Association of baseline soluble immune checkpoints with the risk of relapse in PR3-ANCA vasculitis following induction of remission

Abstract

Objectives: We investigated whether soluble immune checkpoints (sICPs) predict treatment resistance, relapse and infections in patients with antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV).

Methods: Plasma sICP concentrations from available samples obtained during conduct of the RAVE trial were measured by immunoabsorbent assays from patients with either proteinase 3 (PR3) or myeloperoxidase (MPO)-ANCA vasculitis and were correlated with clinical outcomes, a set of biomarkers and available flow cytometry analyses focusing on T cell subsets. Log-rank test was used to evaluate survival benefits, and optimal cut-off values of the marker molecules were calculated using Yeldons J.

Results: Analysis of 189 plasma samples at baseline revealed higher concentrations of sTim-3, sCD27, sLag-3, sPD-1 and sPD-L2 in patients with MPO-ANCA vasculitis (n=62) as compared with PR3-ANCA vasculitis (n=127). Among patients receiving rituximab induction therapy (n=95), the combination of lower soluble (s)Lag-3 (<90 pg/mL) and higher sCD27 (>3000 pg/mL) predicted therapy failure. Twenty-four out of 73 patients (32.9%) in the rituximab arm reaching remission at 6 months relapsed during follow-up. In this subgroup, high baseline values of sTim-3 (>1200 pg/mL), sCD27 (>1250 pg/mL) and sBTLA (>1000 pg/mL) were associated with both sustained remission and infectious complications. These findings could not be replicated in 94 patients randomised to receive cyclophosphamide/azathioprine.

Conclusions: Patients with AAV treated with rituximab achieved remission less frequently when concentrations of sLag-3 were low and concentrations of sCD27 were high. Higher concentrations of sTim-3, sCD27 and sBTLA at baseline predicted relapse in patients treated with rituximab. These results require confirmation but may contribute to a personalised treatment approach of AAV.

Keywords: autoimmune diseases; rituximab; systemic vasculitis.

Figure 1

Nineteen patients included in our analysis had treatment failure, of whom 10 received rituximab and 9 were treated with cyclophosphamide as induction and azathioprine as maintenance therapy. (A) A combination of higher sCD27 and lower sLag-3 associated with treatment failure in the rituximab arm. Only two out of six patients achieved remission in this group (p<0.001). (B) The same combination did not predict treatment failure in patients receiving cyclophosphamide/azathioprine. CD, cluster of differentiation; sLag-3, soluble lymphocyte activation gene 3.

Figure 2

Different baseline plasma concentrations of immune checkpoints comparing patients in sustained complete remission (green) versus relapsed patients (red) receiving rituximab. Significant differences are marked by * for p<0.05 and ** for p<0.005 (A). Kaplan-Meier curves for the sustained remission rates in rituximab (RTX)-treated patients in months from the time point of complete remission achievement. Relapse defined the event. Patients were divided according to the baseline plasma concentrations of (B) sTim-3, (C) sCD27 and (D) sBTLA. (E) displays the combination of those three markers in RTX-treated patients. BTLA, B- and T-lymphocyte attenuator; CD, cluster of differentiation; IDO, indoleamine 2,3-dioxygenase; Lag-3, lymphocyte activation gene 3; PD-1, programmed cell death protein 1; PD-L2, programmed cell death-ligand 2; Tim-3, T-cell immunoglobulin and mucin domain 3.

Figure 3

Occurrence of infections in the rituximab (A) and cyclophosphamide/azathioprine arms (B) grouped into the number of soluble markers (sCD27, sTim-3 and sBTLA). In the rituximab arm, fewer infections (p=0.035) were observed when no marker was high at baseline, in comparison to the groups with at least one highly expressed marker. In comparison, the expression of these three soluble immune checkpoints did not predict occurrence of infections in the cyclophosphamide/azathioprine arm (p=0.643). BTLA, B- and T-lymphocyte attenuator; CD, cluster of differentiation; Tim-3, T-cell immunoglobulin and mucin domain 3.

Figure 4

Spearman correlation matrix of soluble immune checkpoints and T-cell populations (A) (n=87) and of biomarker expressions (n=69) (B), each at baseline in rituximab-treated patients. Blue represents positive correlations and red represents negative correlations. The given flow cytometry legend shows the analysed and gated T-cell subpopulations, and numbers are used accordingly in the correlation matrix (A). ‘Combination’ includes the highly expressed soluble checkpoints (sCD27 (>1250 pg/mL), sTim-3 (>1200 pg/mL) and sBTLA (>1000 pg/mL)). Significant findings are flagged using * for p<0.05, ** for p<0.01 and *** for p<0.001. In matrix (A) red frames highlight significant negative correlations (CD8+ subpopulations) and blue frames the positive correlations (CD4+ subpopulations). In matrix (B), the blue frame highlights significant positive correlations with biomarker expressions. The combination correlated strongly with CD4+ Treg/PD1+ cells (A), as well as tumour necrosis factor receptor II (TNFRII), neutrophil gelatinase-associated lipocalin (NGAL) and osteopontin. None of the biomarkers correlate with sustained remission (SR). BCA, B-cell-attracting chemokine; BNGF, ß-nerve growth factor; BTLA, B- and T-lymphocyte attenuator; CD, cluster of differentiation; FGF, fibroblast growth factor; G-CSF, granulocyte-colony-stimulating factor; GM-CSF, granulocyte macrophage-colony-stimulating factor; ICAM, intracellular adhesion molecule; IDO, indoleamine 2,3-dioxygenase; IFN, interferon; IL, interleukin; IP10, interferon-gamma-induced protein 10; KIM-1, kidney injury molecule-1; Lag-3, lymphocyte activation gene 3; NGAL, neutrophil gelatinase-associated lipoprotein; PAI-1, plasminogen activator inhibitor-1; PD-1, programmed cell death protein 1; PD-L2, programmed cell death-ligand 2; PDGF, platelet-derived growth factor; TARC, thymus- and activation-regulated chemokine; Tim-3, T-cell immunoglobulin and mucin domain 3; TIMP, tissue inhibitor of metalloproteinase; TNFR, tumour necrosis factor receptor; VCAM, vascular cell adhesion protein.