Quantitative and Functional Analyses of Spleen and in Situ Islet Immune Cells Before and After Diabetes Onset in the Nod Mouse

Abstract

Cytofluorometric analysis using specific monoclonal antibodies directed against the T cell antigens Thy-1.2, CD4, CD8, CD4V/J(8.1+8.2+8.3), and the antigen Mac-1 expressed by mature macrophages and NK cells were used to characterize and quantify the phenotypes of (1) unfractionated and Percoll gradient fractionated in situ islet immune cells isolated from prediabetic and diabetic female NOD mouse spleens. We found in prediabetic female mice that the majority (-70%) of the in situ islet immune cells were Thy-1.2 positive T cells. CD4 positive T cells (-40%) were the most abundant phenotype together with double negative T cells (-20%). The percentages of CD8 positive T cells were -10%, and only -4% of the immune cells were Mac-1 positive. The percentages of CD4V/3(8.1+8.2+8.3) positive and double negative T cells in diabetic spleens were significantly higher in comparison to prediabetic spleens. In C57B1/6J control nondiabetic mice the percentage of double negative T cells in the spleens was significantly 4-fold lower when compared to diabetic NOD spleens. The specific cytolytic activity mediated by in situ islet immune cells against 51Cr-labeled dispersed syngeneic single-cell islet cells at an effector to target ratio of 20 was twenty- to thirty-fold higher than that mediated by prediabetic splenic lymphoid cells. It is concluded that prediabetic NOD mouse in situ islet immune cells are mostly CD4 positive and double negative T cells, and that CD4 and CD8 positive T cells in the intra-islet infiltrate warrants further evaluation as potential effector T cells in target J-cell destruction.

Key Words:

• NOD mice
• intra-islet immune cells
• phenotypes
• cytotoxicity