Population dynamics of islet-infiltrating cells in autoimmune diabetes

Abstract

Type-1 diabetes in the nonobese diabetic (NOD) mouse starts with an insulitis stage, wherein a mixed population of leukocytes invades the pancreas, followed by overt diabetes once enough insulin-producing β-cells are destroyed by invading immunocytes. Little is known of the dynamics of lymphocyte movement into the pancreas during disease progression. We used the Kaede transgenic mouse, whose photoconvertible fluorescent reporter permits noninvasive labeling and subsequent tracking of immunocytes, to investigate pancreatic infiltrate dynamics and the requirement for antigen specificity during progression of autoimmune diabetes in the unmanipulated NOD mouse. Our results indicate that the insulitic lesion is very open with constant cell influx and active turnover, predominantly of B and T lymphocytes, but also CD11b(+)c(+) myeloid cells. Both naïve- and memory-phenotype lymphocytes trafficked to the insulitis, but Foxp3(+) regulatory T cells circulated less than their conventional CD4(+) counterparts. Receptor specificity for pancreatic antigens seemed irrelevant for this homing, because similar kinetics were observed in polyclonal and antigen-specific transgenic contexts. This "open" configuration was also observed after reversal of overt diabetes by anti-CD3 treatment. These results portray insulitis as a dynamic lesion at all stages of disease, continuously fed by a mixed influx of immunocytes, and thus susceptible to evolve over time in response to immunologic or environmental influences.

Keywords: Treg; cell tracer; reporter.

Fig. 1.

Dynamic profile of the pancreatic infiltrate during disease progression. (A) Background level of total CD45⁺ cells in pancreas was measured using flow cytometry. (B–E) The immune cell infiltrate in pancreata from mice at the different indicated ages was enumerated and profiled by flow cytometry. B, Inset shows 4- to 8-wk data on expanded scale. (F) Lymphocyte proliferation during disease progression in NOD. BrdU was administered (1.2 mg BrdU in 200 mL, two injections 10 h apart) to female NOD mice from 4 to 14 wk of age. At 22 h after the first injection of BrdU, BrdU incorporation into CD4⁺, CD8⁺ T cells, and CD45R⁺ B cells was measured by flow cytometry for the pancreas, PLNs, and spleen.

Fig. 2.

Monitoring cell trafficking to the pancreas. (A) Sample flow cytometry data for CD45⁺ cells from nonphotoconverted CLNs and 0-h, 1-min, and 24-h time points. (B) The proportion of CD45⁺ cells comprised of recent immigrants was measured by flow cytometry data generated at 0, 1, 3, or 7 d after photoconversion of the CLNs in 12- to 14-wk-old mice. (C) Islets in explanted pancreata from 10- to 12-wk-old mice were imaged by confocal microscopy 36 h after photoconversion of the CLNs. Kaede-red⁺ recent immigrants (red), β-cells (blue), and islet periphery (dashed white line). Representative z-stack projection shown here. Six pancreata per group were imaged.

Fig. 3.

Monitoring lymphoid and myeloid cell trafficking to the pancreas. The proportion of lymphocyte (A and B) or myeloid (C and D) populations that was comprised of recent immigrants in pancreas and inguinal LNs (ILNs) was measured by flow cytometry data generated at 0, 24, 72, or 168 h after photoconversion of the CLNs. (E) Schematic depiction of mathematical modeling of lymphocyte migration into the pancreas. (F) Migration ratios for lymphocyte subsets trafficking to pancreas, PLNs, or spleen vs. control ILNs were calculated from 24-h flow cytometry data. (G and H) Effector memory phenotype (G; CD62L, CD44) and T-cell activation (H; CD69) were assessed in resident and recent immigrant CD4⁺ T cells in pancreas, ILNs, and spleen. Each bar represents an individual mouse.

Fig. 4.

Monitoring Tconv and Treg cell trafficking to the pancreas. (A) The proportion of Tconv and Treg populations that was comprised of photoconverted cells at the site of photoconversion (CLNs). (B and C) Recent immigrants in the pancreas and ILNs were measured by flow cytometry. Data were generated at 0, 24, 72, or 168 h after photoconversion of the CLNs. (D) Migration of Tconv and Treg cells to the pancreas vs. the control ILNs was calculated from same flow cytometry data. ***P < 0.001 (aggregate P value).

Fig. 5.

Does antigen specificity affect migration into the lesions? The stages of disease initiation (4–6 wk) and established insulitis (12–14 wk) were examined in both NOD.Kaede (NOD) and BDC2.5.NOD.Kaede (BDC2.5) mice. Migration of lymphocytes (CD4⁺ T cells, CD8⁺ T cells and B cells) into the pancreas was measured by flow cytometry 72 h after photoconversion of the CLNs. (A) Proportion of cell population comprised of recent immigrants. (B) Migration of lymphocytes to pancreas, vs. control ILNs was calculated. (C) Number of recent lymphocyte immigrants in pancreas. (D) Number of photoconverted vs. total cells. Four mice per group, two separate experiments.

Fig. 6.

Tracing lymphocyte traffic to the pancreas after therapeutic anti-CD3 treatment. NOD.Kaede mice were treated with anti-CD3 at the onset of diabetes. (A) Histology showing insulitis in an anti–CD3-treated mouse at day 30 after first treatment. Image courtesy of J. Nishio. (B and C) On day 30 after diabetes onset, (B) migration, and (C) migration ratio (vs. ILN) of lymphocytes (CD4⁺ T, CD8⁺ T, B cells, Tconv, and Treg cells) into the pancreas was measured by flow cytometry, 72 h after photoconversion of the CLNs. Total of 5–6 mice per group.