Deep Flow Cytometry Unveils Distinct Immune Cell Subsets in Inducible T Cell Co-Stimulator Ligand (ICOSL)- and ICOS-Knockout Mice during Experimental Autoimmune Encephalomyelitis

Abstract

The inducible T cell co-stimulator ligand (ICOSL), expressed by antigen presenting cells, binds to the inducible T cell co-stimulator (ICOS) on activated T cells. Improper function of the ICOS/ICOSL pathway has been implicated in several autoimmune diseases, including multiple sclerosis (MS). Previous studies showed that ICOS-knockout (KO) mice exhibit severe experimental autoimmune encephalomyelitis (EAE), the animal model of MS, but data on ICOSL deficiency are not available. In our study, we explored the impact of both ICOS and ICOSL deficiencies on MOG_35-55 -induced EAE and its associated immune cell dynamics by employing ICOSL-KO and ICOS-KO mice with a C57BL/6J background. During EAE resolution, MOG-driven cytokine levels and the immunophenotype of splenocytes were evaluated by ELISA and multiparametric flow cytometry, respectively. We found that both KO mice exhibited an overlapping and more severe EAE compared to C57BL/6J mice, corroborated by a reduction in memory/regulatory T cell subsets and interleukin (IL-)17 levels. It is noteworthy that an unsupervised analysis showed that ICOSL deficiency modifies the immune response in an original way, by affecting T central and effector memory (T_CM, T_EM), long-lived CD4⁺ T_EM cells, and macrophages, compared to ICOS-KO and C57BL/6J mice, suggesting a role for other binding partners to ICOSL in EAE development, which deserves further study.

Keywords: ICOSL; experimental autoimmune encephalomyelitis; memory cells; multiple sclerosis; regulatory T cells.

Figure 1

Clinical disease course of MOG_35-55-induced EAE in C57BL/6J, ICOSL-KO and ICOS-KO mice. (A) EAE was induced using the established protocol in the laboratory (31). Results from three independent experiments are shown. Data are shown as mean ± SEM (n = 2830 mice/group). (B) Antigen-recall assay to MOG of splenic cells isolated from the three groups (n = 4–5) and cultured for 72 h in the presence/absence (no stimuli) of MOG_35-55 peptide (10 μg/mL). (C) IL-17A protein levels (pg/µg) in brain and spinal cord homogenates from C57BL/6J, ICOSL-KO and ICOSK-KO mice. Data are shown as mean ± SEM (n = 5 mice/group). For statistical analysis, Kruskal–Wallis test with Dunnett multiple comparisons was used as a post-hoc test. The asterisks (*) or pound signs (#) depicted in the vertical line correspond to the statistical significance of the average daily disease score, which was computed from cumulative data across experiments. * p < 0.05; ** p < 0.01 C57BL/6J vs. ICOS-KO; # p < 0.05; ## p < 0.01; ### p < 0.001 C57BL/6J vs. ICOSL-KO.

Figure 2

Multiparametric-flow-cytometry-supervised analysis of splenocytes from MOG_35-55-induced EAE C57BL/6J, ICOSL-KO, and ICOS-KO mice. Each dot plot shows the absolute number (expressed as a power of 10⁶) of immune cell populations in the spleen (day 32 post-immunization) harvested from C57BL/6J, ICOSL-KO, and ICOS-KO mice. The samples were acquired using FACSymphony^TM A5 (Becton and Dickinson, San José, CA, USA) flow cytometer and data were analyzed using FACSDIVA software (Version 9.1, Becton and Dickinson, San José, CA, USA). CM: central memory; EM: effector memory; Tregs: regulatory T cells; MDSC: myeloid-derived suppressor cells. Data are shown as mean ± SEM of three independent experiments (n = 19–24 mice/group). For statistical analysis, Kruskal–Wallis test with Dunnett multiple comparisons as post-hoc test was used, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Unsupervised flow cytometry analysis identified 21 cell clusters. (A) UMAP multidimensionality reduction for C57BL/6J (left), ICOS-KO (center) and ICOSL-KO (right), concatenated and analyzed simultaneously. Data are shown as density dot plots. (B) Heatmap derived from the total median fluorescence intensity (MFI) (column-scaled z-scores) and comparison of the clusters among the three groups (C57BL/6J, ICOS-KO and ICOSL-KO). For statistical analysis, Kruskal–Wallis test with Dunnett multiple comparisons was used, ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

Unsupervised analysis reveals new cell subsets. Each dot plot shows the percentages of the depicted cells in C57BL/6J, ICOSL-KO, and ICOS-KO mice. For statistical analysis, Kruskal–Wallis test with Dunnett multiple comparisons was used. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are shown as mean ± SEM of three independent experiments (n = 19–24 mice/group).