B Lymphocytes in Multiple Sclerosis: Bregs and BTLA/CD272 Expressing-CD19+ Lymphocytes Modulate Disease Severity

Abstract

B lymphocytes contribute to the pathogenesis of Multiple Sclerosis (MS) by secreting antibodies and producing cytokines. This latter function was analyzed in myelin olygodendrocyte protein (MOG)-stimulated CD19+ B lymphocytes of 71 MS patients with different disease phenotypes and 40 age-and sex-matched healthy controls (HC). Results showed that: 1) CD19+/TNFα+, CD19+/IL-12+ and CD19+/IFNγ+ lymphocytes are significantly increased in primary progressive (PP) compared to secondary progressive (SP), relapsing-remitting (RR), benign (BE) MS and HC; 2) CD19+/IL-6+ lymphocytes are significantly increased in PP, SP and RR compared to BEMS and HC; and 3) CD19+/IL-13+, CD19+/IL-10+, and CD19+/IL-10+/TGFβ+ (Bregs) B lymphocytes are reduced overall in MS patients compared to HC. B cells expressing BTLA, a receptor whose binding to HVEM inhibits TcR-initiated cytokine production, as well as CD19+/BTLA+/IL-10+ cells were also significantly overall reduced in MS patients compared to HC. Analyses performed in RRMS showed that fingolimod-induced disease remission is associated with a significant increase in Bregs, CD19+/BTLA+, and CD19+/BTLA+/IL-10+ B lymphocytes. B lymphocytes participate to the pathogenesis of MS via the secretion of functionally-diverse cytokines that might play a role in determining disease phenotypes. The impairment of Bregs and CD19+/BTLA+ cells, in particular, could play an important pathogenic role in MS.

Figure 1. Gating strategies.

An initial assessment of lymphocyte population was performed using a forward/sideward scatterplot (FSC/SSC) (A). A first gate was set around the lymphocytes. The lymphocyte population was gated on SSC/CD19-scatterplot identifying B-cells (CD19+) (B). The percentage of TNFα (Fig. 2B), IL-12 (Fig. 2D), IFNγ (Fig. 2F), IL-6 (Fig. 2H), IL-4 (Fig. 3B) and IL-13 (Fig. 3D) CD19+ cells was then calculated using the B cell gate.

Figure 2. Pro-inflammatory cytokines in MOG-stimulated CD19+ B lymphocytes.

Panel B: CD19+/TNFα+ cells; Panel D: CD19+/IL-12+ cells; Panel F: CD19+/IFNγ + cells; Panel H: CD19+/IL-6+ cells. Representative results obtained in MOG-stimulated CD19+ B lymphocytes of PPMS, SPMS, RRMS and BEMS patients, as well as of age-and-sex- matched HC are presented in panels A (TNFα), C (IL-12), E (IFNγ), and G (IL-6); summary results are shown in panels B (TNFα), D (IL-12), F (IFNγ) and H (IL-6). The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown, *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal- Wallis test.

Figure 3. Anti-inflammatory cytokines in MOG-stimulated CD19+ B lymphocytes.

Panel A: CD19+/IL-14+ cells; Panel C: CD19+/IL-13+ cells. Representative results obtained in MOG-stimulated CD19+ B lymphocytes of PPMS, SPMS, RRMS and BEMS patients, as well as of age-and-sex- matched HC are presented in panels A (IL-4) and C (IL-13); summary results are shown in panels B (IL-4), and D (IL-13). The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown, *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal- Wallis test.

Figure 4. MOG-stimulated CD19+ B regulatory lymphocytes.

IL-10-expressing CD19+ B lymphocytes are shown in panel A; IL-10- and TGFβ- coexpressing B cells are displayed in panel B. MOG-stimulated CD19+ B lymphocytes of PPMS, SPMS, RRMS and BEMS patients as well as of age-and-sex- matched HC are shown. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown, *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal- Wallis test.

Figure 5. BTLA-expressing CD19+ B lymphocytes.

Panel B: CD19+/BTLA+ cells; panel D: CD19+/BTLA+/IL-10+ cells. Representative results obtained in MOG-stimulated CD19 B lymphocytes of PPMS, SPMS, RRMS and BEMS patients, as well as of age-and-sex- matched HC are presented in panels A and C; summary results are shown in panels B and D. The boxes stretch from the 25th to the 75th percentile; the lines across the boxes indicate the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown, *p < 0.05, **p < 0.01, ***p < 0.001, Kruskal- Wallis test.

Figure 6. Effect of Fingolimod (FTY) treatment in B lymphocytes in MS.

TNFα- (A), IL-13- (B), BTLA and IL-10 (C), IL-10- (D) and BTLA- (E) expression within the CD19 B cell population before onset of fingolimod treatment (before FTY) and after 1 month of fingolimod-treatment (on FTY) in RRMS patients. Statistical significance is shown, *p < 0.05, **p < 0.01, Kruskal- Wallis test.

Figure 7. mRNA expression by Real-Time PCR.

Single Real-Time PCR results obtained in MOG- stimulated PBMCs of individuals with a diagnosis of PPMS, SPMS, RRMS and BEMS and of age- and sex-matched Healthy Controls (HC). IL-10 is shown in panel A; TNFα in panel B. The results are shown as fold-change expression from the un-stimulated samples. Gene expression was calculated relative to GAPDH housekeeping gene. Summary results are shown in the bar graphs.