Natural killer cells distinguish innocuous and destructive forms of pancreatic islet autoimmunity

Abstract

In both human patients and murine models, the progression from insulitis to diabetes is neither immediate nor inevitable, as illustrated by the innocuous versus destructive infiltrates of BDC2.5 transgenic mice on the nonobese diabetic (NOD) versus C57BL/6.H-2g7 genetic backgrounds. Natural killer (NK)-cell-specific transcripts and the proportion of NK cells were increased in leukocytes from the aggressive BDC2.5/B6.H-2g7 lesions. NK cell participation was also enhanced in the aggressive lesions provoked by CTLA-4 blockade in BDC2.5/NOD mice. In this context, depletion of NK cells significantly inhibited diabetes development. NOD and B6.H-2g7 mice exhibit extensive variation in NK receptor expression, reminiscent of analogous human molecules. NK cells can be important players in type 1 diabetes, a role that was previously underappreciated.

Fig. 1.

NK-specific genes are up-regulated in BDC2.5/B6.H-2^g7 pancreatic infiltrate. (A) Live CD45⁺ cells and CD4⁺ BDC2.5-clonotype⁺ cells were sorted from the pancreas of 23- to 25-day-old BDC2.5/NOD or BDC2.5/B6.H-2^g7 mice for microarray analysis. (Left) Cumulative number of up- or down-regulated genes in BDC2.5/B6.H-2^g7 versus BDC2.5/NOD pancreatic CD45⁺ cells for the indicated relative variation. The gray line indicates the level of “background” variation as measured from a randomized data set. (Right) Cumulative number of up-regulated genes in BDC2.5/B6.H-2^g7 versus BDC2.5/NOD pancreatic CD45⁺ cells or CD4⁺ BDC2.5-clonotype⁺ cells. (B) Log-scale plot of expression values in pancreatic CD45⁺ cells with highlights on genes characteristic of the indicated cell type. (C) Microarray expression value of NK-specific genes in B6.H-2^g7 purified NK cells versus CD45⁺ pancreatic infiltrate.

Fig. 2.

NK frequency correlates with pancreatic infiltrate aggressivity. (A) NK cell were identified as DX5⁺CD3^– by cytofluorimetric analysis; their frequency was measured in the pancreas of 20- to 24-day-old BDC2.5/NOD and BDC2.5/B6.H-2^g7 mice. One representative plot is shown as well as the value for all individual mice tested (P < 0.03). Spleen values are shown for control. Gated on live CD45⁺ cells. (B) NK cell frequency in 16- to 22-day-old anti-CTLA4 treated versus untreated BDC2.5/NOD mice (P < 0.01).

Fig. 5.

Strain-specific expression of NK-specific genes. (A) Relative variation (fold-change B6.H-2^g7/NOD) in expression values of NK-specific genes are plotted for splenic NK cells and CD45⁺ pancreatic leukocytes. (B) Microarray data for NK-specific gene expression in the pancreatic infiltrate of BDC2.5/NOD and B6.H-2^g7, as well as in splenic NOD and B6.H-2^g7 NK cells. The genes were sorted by chromosomal position. Fold changes (B6.H-2^g7/NOD) are indicated in each tissue. We confirmed the specificity of the probes used in the Affymetrix MuAv2 chip. For some of the Ly49 genes, some cross-reactivity is possible with other Ly49 genes.

Fig. 3.

NK cells are early participants of an aggressive pancreatic lesion. BDC2.5/NOD mice were treated with anti-CTLA4 Ab at days 10, 12, and 14. The number of NK cells and T cells was measured in the pancreas of each mouse from day 12 to day 22. For comparison, the average number of NK cells (and T cells, respectively) found in the insulitis-free pancreas of a young NOD mice (likely arising from blood contamination) is 16 (50) at day 12, 38 (267) at day 15, and 165 (1,120) at day 18.

Fig. 4.

NK depletion impairs α-CTLA4 Ab-induced diabetes development. Arrows indicate injections with αCTLA-4 Ab. (A) BDC2.5/NOD mice treated with anti-Asialo GM1 or rabbit IgG as control. (B) BDC2.5/NOD or BDC2.5/NOD.NK1.1 were treated with PK136 (300 μg three times a week).

Fig. 6.

Wide differences in NK surface receptor expression between NOD and B6.H-2^g7 mice. (A) Real-time PCR analysis of Ly49a, Ly49p, and Nkrp1-a in sorted splenic NK cells from 4- to 5-week-old NOD, NOD.NK1.1, and B6.H-2^g7 mice. (B) Cytofluorimetric analysis of NK surface receptors on NOD, NOD.NK1.1, and B6.H-2^g7 splenocytes. Bold numbers indicate the average percentage of NK cells (±SD) positive for the indicated surface molecule (when gated on DX5⁺CD3^–). Numbers in parenthesis indicate the mean fluorescence intensity on the x axis of the designated population. The mAb used to stain Ly49A^B6 (clone A1) has been showed to cross-react with Ly49A^NOD and partially to Ly49P^NOD (40). The preferential expression of Ly49P by NOD NK cells might be reflected by the slight but clear staining observed on Ly49A^– NOD NK cells versus Ly49A^– B6.H-2^g7 NK cells.