Endocrine self and gut non-self intersect in the pancreatic lymph nodes

Abstract

The autoimmune cascade that culminates in diabetes initiates within pancreatic lymph nodes (PLNs). Here, we show that developmentally controlled lymphogenesis establishes a preferential trafficking route from the gut to the PLN, where T cells can be activated by antigens drained from the peritoneum and the gastrointestinal tract. Furthermore, intestinal stress modifies the presentation of pancreatic self-antigens in PLNs. The convergence of endocrine and intestinal contents within PLNs has significant implications for type 1 diabetes and may help to explain the link between autoimmune pathogenesis and environmental provocation.

Fig. 1.

Preferential migration to PLNs. (A) Distribution of transferred cells was examined in PLNs, MLNs, inguinal LNs, and axillary LNs of 4-week-old NOD mice 24 h after i.p. injection with 10 × 10⁶ CFSE-labeled splenocytes from 4-week-old NOD donors. Shown is representative fluorescence in recipient LNs as captured by confocal microscopy (magnification ×250). Data are representative of 10-20 mice. The donor cell proportion among total LN cells is indicated. (B) Cytofluorimetric enumeration of donor cells in recipient LNs after i.p. or i.v. transfer. The migration index is calculated as the ratio of donor cell density in PLNs to the donor cell density in SLNs (filled columns) or MLNs (open columns). Shown is data from 4-10 mice. (C) Kinetics of donor cell trafficking to PLNs, MLNs, and SLNs was determined by cytofluorimetry. Data are shown as the number of CFSE⁺ donor cells (¢) × 10^-3 per 1 × 10⁶ recipient LN cells. Three or four mice were examined at each time point. (D) Donor cells were pretreated with 100 ng/ml Pertussis toxin, washed, transferred i.p. into age- and strain-matched recipients, and analyzed in recipient LNs 24 h later by flow cytometry. Data are shown as the number of CFSE⁺ donor cells (¢) × 10^-3 per 1 × 10⁶ recipient LN cells in PLNs (filled column) and MLNs (open column). Three to four mice were used per condition. (E) Proliferation of transferred naive OT-I T cells (gated on CD8⁺Vα2⁺ cells) in PLNs, MLNs, and SLNs after i.p. injection of BSA-coated beads (total of 20 μg of BSA per recipient) or OVA-coated beads (total of 20 μg of OVA per recipient) into adult B6 mice. Proliferation of OT-I T cells was assessed 48 h after transfer by CFSE dilution in donor T cells. Values depict the frequency of divided OT-I T cells among the donor CD8⁺ T cells. Data are representative of four to five mice per condition. (F) Proliferation of transferred, naive BDC2.5 T cells (gated on CD4⁺Vβ4⁺ cells) in PLNs and SLNs after i.p. injection of graded doses of pancreatic islets or PBS into juvenile NOD mice. Proliferation of BDC2.5 T cells was assessed in all experiments 70 h after transfer by CFSE dilution in CD4⁺ T cells. Values depict the frequency of divided BDC2.5 T cells among the donor CD4⁺ T cells.

Fig. 2.

Lymphatic access to PLNs is developmentally regulated. (A) Fluorescently labeled splenocytes from adult NOD donors were transferred i.p. into infant and adult NOD recipients and then enumerated 24 h later in PLNs and SLNs by cytofluorimetry. Data are shown as the number of CFSE⁺ donor cells (¢) × 10^-3 per 1 × 10⁶ recipient LN cells in PLNs and SLNs of both recipients. Shown are data from seven to eight mice per age group. (B) Donor cell trafficking to PLNs, MLNs, and SLNs in recipients of different ages was determined by flow cytometry. The migration index is calculated as the ratio of donor cell density in PLNs to the donor cell density in MLNs (filled circles) or the ratio of donor cell density in SLNs to the donor cell density in MLNs (open circles). Data are representative of 3-15 mice. (C) Fluorescently labeled splenocytes from adult and infant NOD donors were transferred i.p. into adult and infant recipients and then enumerated 24 h later in recipient LNs by cytofluorimetry. The migration index is calculated as the ratio of donor cell density in PLNs to the donor cell density in SLNs.

Fig. 3.

Gut antigens are presented to CD8⁺ and CD4⁺ T cells in PLNs. (A) Proliferation of transferred naive OT-I T cells (gated on CD8⁺Vα2⁺ cells) in SLNs, Peyer's patches (PPs), MLNs, and PLNs after intragastric administration of 60 mg of OVA (IG OVA^hi) or 2 mg of OVA (IG OVA^lo) or i.v. injection of 2 mg of OVA (IV OVA) into adult B6 mice as compared with controls. Proliferation of OT-I T cells was assessed 48 h after transfer by CFSE dilution in donor T cells. Each symbol represents an individual mouse. (B) Proliferation of transferred naive OT-II T cells (gated on CD4⁺Vα2⁺ cells) in MLNs and PLNs after intragastric administration of 60 mg of OVA (IG OVA^hi) into adult B6 mice. Proliferation of OT-II T cells was assessed 48 h after transfer by CFSE dilution in donor T cells. Each symbol represents an individual mouse.

Fig. 4.

Intestinal alterations influence the autoimmune response in PLNs. (A) Proliferation of transferred naive BDC2.5 T cells (gated on CD4⁺Vβ4⁺ cells) in PLNs of NOD mice on a LP or control (Ctl) diet or pretreated with 85 mg/kg INDO, 5% DSS (DSS^hi), or 2% DSS (DSS^lo) as compared with control littermates (Ctl). T cell proliferation, assessed by CFSE dilution in BDC2.5 T cells, was reduced 47% by LP (P = 0.002), 40% by INDO (P = 0.004), and 54% by 5% DSS (P = 0.001) and was enhanced 22% by 2% DSS (P = 02). Data are shown as the percentage of divided BDC2.5 T cells among the donor CD4⁺ T cells. Values in columns depict number of mice per condition. (B) Proliferation of transferred naive BDC2.5 T cells (gated on CD4⁺Vβ4⁺ cells) in popliteal LNs of NOD mice on a LP diet or pretreated with 5% DSS (DSS^hi) or 2% DSS (DSS^lo) after footpad immunization with BDC2.5 peptide mimic. Data are shown as the percentage of divided BDC2.5 T cells among the donor CD4⁺ T cells. Values in columns depict number of mice per condition. (C) Representative histograms of CD44 staining on donor BDC2.5 T cells in PLNs and MLNs of NOD mice after transfer into mice on 2% DSS or control (CTL) littermates. (D) Frequency of pancreatic islets with heavy leukocyte infiltration in BDC2.5/NOD mice treated with low-dose DSS. Each symbol represents an individual mouse. Data shown as the percentage of islets with heavy leukocyte infiltration among total islets. Each symbol represents an individual mouse.