Rotavirus-specific T cell responses and cytokine mRNA expression in children with diabetes-associated autoantibodies and type 1 diabetes

Abstract

Rotavirus infections have been implicated as a possible trigger of type 1 diabetes. We elucidated this connection by comparing peripheral blood T cell responses to rotavirus between children with newly diagnosed type 1 diabetes (n = 43), healthy children with multiple diabetes-associated autoantibodies (n = 36) and control children carrying human leukocyte antigen (HLA)-conferred susceptibility to type 1 diabetes but without autoantibodies (n = 104). Lymphocyte proliferation assays based on stimulation with an antigen were performed using freshly isolated peripheral blood mononuclear cells (PBMC) and IgG and IgA class rotavirus antibodies were measured using plasma samples collected from the children. The expression of interferon (IFN)-gamma, interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-beta in PBMC was studied with real-time polymerase chain reaction (PCR) in a subgroup of 38 children. No differences were observed in the strength or frequency of positive T cell responses to rotavirus between children with overt diabetes, children with multiple autoantibodies and control children. Children with diabetes-associated autoantibodies had, instead, stronger T cell responses to purified coxsackie B4 virus than control children. Rotavirus-stimulated lymphocytes from autoantibody-positive children produced more IL-4 and phytohaemagglutinin (PHA)-stimulated lymphocytes more IL-4 and IFN-gamma than lymphocytes from control children. PHA-stimulated lymphocytes from children with diabetes also produced more IL-4 and purified protein derivative (PPD)-stimulated lymphocytes less TGF-beta than lymphocytes from autoantibody-negative control children. In conclusion, our lymphocyte proliferation studies did not provide evidence supporting an association between rotavirus infections and the development of type 1 diabetes or diabetes-associated autoantibodies in young children.

Fig. 1

T cell responses to human rotavirus lysate expressed as stimulation indices (SIs) showed a moderate correlation with age (r = 0·32, P < 0·0001). The dotted line was drawn at SI ≥ 3 as the cut-off limit of a positive response.

Fig. 2

T cell responses shown as stimulation indices (SIs) to a panel of antigens [purified protein derivative (PPD), tetanus toxoid (TT); purified human rotavirus (PRV); human rotavirus lysate (RV); Nebraska calf diarrhoea virus (NCDV); purified coxsackie B4 virus (PCB); lysate coxsackie B4 virus (CBV)] in children with diabetes-associated autoantibodies (a) and in children with type 1 diabetes (b). T cell responses to PCB were stronger in children with clinical diabetes and autoantibodies (grey boxes, P = 0·0012 and P = 0·0003, respectively) than in control children (white boxes) as well as responses to CBV in children with T1D (P = 0·013) and responses to PPD in children with diabetes-associated autoantibodies (P = 0·026). Median value in each box is shown with a horizontal line in the box. The box plots delineate values between the 25th and 75th percentiles and the whiskers values between the 10th and the 90th percentiles. The values outside this range are indicated with circles.