PD-1 deficiency reveals various tissue-specific autoimmunity by H-2 b and dose-dependent requirement of H-2 g7 for diabetes in NOD mice

Yoshida et al. 10.1073/pnas.0710951105.

Supporting Information

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SI Figure 6
SI Movie 1
SI Figure 7
SI Table 1
SI Figure 8
SI Figure 9
SI Figure 10
SI Materials and Methods

SI Figure 6

Fig. 6. Slight yet detectable insulitis in NOD-H2^b/bPdcd1^-/- mice. The percentage of islets with each grade of insulitis is shown for NOD-H2^b/bPdcd1^+/+ (Left) and NOD-H2^b/bPdcd1^-/- (Right) mice at 22-25 weeks of age. White, pink, red, and black bars represent grade 0, 1, 2, and 3 insulitis, respectively.

SI Figure 7

Fig. 7. Multiorgan inflammation in NOD-H2^b/bPdcd1^-/- and NOD-H2^b/g7Pdcd1^-/-mice. Representative H&E staining of salivary gland (a-d), stomach (e-h), and pancreas (i-l) is shown for 22- to 25-wk-old NOD-H2^b/g7Pdcd1^+/+ (a, e, and i), NOD-H2^b/g7Pdcd1^-/- (b, f, and j), NOD-H2^b/bPdcd1^+/+ (c, g, and k), and NOD-H2^b/bPdcd1^-/- (d, h, and l) mice. (Original magnification, ×40.)

SI Figure 8

Fig. 8. PD-1 deficiency does not affect the cellular composition in thymus and spleen. (a) The absolute numbers of total, CD4^-CD8^- (DN), CD4⁺CD8⁺ (DP), CD4⁺CD8^- (CD4), and CD4^-CD8⁺ (CD8) thymocytes are shown for 25-wk-old NOD-H2^b/bPdcd1^+/+ (white bars) and NOD-H2^b/bPdcd1^-/- (black bars) mice. (b) The numbers of B cells, CD4⁺ T cells, and CD8⁺ T cells in spleen are shown. Symbols are the same as in a.

SI Figure 9

Fig. 9. PD-1 deficiency does not affect the production of Tregs. (a) Representative FACS profiles of CD4 vs. CD25 staining (Left) for total spleen cells, and CD25 vs. CD62L staining (Center) and CD25 vs. Foxp3 staining (Right) for CD4-gated spleen cells from NOD-H2^b/bPdcd1^+/+ (Upper) and NOD-H2^b/bPdcd1^-/- (Lower) mice are shown. (b) The mean percentage of CD4⁺CD25⁺ (Left), CD4⁺CD25⁺CD62L⁺ (Center), and CD4⁺CD25⁺Foxp3⁺ (Right) cells is shown for NOD-H2^b/bPdcd1^+/+ (white bars) and NOD-H2^b/bPdcd1^-/- (black bars) mice. Data are the mean + SEM. Representative data from more than three experiments are shown. (c) CD4⁺CD25⁺ Tregs from NOD-H2^b/bPdcd1^-/- mice retain suppressive activity in vitro. CD4⁺CD25⁺ Tregs from NOD-H2^b/bPdcd1^+/+ (open symbols) and NOD-H2^b/bPdcd1^-/- (filled symbols) mice were examined for their suppressive activity against CD4⁺CD25^- effector T cells from NOD-H2^b/bPdcd1^+/+ (circles) and NOD-H2^b/bPdcd1^-/- (squares) mice. Data are the mean ± SEM of triplicate cultures. Representative data of more than three experiments are shown.

SI Figure 10

Fig. 10. Augmented expression of B7-2 and CTLA-4 in the inflamed nerves of neuropathic Pdcd1^-/- mice. Relative quantities of mRNAs for the indicated genes in inflamed nerve in relation to control nerve were calculated as described in Materials and Methods. Data are the mean + SEM of three mice.

SI Movie 1

Movie 1. Representative movie of a mouse with severe paralysis of the limbs.

Table 1. Multiorgan inflammation in NOD-H2^b/bPdcd1^-/- and NOD-H2^b/g7Pdcd1^-/- mice

Genotype	Age, wk	n	Sialoadenitis, % (grade)	Gastritis, % (grade)	Pancreatitis, % (grade)
g7/g7 WT	4-6	10	0 (0)	0 (0)	0 (0)
g7/g7 WT	20	14	64 (0.78 ± 0.13)	0 (0)	0 (0)
g7/g7 KO	4-6	13	62 (0.69 ± 0.13)	8 (0.08 ± 0.06)	0 (0)
b/g7 WT	20-24	9	44 (0.56 ± 0.24)	0 (0)	0 (0)
b/g7 KO (DM+)	15-20	15	100 (2.07 ± 0.21)	100 (1.81 ± 0.18)	33 (0.51 ± 0.21)
b/g7 KO (DM-)	20-24	15	100 (2.51 ± 0.13)	100 (2.53 ± 0.12)	14 (0.33 ± 0.22)
b/b WT	20-24	23	38 (0.36 ± 0.12)	0 (0)	0 (0)
b/b KO	20-24	24	100 (2.13 ± 0.16)	100 (2.24 ± 0.11)	44 (0.77 ± 0.24)

