Differential inhibition of autoreactive memory- and alloreactive naive T cell responses by soluble cytotoxic T lymphocyte antigen 4 (sCTLA4), CTLA4Ig and LEA29Y

Abstract

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a potent inhibitory co-stimulatory molecule believed to be involved in type 1 diabetes and other autoimmune diseases. An association has been reported of both mRNA expression and serum levels of the soluble splice variant of CTLA4 (sCTLA4) with type 1 diabetes. Furthermore, recombinant fusion proteins CTLA4Ig and LEA29Y have been proposed as therapies for type 1 diabetes. We studied the role of (s)CTLA4 in islet autoimmunity. Binding capacity of the proteins to antigen-presenting cells was determined by flow cytometry in competition and binding assays. Functionality of sCTLA4 as well as the therapeutic inhibitory fusion proteins CTLA4Ig and LEA29Y was measured in a dose-response lymphocyte stimulation test, using a panel of diabetes-associated T cell clones reactive to islet autoantigens. As controls, mixed lymphocyte reactions (MLR) were performed to assess functionality of these proteins in a primary alloreactive setting. All three CTLA4 molecules were able to bind to antigen-presenting cells and inhibit the expression of CD80/CD86. sCTLA4 was able to suppress proliferation of different committed autoreactive T cell clones in a dose-dependent manner, whereas CTLA4Ig and LEA29Y were not. Conversely, CTLA4Ig and LEA29Y, rather than sCTLA4, were able to suppress naive alloreactive proliferation in a MLR. Our results indicate a differential role for sCTLA4, CTLA4Ig and LEA29Y proteins in memory versus primary immune responses with implications for efficacy in intervention therapy.

Fig. 1

Schematic representation of cytotoxic T lymphocyte antigen 4 (CTLA4) binding and competition assay (a). Antigen-presenting cells (APCs) were incubated with CTLA4Ig, LEA29Y or sCTLA4. Subsequently, the cells were incubated with antibodies to CD3, CTLA4 (bound to the APC) and to its ligands CD80 and CD86 and expression was measured by fluorescence-activated cell sorter. Histograms show a representative example (of three independent experiments) of the expression of CD80 (b), CD86 (c) and CTLA4 (d) on APCs (CD3-negative peripheral blood mononuclear cells). Untreated APCs are represented by the grey area, APCs incubated with CTLA4Ig by the thick line, with LEA29Y by the thin line and with sCTLA4 by the dotted line. The bar graphs on the right represent the expression of the respective markers.

Fig. 2

Proliferation of autoreactive T cell line specific for IA-2 (a) and T cell clones specific for glutamic acid decarboxylase (b) and rat insulinoma (RIN) (c). The x-axis indicates the added concentration of protein, the y-axis indicates proliferation in counts per minute (cpm). □ = without peptide (negative control), ▪ = with peptide (positive control), • = cytotoxic T lymphocyte antigen 4 (CTLA4)Ig, ▴ = LEA29Y, ▾ = soluble CTLA4. Shown are mean ± standard error. All data were reproduced once. (d) Average suppression of the highest concentration per protein in all six experiments performed. (e) Cytokine production (interferon-γ/interleukin-10 ratio) of the anti-RIN clone treated with the highest concentrations. ***P-value < 0·001.

Fig. 3

One-way mixed lymphocyte reaction (MLR), assessing the inhibitory capacity of soluble cytotoxic T lymphocyte antigen 4 (CTLA4) proteins in an unprimed alloreactive setting. Stimulator cells were human leucocyte antigen (HLA) DR/DR 4/4, responder cells were D-related 3/3. The x-axis indicates the added concentration of protein, the y-axis indicates proliferation in counts per minute (cpm). ▪ = MLR without addition (positive control), □ = medium without cells (negative control), • = CTLA4Ig, ▴ = LEA29Y, ▾ = soluble CTLA4. Shown are mean ± standard error. These results were reproduced twice.