Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3

Abstract

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells. Lag3(-)(/)(-) mice exhibited accelerated, invasive insulitis, corresponding to increased CD4(+) and CD8(+) T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4(+) T cells and islet specific glucose-6-phosphatase-specific CD8(+) T cells were significantly increased in the islets of Lag3(-)(/)(-) mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4(+) and CD8(+) T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

Figure 1

Accelerated diabetes in the absence of LAG-3. Diabetes incidence was monitored in Lag3^−/− NOD females (A) and males (B) together with littermate heterozygous Lag3^+/– and Lag3^+/+(WT) mice (♀ +/+ n=53, +/− n=46, −/− n=29; ♂ +/+ n=40, +/− n=39, −/− n=31). C, NOD WT female mice (7 weeks old) were treated with blocking antibodies to LAG-3, PD-1, control IgG1, or PBS on days 0, 5, and 7 and monitored for diabetes (n=9). D-E, Splenocytes from 5-6 week old mice (D - WT; E - Lag3^−/−) were MACS depleted of CD4⁺ or CD8⁺ T cells, reconstituted with either CD4⁺ or CD8⁺ T cells from age-matched Lag3^−/− (D) or WT (E) female mice, and transferred into female NOD.scid recipients (n=11-14).

Figure 2

Increased insulitis and numbers of infiltrating CD4⁺ and CD8⁺ T cells in the islets of Lag3^−/− mice. Histological assessment of insulitis performed in female (A) and male (B) Lag3^−/− and WT mice at three different ages (3-4 weeks, ♀ n=2-6 ♂ n=2-4; 4-5 weeks, ♀ n=3-11 ♂ n=4-9; and 6 weeks old, ♀ n=16-18 ♂ n=20-22). In the top panel: percent of islets exhibiting no insulitis (white), peri-insulitis (grey) and invasive insulitis (black) (***p≤0.0009, **p=0.0014). Percent and number of CD4⁺ (C; n=14-23), CD8⁺ (D; n=5-12), and CD4⁺Foxp3⁺ (E; n=9-18) T cells based on flow cytometric analysis of pancreatic LN (PLN) and islets from 6 week old wild type (white), 6 week old Lag3^−/− (black) or 10 week old Lag3^−/− (grey) female mice (***p≤0.0004, **p≤0.088, *p=0.0399).

Figure 3

CD4⁺ and CD8⁺ T cells exhibit increased proliferation in the islets of Lag3^−/− mice. A, Proliferation was assessed based on intracellular Ki67 staining of CD8⁺, CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ T cells in 6 week old Lag3^−/− female mice (n=7) and 6 (n=10) or 10 week old (n=5) WT female mice (*p≤0.0229). B, CXCR3 cell surface expression on CD4⁺ T cells was measured by flow cytometric analysis of 6-7 week old female Lag3^−/− mice (n=7; *p=0.0262). C, Intracellular IFNγ expression was assessed in the organs of 6 week old female and 7 week old male Lag3^−/− and WT mice after 5 hour stimulation with PMA and ionomycin (n=7-10).

Figure 4

Accumulation of islet antigen specific clones in the islets of Lag3^−/− NOD mice. Frequency of chromogranin A mimotope (BDC2.5mi) tetramer positive CD4⁺ (A; n=14-15; *p=0.0495) and IGRP mimotope (NRP-V7) tetramer positive CD8⁺ (B; n=9-11; **p=0.0024) T cells was assessed in the organs of Lag3^−/− mice. Representative islet FACS plots are shown. Numbers of CD44^lo precursor (C) and CD44^hi activated/expanded (D) IGRP reactive CD8⁺ T cells were calculated after enrichment for NRP-V7 tetramer⁺ cells via anti-PE MACS beads and flow cytometric analysis (n=5-9; **p≤0.0022, *p=0.04).