SI Materials and Methods

Reagents. APC- and PE-conjugated streptavidin, APC-conjugated Abs against CD3, CD8, B220, Gr1, and IFNg, Cy5-conjugated Ab against CD8, PE-conjugated Abs against CD4, B220, CD44, CD62L, CD69, CD122, PD-L1, Foxp3, Mac1, IL-4, and IL-17, FITC-conjugated Abs against CD4, CD8, CD44, CD11c, and CD3, and biotinylated Ab against CD25 were purchased from eBioscience. Polyclonal Abs against PD-L1 and PD-L2 were purchased from R & D Systems. Rabbit antineuronal class b-tubulin and rabbit anti-human MBP Abs were purchased from Covance and Chemicon, respectively. FITC and TxRed-conjugated polyclonal Abs against goat IgG, and TxRed-conjugated polyclonal Abs against mouse IgM were purchased from Jackson. PerCP-conjugated Ab against CD4 was purchased from Pharmingen.

Histological Analysis. Organs were fixed with 10% buffered formalin, processed, and embedded in paraffin. Sections were stained with hematoxylin and eosin by using standard techniques. Insulitis was scored as previously described [Wang J, et al. (2005) Establishment of NOD-Pdcd1^-/- mice as an efficient animal model of type I diabetes. Proc Natl Acad Sci USA 102:11823-11828]. Pancreatitis, sialoadenitis, and gastritis were evaluated according to published criteria [Kuroda N, et al. (2005) Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice. J Immunol 174, 1862-70].

Real-Time PCR Using TaqMan Low-Density Arrays. Total RNA was isolated from sciatic nerves by using TRIzol (Invitrogen). Single-strand cDNA was synthesized by using Quantitect Reverse Transcription (Qiagen), mixed with TaqMan Universal PCR Master Mix (Applied Biosystems), and loaded into 384 wells of a TaqMan Low-Density Array Immuno Profiling Plate (Applied Biosystems). PCRs were performed on an ABI PRISM 7900 HT system and analyzed by using SDS2.2 software according to the manufacturer's protocol. Gene expression profiling was achieved by using the comparative cycle threshold (C_t) method of relative quantification. The b-actin reference gene Actb was used as an endogenous control, and the calibrator samples were from healthy subjects. Genes with C_t > 35 were eliminated for lack of reproducibility. DC_t represents the C_t of the target minus that of the endogenous control, and DDC_t represents the DC_t of each target minus that of the calibrator. Relative quantities were determined by using the formula; relative quantity = 2^-DDCt.

Immunohistology. Sciatic nerve, stomach, and pancreas were collected from the indicated mice and snap-frozen in OCT compound. Cryosections were fixed with CytoFix (Pharmingen) and stained with the indicated Abs. Sections of sciatic nerve were fixed in 95% ethanol at 4°C for 30 min, followed by 100% acetone at room temperature for 1 min. AutoAbs were evaluated as described [Okazaki T, et al. (2005) Hydronephrosis associated with antiurothelial and antinuclear autoantibodies in BALB/c-Fcgr2b^-/-Pdcd1^-/- mice. J Exp Med 202:1643-1648]. Signals were observed with Leica Application Suite Advanced Fluorescence (Leica).

In Vitro Treg Assay. CD4⁺CD25^- effector T cells and CD4⁺CD25⁺ Tregs were isolated from 8-wk-old NOD-H2^b/bPdcd1^+/+ and NOD-H2^b/bPdcd1^-/- mice, and in vitro Treg assay was performed as described [Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155, 1151-1164]. Briefly, CD8 T, B, and adherent cells were removed from cell suspensions to enrich for CD4 T cells by panning using anti-CD8 (3.155) and anti-CD24 (J11D) Abs (kindly provided by Dr. Shimon Sakaguchi, Kyoto University, Kyoto, Japan). CD4⁺CD25^- effector T cells and CD4⁺CD25⁺ Tregs were separated on autoMACS (Miltenyi Biotec) by using an anti-CD25 Ab. T cell-depleted spleen cells from 8-wk-old NOD-H2^b/bPdcd1^+/+ mice were treated with mitomycin C and used as APCs. The purity of the prepared cells was >95%. CD4⁺CD25^- effector T cells (2.5 ´10⁴ cells) and 5 ´ 10⁴ APCs were cultured in 96-well plates for 3 days with 2.5 mg/ml anti-CD3 Ab (2C11) in RPMI medium 1640 supplemented with 10% FCS, penicillin (100 units/ml), streptomycin (100 mg/ml), and 50 mM 2-ME. CD4⁺CD25⁺ Treg cells were added to the culture at the indicated ratio of CD25^- T cells : Treg cells. The incorporation of [³H]thymidine by proliferating effector cells during the last 6 h of culture was measured by a scintillation counter.

Cytoplasmic Staining. Spleen cells and sciatic nerve-infiltrating cells were stimulated with 10 ng/ml PMA and 1 mg/ml ionomycin for 5 h with Goldi-stop (Pharmingen) before staining with Abs against CD4, CD8, IFNg , IL-4, and IL-17. Cells were analyzed by FACSCalibur (Becton Dickinson).