CD226 Deletion Reduces Type 1 Diabetes in the NOD Mouse by Impairing Thymocyte Development and Peripheral T Cell Activation

Abstract

The costimulatory molecule CD226 is highly expressed on effector/memory T cells and natural killer cells. Costimulatory signals received by T cells can impact both central and peripheral tolerance mechanisms. Genetic polymorphisms in CD226 have been associated with susceptibility to type 1 diabetes and other autoimmune diseases. We hypothesized that genetic deletion of Cd226 in the non-obese diabetic (NOD) mouse would impact type 1 diabetes incidence by altering T cell activation. CD226 knockout (KO) NOD mice displayed decreased disease incidence and insulitis in comparison to wild-type (WT) controls. Although female CD226 KO mice had similar levels of sialoadenitis as WT controls, male CD226 KO mice showed protection from dacryoadenitis. Moreover, CD226 KO T cells were less capable of adoptively transferring disease compared to WT NOD T cells. Of note, CD226 KO mice demonstrated increased CD8⁺ single positive (SP) thymocytes, leading to increased numbers of CD8⁺ T cells in the spleen. Decreased percentages of memory CD8⁺CD44⁺CD62L^- T cells were observed in the pancreatic lymph nodes of CD226 KO mice. Intriguingly, CD8⁺ T cells in CD226 KO mice showed decreased islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-tetramer and CD5 staining, suggesting reduced T cell receptor affinity for this immunodominant antigen. These data support an important role for CD226 in type 1 diabetes development by modulating thymic T cell selection as well as impacting peripheral memory/effector CD8⁺ T cell activation and function.

Keywords: CD226; CD8+ T cells; Sjögren’s disease; costimulatory receptors; type 1 diabetes.

FIGURE 1

CRISPR/Cas9 targeting methods for generation of CD226 KO strain. (A) A guide RNA was used to target the fourth exon of Cd226, inducing a single base pair deletion (red) that led to a frameshift and premature termination of CD226 protein translation. Modified image from Gene NCBI. Histograms showing CD226 expression in splenic (B) CD4⁺ T cells, (C) CD8⁺ T cells, and (D) NK cells of 12-week-old female, pre-diabetic WT (black), HET (gray), and KO (light gray) mice.

FIGURE 2

CD226 KO NOD mice are protected from type 1 diabetes. Disease incidence was monitored weekly, with diabetes defined as two consecutive daily blood glucose readings >250 mg/dL. CD226 KO (dotted lines) significantly lowers disease incidence in (A) females and (B) males as compared to WT (solid black) or HET (solid gray) mice. Log-rank (Mantel–Cox) test; *p < 0.05; **p < 0.01. Female: +/+, n = 20; +/-, n = 26; –/–, n = 21; Male: +/+, n = 19; +/-, n = 23; –/–, n = 16.

FIGURE 3

CD226 KO mice have reduced insulitis and dacryoadenitis. At least 45 islets per mouse were analyzed from three sections 250 microns apart. (A) Representative images of hematoxylin and eosin-stained pancreata from 12-week old female CD226 WT and KO mice. Yellow arrows denote islets. (B) Total number of islets categorized by insulitis score for pre-diabetic 12-week old females or (C) pre-diabetic 16-week old males. Chi-square test; ****p < 0.0001. Female: N = +/+, 10; –/–, 10. Male: N = +/+, 7; N = –/–, 10. (D) Representative images of hematoxylin and eosin-stained lacrimal glands showing numerous inflammatory foci in CD226 WT, but not KO lacrimal glands. Bars are 1 mm. (E) Dacryoadenitis scoring from 10–16 week old male CD226 WT and KO mice. (F) Sialoadenitis scoring in 10–14 week old female CD226 WT and KO mice. Symbols represent individual mice. Lines are medians. Mann–Whitney test; ****p < 0.0001.

