Increased expression of CD40 ligand (CD154) on CD4+ T cells as a marker of disease activity in rheumatoid arthritis

Abstract

Objectives: The interaction between the activation induced surface glycoprotein CD40L (ligand) (CD154) on CD4+ T cells and its receptor CD40, which is expressed on various cell types, plays a crucial part in numerous cell mediated and humoral immune reactions that may be of pathogenetic importance in rheumatoid arthritis (RA). To further evaluate the pathogenetic role of CD40L in RA, expression of CD40L and various other T cell activation antigens as well as costimulatory molecules was investigated on CD4+ T cells in RA by flow cytometry.

Methods: Two colour flow cytometry was used to determine the percentage of CD4+ T cells expressing CD40L, CD69, CD25, HLA-DR, CD39, CD27 and CD28 in peripheral blood (PB) of 62 RA patients in comparison to 20 healthy controls (HC). Disease activity was assessed by clinical, laboratory and radiological examination. Status of clinical remission of RA was evaluated according to the ACR preliminary criteria for complete clinical remission of RA.

Results: CD40L was expressed on > 10% of CD4+ T cells in 29% of RA patients thus defining a CD40L(high+) patient group. Disease activity as estimated by C reactive protein, rheumatoid factor and status of clinical remission of disease (p = 0.049) was higher in this subgroup than in the RA CD40L(low+) group. Expression of CD69, CD25, and HLA-DR was significantly increased in both RA patient groups in comparison with HC. However, the percentage of CD39+ CD4+ T cells was increased only in the RA CD40L(high+) subgroup (versus HC p = 0.019, versus RA CD40L(low+) p = 0.044). Furthermore, expression of CD40L and CD39 on CD4+ T cells correlated positively as estimated by Spearman rank correlation (p<0.001). The percentage of CD4+ T cells lacking the costimulatory molecules CD27 (p = 0.002) and CD28 (p = 0.026) was increased in RA CD40L(low+) patients in comparison with HC.

Conclusions: These data suggest that increased expression of CD40L on CD4+ T cells in RA indicates prolonged and increased activation of CD4+ T lymphocytes and is associated with active disease and possibly an unfavourable prognosis. Whether this phenotypically defined RA CD40L(high+) subgroup will preferentially respond to an anti-CD40L antibody treatment remains to be elucidated.

Figure 1

Percentage of PB CD4+ T cells expressing CD40L in healthy controls (HC, n = 20), RA CD40L^low+ (n = 44) and RA CD40L^high+ (n = 18) patients as well as percentage of SF CD4+ CD40L+ T cells in RA patients (SF, n = 10). The bars indicate the medians.

Figure 2

Fluorescence activated cell sorter histograms of CD40L expression (gating on CD4+ T cells) demonstrating the intensity and density of CD40L antigen expression patterns (thin line) defined as CD40L^high+ (A) and CD40L^low+ (B) expression. The negative control was obtained by using a isotype monoclonal antibody (thick line).

Figure 3

Intraindividual variability of CD40L expression in 8/18 RA CD40L^high+ patients. Under intensified immunosuppressive treatment the percentage of CD40L+ CD4+ T cells declined in seven of eight patients, but remained in seven of eight patients > 10%. The mean time (t1-t2) was eight months (ranging from 3 to 20 months).

Figure 4

The percentage of CD39+ CD4+T cells correlated significantly (p < 0.001) with the expression of CD40L in the RA CD40L^high+ group as determined by Spearman rank correlation. Data are shown in logarithmic scale.