The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

Abstract

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.

Fig 1. CD40 mRNA expression in peripheral blood immune cell subsets.

CD40 mRNA expression was determined by RT-PCR in freshly purified immune cell subsets or in vitro differentiated subsets (Th1, Th2, Th17; differentiated from fresh CD4CD45RA) from healthy controls (n = 3, or n = 2 for pDC).

Fig 2. Genotype dependent CD40 protein expression in peripheral B-lymphocyte subsets of MS patients and healthy controls.

B lymphocyte subsets from healthy controls (n = 86) and MS patients (n = 21) were identified by flow cytometry (A) and CD40 expression determined relative to an isotype control (relative fluorescence intensity; RFI). Regulatory B cells were identified as CD19+CD38hiCD24hi (data not shown) (B). Association of rs1883832 genotype with CD40 expression in healthy controls (CC = 49, CT = 27, TT = 10) was examined in total B lymphocytes (C), naïve B-lymphocytes (D) and classical memory B-lymphocytes (E), and in total B lymphocytes (F), naïve B lymphocytes (G) and classical B lymphocytes (H) of MS patients (CC = 12, CT = 7, TT = 2). p values were determined by Mann-Whitney U test comparison of each group.

Fig 3. CD40 protein is under- expressed in B lymphocytes of MS patients.

B lymphocyte subsets from healthy controls (n = 86) and MS patients (n = 24) were identified by flow cytometry and CD40 expression determined relative to an isotype control (relative fluorescence intensity; RFI). Surface levels of CD40 were compared in total B-lymphocytes (A), B lymphocytes from rs1883832 CC individuals (B; n = 49 healthy controls, n = 12 MS patients), naïve B lymphocytes (C), classical memory B lymphocytes (D) and IgM memory B lymphocytes (E). P-values were determined using Mann—Whitney test.

Fig 4. Genotype dependent CD40 protein expression in peripheral monocyte subsets of MS patients and healthy controls.

Monocyte subsets were identified by flow cytometry (A) and CD40 expression determined relative to an isotype control (relative fluorescence intensity; RFI) (B). Association of rs1883832 genotype with CD40 expression was examined in classical CD14+CD16- (C), intermediate CD14+CD16+ (D) and non-classical CD14lowCD16++ monocytes (E) from healthy controls (CC = 49, CT = 27, TT = 10), and classical CD14+CD16- (F), intermediate CD14+CD16+ (G) and non-classical CD14lowCD16++ monocytes (H) from MS patients (CC = 12, CT = 7, TT = 2). P—values were determined by Mann-Whitney test.

Fig 5. CD40 expression is higher in differentiated dendritic cell subsets.

CD40 expression was determined in freshly purified immune cell subsets (B cells, monocytes) or in vitro differentiated dendritic cells (DC1, DC2) from healthy controls. Gene expression by RT-PCR (A; n = 49) and relative fluorescence intensity (RFI) by flow cytometry (B; n = 41) are shown; *significantly different from monocytes and DCs; **significantly different from B cells and monocytes (A) or from monocytes (B); p < 0.05 by Mann-Whitney test.

Fig 6. The CD40 risk allele is under expressed in dendritic cell subsets.

Association of rs1883832 genotype with CD40 expression was determined in in vitro differentiated dendritic cells (DC1, DC2) from healthy controls (non-carriers, CC, or carriers, CT/TT, of the risk allele). Gene expression by RT-PCR (A; n = 49) and relative fluorescence intensity (RFI) by flow cytometry (B; n = 41) are shown; p values by Mann-Whitney test.

Fig 7. Lower proportions of the full-length isoform expressed from the CD40 risk allele in monocytes and dendritic cells.

Association of rs1883832 genotype (CC, TC, TT) with the proportion of full-length isoform of CD40 (%FL) expressed in in vitro differentiated dendritic cells (DC1, DC2) from healthy controls (n = 49). Molar ratios of isoforms were quantitated by RT-PCR and amplification of a region spanning CD40 exon 4–10, followed by electrophoretic separation and fluorescent detection (Bioanalyzer, Agilent); p values by Mann-Whitney test.

Fig 8. Proportions of the full-length isoform expressed from the CD40 risk allele in whole blood.

Association of rs1883832 genotype (CC, TC, TT) with the proportion of full-length isoform of CD40 (%FL) expressed in whole blood from healthy controls (A; n = 38) and MS (B; n = 32). Molar ratios of isoforms were quantitated by RT-PCR and amplification of a region spanning CD40 exon 4–10, followed by electrophoretic separation and fluorescent detection (Bioanalyzer, Agilent). Trends were observed for CC > CT in controls (p < 0.13) and for CT > TT in MS, (p < 0.056); p values by Mann-Whitney test.