FIGURE 4

CD226 expression in T cells exacerbates type 1 diabetes induction. (A) Schematic of CD4⁺ and CD8⁺ T cell transfer from pre-diabetic CD226 WT or KO mice to female NOD.SCID recipients. Created with BioRender. (B) Diabetes induction was monitored for up to 20 weeks post-transfer, with incidence significantly decreased in recipients of KO CD4⁺ and KO CD8⁺ (dashed gray) T cells as compared to WT CD4⁺ and KO CD8⁺ (solid black) or WT CD4⁺ and WT CD8⁺ (solid gray) T cell recipients. Mice receiving KO CD4⁺ and WT CD8⁺ (dashed black) also showed significantly decreased incidence as compared to WT CD4⁺ and WT CD8⁺ T cell recipients. Log-rank (Mantel–Cox) test; *p < 0.05; **p < 0.01. WT CD4⁺ and WT CD8⁺, n = 10; WT CD4⁺ and KO CD8⁺, n = 10; KO CD4⁺ and WT CD8⁺, n = 12; KO CD4⁺ and KO CD8⁺, n = 11.

FIGURE 5

CD226 and ligand expression are enriched on CD8⁺ SP thymocytes and thymic dendritic cells (DCs), respectively. Cells were analyzed from 12-week old pre-diabetic female mice. (A) Representative histograms of CD226 geometric mean fluorescence intensity (gMFI) in thymocytes showing that (B) CD8 SP thymocytes have higher CD226 expression than DN, DP, and CD4 SP subsets. (C) Representative histogram of CD226 expression in spleen. (D) gMFI of CD226 elevated on CD8⁺ as compared to CD4⁺ T cells and NK cells. (E) Representative histograms of CD155 on CD11c⁺MHCII⁺ DCs from various organs. Fluorescence minus one (FMO) control shown. (F) CD155 gMFI is elevated on thymic DCs as compared to DCs from all other organs surveyed. (G) Representative histograms of CD112 reveal that (H) CD112 expression is highest on thymic DCs. Friedman test with Dunn’s multiple comparisons test; *p < 0.05; **p < 0.01; ***p < 0.001. N = +/+, 6 for thymic CD226 and CD155, N = +/+, 5 for thymic CD112, N = +/+, 3 for splenic CD226 staining.

FIGURE 6

CD226 KO mice have increased thymic output of CD8⁺ T cells. Thymus and spleen were analyzed from 12-week old pre-diabetic female mice. (A) Representative flow cytometry plots showing percentage of (B) DN, (C) DP, and (D) CD4 SP thymocytes are similar between WT and KO mice. (E) Increased percentage of CD8 SP thymocytes in CD226 KO. Unpaired t test; *p < 0.05. N = +/+, 17; –/–, 19. (F) Representative flow cytometry plots from spleen, gated on live CD3⁺ T cells. (G) Decreased percentage of CD4⁺ and (H) increased percentage of CD8⁺ T cells, resulting in (I) decreased CD4⁺:CD8⁺ ratio in CD226 KO mice. Kruskal–Wallis with Dunn’s multiple comparisons test; *p < 0.05; **p < 0.01. N = +/+, 7; +/–, 8; –/–, 9.

FIGURE 7

Loss of memory CD8⁺ T cells in the pancreatic-draining lymph nodes of CD226 KO mice. (A) Representative flow cytometry plots from 12-week old pre-diabetic females, gated on live CD3⁺ T cells. While percentages of CD62L⁺CD44^– (B) naïve CD4⁺, CD62L^–CD44⁺ (C) memory CD4⁺ T cells, and (D) naïve CD8⁺ T cells remained unaltered in CD226 KO mice, the percentage of (E) memory CD8⁺ T cells was reduced in the pancreatic lymph nodes of CD226 KO mice. Kruskal–Wallis with Dunn’s multiple comparisons test; **p < 0.01. N = +/+, 7; +/–, 8; –/–, 9.

FIGURE 8

Decreased IGRP avidity in CD226 KO mice. (A) Representative flow cytometry plots from 12-week old females, pancreatic-draining lymph node (pLN) gated on live CD8⁺CD44⁺ T cells and pancreas (Pan) gated on live CD8⁺ T cells. (B) While percentages of IGRP-tetramer⁺ cells are similar between WT and KO mice for all organs surveyed, (C) gMFI of the tetramer within the tetramer⁺ gate is significantly reduced in CD226 KO mice. Mann–Whitney test; *p < 0.05. N = +/+, 6; –/–, 12. (D) Representative histograms of CD5 expression on naïve IGRP-tetramer⁺ CD8⁺ T cells in the spleen. (E) CD5 gMFI on naïve IGRP-reactive cells is significantly decreased in CD226 KO mice. Unpaired t-test; *p < 0.05. N = +/+, 5; –/–, 12.