A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease

Figure 1. Study overview. The pilot study included two participants prior to commencement of the Nexvax2 Phase 2 Sub-study.

Figure 2. CD4+ T cell proliferation assessed by CTV-dilution in fresh PBMC from two HLA-DQ2.5+ CD patients incubated for 8 days with peptide pools containing Nexvax2 peptides (NX), non Nexvax2 peptides (xNX) or both, or CEFT positive control peptides. Mean (SEM) percent proliferating CD4+ T cells (A, B) and mean fold-difference in proliferation compared to control (C, D) is shown for PBMC incubated with peptide mixes, data represent mean of triplicates.

Figure 3. Cytokine release in fresh PBMC from two HLA-DQ2.5+ CD patients. Concentrations (A, B) and fold-change relative to control (C, D) are shown for IL-2, IFN-γ, and IL-10 in media after 24-hour incubation of PBMC with gluten peptides or CEFT. Data are normalized for the number of live CD3+CD4+ and CD3+CD8+ T lymphocytes in PBMC and represent the mean + SEM of six replicate wells; Mann-Whitney U test was used to compare cytokine release for peptide mixes versus Control (statistical significance is indicated by *p < 0.05, or **p < 0.01). IL-2 release in PBMC is shown following depletion of B cells, CD4+ T cells or CD8+ T cells, or pre- and co-incubation with antibody that blocks epitope presentation by HLA-DQ or HLA-DR, data represent mean of duplicates (E, F).

Figure 4. Concentration and fold-difference relative to control for Nexvax2 (NX)-stimulated IL-2 (A, B), IFN-γ (C, D) and IL-10 (E, F) release in PBMC and whole blood normalised for the number of live CD3+CD4+ T cells. Unseparated blood was allowed to stand at room temperature while PBMC were separated from a matched sample. Three hours after blood was collected, 225 μl aliquots of whole blood or 0.5 million PBMC in media were dispensed into microwells containing 25 ul PBS and test antigen. Plasma or media were separated after 24-hour incubation. For whole blood incubations, cytokine concentrations in plasma separated from whole blood before incubation with NX or control is also shown. Data points represent cytokine levels for each of six replicates assessed for each condition with blood collected before and six days after commencing gluten challenge for two coeliac disease patients. Mann-Whitney U test was used to compare NX-stimulated cytokine release in PBMC versus fresh whole blood; statistical significance is indicated by *p < 0.05, or **p < 0.01.

Figure 5. Gluten peptide-stimulated 24-hour fresh whole blood IL-2, IFN-γ, and IL-10 release normalised for the number of live CD3+CD4+ and CD3+CD8+ T cells in individual microwells were compared to the negative control in samples from two HLA-DQ2.5+ CD patients: after gluten challenge (A, B) or at baseline before gluten challenge (C, D). Bars represent the mean and SEM of six replicate wells. Mann-Whitney U test was used to compare gluten peptide-stimulated cytokine release versus Control; statistical significance is indicated by *p < 0.05, or **p < 0.01.

Figure 6. Shortening the incubation period (A–C), and varying the period blood was kept in collection tubes at room temperature (D–F) were assessed for their effects on gluten peptide-stimulated fresh whole blood IL-2, IFN-γ, and IL-10 release. Blood samples from two HLA-DQ2.5+ CD patients collected after gluten challenge were studied. Bars represent the mean and SEM of six replicate wells. Statistical significance by Mann-Whitney U test *p < 0.05, **p < 0.01.

Figure 7. Gluten-specific T cell responses prior to Nexvax2 administration. Gluten peptide-stimulated whole blood CRA (A–C) and CD4+ T cell proliferation measured by 8-day CTV-dye dilution assay before treatment (D) at Visit 5 in 21 patients randomized in the RESET CeD Study. Bars represent the mean and SEM. Wilcoxon signed-rank test was used to compare gluten peptide-stimulated cytokine release versus Control; statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001.

Figure 8. Change in gluten peptide pool-stimulated whole blood CRA (A–C) and CD4+ T cell proliferation measured by 8-day CTV-dye dilution assay (D) over 12 weeks from before treatment at Visit 5 to Visit 28 during maintenance treatment in patients treated with Nexvax2 (n = 12) or placebo (n = 9) in the RESET CeD Study. Medians and interquartile ranges are indicated. Wilcoxon signed-rank test was used to compare Visit 5 to Visit 28; statistical significance is indicated by *p < 0.05, or **p < 0.01.