pictures. Shown is one representative donor out of four.
Fig 3.
One representative donor’s response to KLH (30 μg/ml), CMV (2 μl/ml), infliximab, rituximab, adalimumab and natalizumab (all 45 μg/ml) was assessed in the T cell:PBMC assay. The ability to elicit an Ag-specific response was detected by proliferation and IL-2 ELISpot. The response to untreated is included as a negative control. Proliferation was measured by 3[H]-thymidine incorporation in sextuplet at (A) day six and (B) day eight, shown as stimulation index. IL-2-producing T cells were identified by ELISpot analysis as shown by (C) stimulation index and as (D) visualized by pictures. The statistical difference indicated is based on raw data; cpm for T cell proliferation (n = 6) or spw for ELISpot (n = 3) using an unpaired student t test.
Table 1.
Summary of research data and characteristics of BPs from European clinical trials and studies.
Fig 4.
Mean magnitude of the BPs in the test population.
Scatter plot summarizing the mean stimulation index value of all the donors in the study population. Shown is (A) proliferation day six, (B) proliferation day eight and (C) IL-2 secretion. The dotted line indicates SI = 2, which is the cut-off value for a positive response.
Fig 5.
Frequencies of CD4+ T cells specific to the BPs.
The frequency of CD4+ T cell specific for KLH, CMV, Infliximab, Rituximab, Adalimumab and Natalizumab expressed per 106 naïve CD4+ T cell for the individual donors as well as the mean (± SEM) is indicated. The data is based on pooled data from proliferation at day six and eight from all 26 donors. The mean number of BP-specific CD4+ T cells identified is specified at the top of each histogram.
Fig 6.
Comparison of the frequency of high resolution HLA-DRB1 allotypes expressed in the test population versus the; A) the European population and B) the North American population. The correlation was calculated using Pearson correlation coefficient.
Table 2.
A summary of the high resolution HLA-DR, DQ and DP haplotypes of the included donors in the assay.
Fig 7.
Frequency of responding donors.
Summary of CD4+ T cell response to BPs among 26 donors. Donors were considered to be positive responders if one of both of the two proliferation assays, and/or the IL-2 secretion assay, showed a SI>2 with p<0.05 for a given donor’s response to a given BP compared to the respective control assay result.
Fig 8.
Plot of T cell response to the BPs.
The frequency of responding donors and their mean IL-2 secretion and proliferation stimulation index (SI) levels. Donors were considered to be positive responders if one or both of the two proliferation assays, and/or the IL-2 secretion assay, showed an SI>2 with P<0.05 for a given donor’s response to a given BP compared to the respective control assay result. The mean SI for IL-2 secretion and proliferation levels for these positive responders are plotted against the % of positive responders for each BP.