Extended Data Figure 3. Identification and validation of genes involved in CD4 costimulation that correlate with clinical outcome, and how that relationship changes after treatment.
(A) A knowledge-based network analysis of 336 probes comprising the ‘black’ expression module (Fig.1E) identifies a network of costimulation signaling (Supplementary Table 3). Individual genes are shown in circles with the ‘strength’ of their connections indicated by the weight of the black bar linking them. Pathways of TCR signaling, ICOS-ICOSL signaling and CD28 signaling all significantly enriched in this module (FDR p < 0.05). (B-E) Scatterplots showing the outcome of multiple linear regression models testing the association of 4 signatures (red symbols) as indicated, directly compared to clinical markers of disease activity (black symbols). x-axis = magnitude of association (regression coefficient, change in normalized flare rate (flares/days follow-up) per unit change in each variable tested). y-axis = significance of association in multiple regression model, P. significance threshold (dashed red line, P = 0.05). (B) CD8 turquoise module eigengene in AAV, (C) CD4 costimulation (black) module eigengene in AAV, (D, E) CD8 exhaustion signature (Supplementary Table 6) in AAV/SLE (D) and IBD (E). Clinical variables incorporated vary due to differing relevance in each case but include some of: disease activity score (BVAS/BILAG/CDAI/Harvey-Bradshaw score), CRP, autoantibody titer (PR3/MPO, dsDNA), Lymphocyte count, neutrophil count, platelet count, IgG, IgA, IgM, ESR, age. (F) Line plot showing mean expression of a CD8 T cell exhaustion signature in 38 AAV patients measured at presentation during active, untreated disease (t0) and again 12 months later when disease activity was quiescent and patients were on maintenance immunosuppressive therapy (t12). Patients are grouped into those falling above (red) and below (blue) median expression of the exhaustion signature eigengene at entry. P = Mann-Whitney test comparing t12 and t0 values. The difference between the groups that is easily apparent at enrolment with active, untreated disease (t0) is no longer apparent when disease is treated and quiescent twelve months later (t12). (G-I) Scatterplots showing inverse correlation between individual eigenvalues of the CD4 costimulation signature (x-axis, red) and the CD8 exhaustion signature (y-axis, blue) defined as in Fig. 2, for AAV (G), SLE (H) and IBD (I) cohorts. Correlation = Pearson, r2, 2-tailed significance.