Platelet-Derived Soluble CD40L and Its Impact on Immune Modulation and Anti-IL6R Antibody Treatment Outcome in Rheumatoid Arthritis

Abstract

Background: Platelets (PLTs) from healthy donors (HD) modulate T lymphocyte responses but PLTs from rheumatoid arthritis (RA) patients contribute to persistent systemic inflammation. This suggests that PLTs from RA patients and HD have different immunomodulatory effects.

Methods: Using cell culture, flow cytometry, proteomics, and ELISA, we compared PLTs from HD and RA patients and their effects on T lymphocyte activation and cytokine production.

Results: HD PLTs suppressed T lymphocyte proliferation and IFNγ and TNF production, while RA PLTs exhibited reduced suppressive capacity. In the presence of RA PLTs, IFNγ levels correlated with T lymphocyte proliferation, greater disease activity, and anti-citrullinated protein antibodies (ACPA). Proteomic analysis revealed that RA PLTs show upregulation of proteins linked to acute-phase response and complement activation. RA PLTs secreted higher levels of soluble CD40L (sCD40L) and PDGF-BB that correlated with enhanced IFNγ production. Seropositive RA patients had higher levels of sCD40L, and these levels were predictive of disease remission in RA patients treated with anti-IL6R. sCD40L was found to enhance T lymphocyte activation and to contribute to increased pro-inflammatory cytokine production.

Conclusions: This study highlights the diminished ability of RA PLTs to suppress T lymphocyte activation and that sCD40L can be a potential biomarker and therapeutic target in RA.

Keywords: CD40L; T lymphocyte activation; cytokine production; platelet immunomodulation; rheumatoid arthritis.

Figure 1

Proliferative response of PBMCs and PLT aggregation in HD and RA patients. (A) The proliferative profile of PBMCs from HD activated for 72 h in the presence of HD PLTs, RA PLTs, or in the absence of PLTs (w/o). (B) Individual representation of blasts or proliferative PBMCs using HD PBMCs or RA PBMCs activated in the presence of RA PLTs or HD PLTs. (C) Proportion of aggregated PLTs in whole blood from HD and RA patients, accompanied by representative dot plots from flow cytometry analysis showing the proportion of PMPs, PLTs, and aggregated PLTs. Statistical analysis was performed using Wilcoxon (B) or Mann–Whitney test (C), * p < 0.05.

Figure 2

Cytokine production and correlations in PBMC-PLT co-cultures from HD and RA patients. (A) Percentage of cells producing IFNγ, TNF, IL-17, and IL-10 was determined in PBMCs from HD and RA patients after activation in the presence of PLTs from HD or RA donors. (B) Correlations between proliferation levels, CD4+ blasts, and the percentage of TNF with IFNγ were analyzed in PBMCs from RA patients in the presence of RA PLTs. (C) Ratios of IFNγ percentages in PBMC cultures with RA PLTs versus HD PLTs were compared, stratified by the RA disease activity index and the presence of ACPA or RF. Statistical analysis was performed using the Mann–Whitney test (C), Wilcoxon test (A), and Spearman correlation (B). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

Proteomic and functional analysis of differentially expressed proteins in PLTs from HD and RA patients. (A) Heatmap showing the proteomic profiles of PLTs from HD and RA patients. (B) Analysis of the compartmental origin of differentially expressed proteins. (C) Gene ontology analysis of the top differentially expressed proteins.

Figure 4

Proliferation and cytokine production in activated PBMCs in the presence of supernatants (sup) or PMPs from PLTs of HD and RA patients. (A) Percentage of proliferating and cytokine-producing cells (IFNγ, TNF, IL-17, and IL-10) from activated PBMCs from HD and RA patients in the presence of supernatants from collagen-activated PLTs of HD and RA donors. (B) Percentage of proliferating and cytokine-producing cells from activated PBMCs from HD and RA patients in the presence of PMPs from collagen-activated PLTs of HD and RA. Statistical analysis was performed using the Wilcoxon test. * p < 0.05.

Figure 5

PLT-derived molecules and their immunological correlations in supernatants and plasma from HD and RA patients. (A) Concentration of PLT-derived molecules in supernatants of collagen-activated HD PLTs and RA PLTs. (B) Correlation between the levels of sCD40L and PDGF-BB in RA PLT supernatants and the percentage of CD4+ T lymphocytes producing IFNγ in PBMCs from HD cultured with RA PLTs. (C) Comparison of sCD40L levels in supernatants of activated PBMCs in the presence of PLTs, and plasma from RA patients, stratified by the presence of ACPA and RF. Statistical analysis was performed using the Mann–Whitney test and Spearman correlation. * p < 0.05 and ** p < 0.01.

Figure 6

Plasma concentration of sCD40L and its predictive value for remission in patients treated with anti-IL6R antibodies. (A) Comparison of plasma concentrations of sCD40L in patients who reached remission versus those who did not after treatment. (B) ROC analysis to determine the optimal concentration of sCD40L for predicting remission (cutoff). (C) Comparison of baseline characteristics of patients stratified by the sCD40L cutoff. (D) Comparison of patient frequencies in the remission and non-remission groups based on this concentration. (E) IFNγ concentration in the plasma of patients stratified by sCD40L cutoff. Statistical analysis was performed using the Mann–Whitney test and chi-square. *** p < 0.001.

Figure 7

Effect of recombinant CD40L on proliferation and cytokine expression of PBMCs activated in the presence of PLTs. PBMCs were activated in the presence of HD PLTs or supernatant from PLT (SPN PLT) from HD, with two different concentrations of recombinant CD40L (sCD40L) added to the culture for 72 h. The effects of the different concentrations of sCD40L on CD4+ proliferation, blast differentiation, and cytokine expression (IFNγ, TNF, IL-17, and IL-10) were measured and are shown relative to the levels observed in the absence of PLTs. Statistical analysis was performed using the Wilcoxon test for paired samples. * p < 0.05 and ** p < 0.01.