Increased expression of CTLA-4 (CD152) by T and B lymphocytes in Wegener's granulomatosis

Abstract

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4-/CD8- T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease.

Fig. 1

Spontaneous CTLA-4 expression by PBMC. Cytospin preparations from freshly isolated PBMC from a WG patient stained for CD3 (moAb UCHT1, green) or CD20 (moAb L26, green) and for CTLA-4 (CD152, moAb BNI3 or BNI8, red). Cells coexpressing CD3/CD20 and CTLA-4 appear yellow on double exposure. (a) Unstimulated PBMC stained for CD3. (b) Same preparation stained for CTLA-4 (BNI3). (c) Double exposure, CD3 stained green and CTLA-4 red; cells coexpressing CD3 and CTLA-4 appear yellow. CTLA-4 expressing cells, which did not coexpress CD3 are indicated by arrows. These cells were CD20⁺ B cells. (d) Unstimulated PBMC stained for CD20. (e) Same preparation stained for CTLA-4 (BNI8). (f) Double exposure, CD20 stained green and CTLA-4 red; cells coexpressing CD3 and CTLA-4 appear yellow. Isotype controls remained negative, original magnification × 100.

Fig. 2

Spontaneous CTLA-4 expression by T lymphocytes (CD3⁺) freshly isolated from WG patients and control subjects. Medians and statistically significant differences are indicated in the figure.

Fig. 3

CTLA-4 expression by CD4⁺ and CD8⁺ T cell subpopulations freshly isolated from WG patients and control subjects. Medians and statistically significant differences are indicated in the figure.

Fig. 4

Spontaneous CTLA-4 expression by T lymphocytes (CD3⁺) freshly isolated from blood donors of two different age groups. Medians are indicated in the figure.

Fig. 5

Mitogen induction of CTLA-4 expression by T cells (CD3⁺). Peripheral blood mononuclear cells (PBMC) from WG patients and control subjects were incubated for 48 h in the presence of suboptimal and plateau concentrations of phytohaemagglutinin (PHA); medium alone served as control. CTLA-4 induction by plateau concentrations of PHA was significantly less with PBMC from WG patients (•) compared with controls (○). Medians and statistically significant differences are indicated in the figure.

Fig. 6

Induction of CTLA-4 expression by B lymphocytes. T cells were depleted from PBMC from healthy donors and the cells were stimulated with 10 ng/ml phorbol ester (PMA) for 48 h in vitro. Cytospin preparations were stained for (a) CD20 (moAb L26, green) and (b) CTLA-4 (moAb BNI8, red). (c) Cells coexpressing B20 and CTLA-4 appear yellow on double exposure. Original magnification × 100.