Cytotoxic B Cells in Relapsing-Remitting Multiple Sclerosis Patients

Abstract

Background: Emerging evidence of antibody-independent functions, as well as the clinical efficacy of anti-CD20 depleting therapies, helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis.

Objective: To investigate whether CD19⁺ B cells may share expression of the serine-protease granzyme-B (GzmB), resembling classical cytotoxic CD8⁺ T lymphocytes, in the peripheral blood from relapsing-remitting MS (RRMS) patients.

Methods: In this study, 104 RRMS patients during different treatments and 58 healthy donors were included. CD8, CD19, Runx3, and GzmB expression was assessed by flow cytometry analyses.

Results: RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8⁺GzmB⁺ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19⁺GzmB⁺ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA), untreated RRMS patients, and healthy donors but not when compared to interferon-β (IFN). Moreover, regarding Runx3, the transcriptional factor classically associated with cytotoxicity in CD8⁺ T lymphocytes, the expression of GzmB was significantly higher in CD19⁺Runx3⁺-expressing B cells when compared to CD19⁺Runx3^- counterparts in RRMS patients.

Conclusions: CD19⁺ B cells may exhibit cytotoxic behavior resembling CD8⁺ T lymphocytes in MS patients during different treatments. In the future, monitoring "cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions.

Keywords: MS treatment; cytotoxicity; granzyme B; neurodegeneration; neuroinflammation.

Figure 1

Cytotoxic CD8⁺ T lymphocytes in relapsing-remitting multiple sclerosis (RRMS) patients. (A) Gate strategy for total CD8⁺ and CD8⁺GzmB⁺ T lymphocytes from healthy donors, untreated RRMS, and treated [Glatiramer Acetate (GA), Interferon-β (IFN), Fingolimod (FTY), and Natalizumab (NTZ)] RRMS patients. (B) Proportion (%) of total CD8⁺ T lymphocytes in healthy donors (blue) and RRMS patients (red). (C) Proportion (%) of CD8⁺ T lymphocytes in healthy donors (blue), untreated RRMS (red), and treated RRMS patients (GA, IFN, FTY, NTZ) (red). (D) Proportion (%) of circulating CD8⁺GzmB⁺ T lymphocytes in healthy donors (blue) and RRMS patients (red). (E) Proportion (%) of circulating CD8⁺GzmB⁺ T lymphocytes in healthy donors (blue), untreated RRMS (red), and treated RRMS patients (GA, IFN, FTY, NTZ) (red). Bars represent mean values. Each column represents mean (95% CI). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (F) Uniform Manifold Approximation and Projection (UMAP) gated in CD3⁺CD8⁺ T lymphocytes from RRMS patients with different conditions non-identified and based on the Arcsinh-transformed expression of the markers. Gate strategy and proportion (%) of granzyme B (GzmB) derived from circulating (G) CD8⁺CD27⁺ vs. CD8⁺CD27^-, (H) CD8⁺CD28⁺ vs. CD8⁺CD28^-, (I) CD8⁺CD57⁺ vs. CD8⁺CD57^-, (J) CD8⁺CD94⁺ vs. CD8⁺CD94^- T lymphocytes in RRMS patients (red). Bars represent mean values. Each column represents mean (95% CI). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (K) Heatmap of the expression of the markers in subpopulations manually identified in CD3⁺CD8⁺ T lymphocytes from RRMS patients. (L) Barplot representing the frequency of each subpopulation in CD3⁺CD8⁺ T lymphocytes.

Figure 2

Cytotoxic CD19⁺ B cells in relapsing-remitting multiple sclerosis (RRMS) patients. (A) Gate strategy for total CD19⁺ and CD19⁺GzmB⁺ B cells from healthy donors, untreated RRMS, and treated [Glatiramer Acetate (GA), Interferon-β (IFN), Fingolimod (FTY), and Natalizumab (NTZ)] RRMS patients. (B) Proportion (%) of total CD19⁺ B cells in healthy donors (blue) and RRMS patients (red). (C) Proportion (%) of total CD19⁺ B cells in healthy donors (blue), untreated RRMS (red), and treated RRMS patients (GA, IFN, FTY, NTZ) (red). (D) Proportion (%) of circulating CD19⁺GzmB⁺ B cells in healthy donors (blue) and RRMS patients (red). (E) Proportion (%) of circulating CD19⁺GzmB⁺ B cells in healthy donors (blue), untreated RRMS (red), and treated RRMS patients (GA, IFN, FTY, NTZ) (red). Bars represent mean values. Each column represents mean (95% CI). *p < 0.05; **p < 0.01; ****p < 0.0001. (F) Uniform Manifold Approximation and Projection (UMAP) gated in CD3^-CD19⁺ B cells from RRMS patients with different conditions non-identified and based on the Arcsinh-transformed expression of the markers. (G) Gate strategy for granzyme B (GzmB)-derived CD19⁺Runx3⁺ B cells. (H) Proportion (%) of GzmB-derived from circulating CD19⁺Runx3⁺ vs. CD19⁺Runx3^- B cells in healthy donors (blue) and RRMS patients (red). (I) Proportion (%) of GzmB derived from circulating CD19⁺Runx3⁺ vs. CD19⁺Runx3^- in RRMS patients (red). Each column represents mean (95% CI). *p < 0.05; **p < 0.01; ****p < 0.0001. (I) Heatmap of the expression of the markers in subpopulations manually identified in CD3^-CD19⁺ B cells from RRMS patients. (J) Barplot representing the frequency of each subpopulation in CD3^-CD19⁺ B cells. (K) Gate strategy for isolated CD19⁺ B cells. (L) Quantitative PCR for PRF1, GzmB, and Runx3 in isolated B cells from healthy donors (blue) and RRMS patients (red). (M) Concentration (pg/ml) of GzmB in supernatants of stimulated CD19⁺ B cells from healthy donors (blue) and RRMS patients (red). Each column represents mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.