Development of autoreactive diabetogenic T cells in the thymus of NOD mice

Journal of Autoimmunity 24 (2005) 11e23 www.elsevier.com/locate/issn/08968411

Development of autoreactive diabetogenic T cells in the thymus of NOD mice Hyokjoon Kwona, Hee-Sook Juna,b,), Yang Yanga, Conchi Morac,1, Sanjeev Mariathasand, Pamela S. Ohashid, Richard A. Flavellc, Ji-Won Yoona,b,c,) a

Julia McFarlane Diabetes Research Centre, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1 Canada b Rosalind Franklin Diabetes Center, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064-3095, USA c Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA d Department of Medical Biophysics and Immunology, Ontario Cancer Institute, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada Received 25 May 2004; revised 14 October 2004; accepted 15 October 2004

Abstract Type 1 diabetes results from destruction of pancreatic b cells by b cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential b cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4CCD8C DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69CCD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4ÿCD8C T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Autoimmune diabetes; Cytotoxic T lymphocytes; Immune tolerance; Cell-mediated immunity; Cell differentiation

1. Introduction Autoimmune disease is generally considered to result from failure to establish self-tolerance. T cell tolerance to self may be established and maintained by clonal

) Corresponding authors. Tel.: C1 847 578 3436; fax: C1 847 578 3432. E-mail addresses: [email protected] (H.-S. Jun), yoon@ ucalgary.ca (J.-W. Yoon). 1 Present address: Laboratori Experimental de Diabetis, Hospital Clinic Universitari, Barcelona, Spain. 0896-8411/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2004.10.002

deletion, clonal anergy and suppression of autoreactive T cells. The development of the T cell repertoire is shaped through positive and negative selection [1e4]. Low affinity/avidity interactions elicit positive selection, whereas high affinity/avidity interactions elicit negative selection. Clonal deletion of T cells reactive to self antigens by negative selection in the thymus is a major mechanism of central tolerance. Type 1 diabetes is an autoimmune disease characterized by T cell-mediated destruction of pancreatic b cells [5e7]. Both genetic predisposition and environmental triggers are believed to affect both the immune system

12

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

and the target b cells, resulting in a loss of T cell tolerance to b cells and the consequent development of type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops diabetes with many characteristics similar to human type 1 diabetes [5e9]. Many immunological defects have been reported in NOD mice, including reduced production of inhibitory cytokines [10], defective function of natural killer cells [11], impaired function of antigen-presenting cells due to the expression of the H-2g7 haplotype [12,13], reduced expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) [14], reduced number of regulatory CD25CCD4C T cells [15], defects in CD8C T cell peripheral tolerance [16], and hypo-responsiveness of T cells after T cell activation [17]. It has been suggested that defects in central tolerance may result in the failure to delete b cell-reactive T cells in the thymus, contributing to the development of autoimmune diabetes in the NOD mouse. This investigation was initiated to determine whether there are any defects in negative selection of DP thymocytes in NOD mice as compared with diabetes-resistant strains of mice. We found no evidence for defects in negative selection of DP thymocytes in the thymic cortex of NOD mice. In contrast, strongly activated immature DP thymocytes are rapidly deleted by negative selection and weakly activated immature DP thymocytes efficiently differentiate into CD69CCD3high DP thymocytes by positive selection. However, positively selected SP thymocytes that develop from efficiently activated DP thymocytes, possibly in the corticomedullary junction, may escape deletion, probably due to defects in negative selection in the medulla of the thymus in NOD mice. On the basis of these observations, we suggest that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes and may be further augmented by escaping deletion due to defects in negative selection in the medulla of the thymus in NOD mice.

2. Materials and methods 2.1. Animals NOD, NOD.scid, C57BL/6, C57BL/6.scid, and Balb/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). NODlpr/lpr mice were kindly provided by Dr. M. Lee from the Samsung Medical Research Centre (Seoul, Korea). The animals were bred and maintained under specific pathogen-free conditions. 2.2. Western blot analysis Cell lysates from total thymocytes or sorted DP thymocytes were prepared, and phosphorylation of

ERK and JNK protein was determined with antiphospho-ERK and anti-phospho-JNK Abs (Cell Signaling Technology, Beverly, MA) by western blot. The membrane was stripped with Re-Blot Plus (Chemicon International, Temecula, CA) and total ERK and JNK protein levels were determined with anti-ERK and antiJNK Abs (Cell Signaling Technology). Proteins were detected by enhanced chemiluminescence (ECL Plus, Amersham Bioscience). Protein band intensity was determined by scanning with Scion Image (Scion Corp, Frederick, MD). The ratio of phosphorylated ERK and JNK was determined by dividing by the total amount of ERK and JNK, respectively. 2.3. Administration of anti-CD3 Ab into NOD and C57BL/6 mice Four- to 5-week-old female mice were injected intraperitoneally with various doses of anti-CD3 Ab or isotype IgG (hamster, PharMingen, San Diego, CA) in 200 ml PBS. Animals were killed at various times after Ab treatment and thymocytes were collected. 2.4. Flow cytometric analysis All Abs used in FACS analysis were purchased from Pharmingen (San Diego, CA); streptavidin-PerCP was obtained from Beckton Dickinson (Mountain View, CA). The cells were incubated with the appropriate fluorochrome-labeled Abs, washed twice with FACS buffer, and analyzed by FACScan. Data files were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA). 2.5. DNA fragmentation assay and TUNEL staining Apoptosis of thymocytes was measured by DNA fragmentation as described previously [18]. For TUNEL staining, thymocytes (2 ! 106) were stained with PElabeled anti-CD8 and biotin-labeled anti-CD4 Abs and then with streptavidin-PerCP. After permeabilising the cells with 0.1% Triton X-100 in 0.1% sodium citrate (pH 5.7), the cells were incubated with FITC-labeled TUNEL reagent (In Situ Cell Death Detection Kit, Roche, Laval, PQ, Canada), washed, and analyzed by FACScan. 2.6. Fetal thymic organ culture (FTOC) Fetal thymic lobes were prepared from C57BL/6 and NOD mice at embryonic day 17.5. Fetal thymic lobes were placed on 0.8 mm polycarbonate filters (Costar, Cambridge, MA), which floated on DMEM supplemented with 12% FCS, 2 mM glutamine, 5 ! 10ÿ5 M 2mercaptoethanol, 100 U/ml penicillin and 100 mg/ml

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

streptomycin, and incubated for 4 days at 37  C. The thymic lobes were then treated with anti-CD3 Ab in the presence or absence of zVAD-fmk (Enzyme Systems Products, Livermore, CA). Thymocytes were prepared and stained for FACS analysis. 2.7. Reaggregation thymic organ culture (RTOC) To prepare thymic stromal cells, thymuses from neonatal NOD.scid and C57BL/6.scid mice were incubated with 0.25% trypsin and 0.1% EDTA (Gibco/ BRL, Gaithersburg, MD) in PBS for 30 min at 37  C, and the cells were dissociated by gentle pipetting. DP thymocytes from 1- to 2-week-old NOD and C57BL/6 mice were sorted using micromagnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. Reaggregates were formed by mixing the thymic stromal cells from NOD.scid or C57BL/6.scid mice and DP thymocytes from NOD or C57BL/6 mice, respectively, in a 1:1 ratio (5 ! 105 cells of each type). After centrifugation, the cells were placed on 0.8 mm polycarbonate filters (Costar), which floated on DMEM supplemented as described for FTOC. Reaggregates were incubated for various times at 37  C, during which time the medium was changed daily. 2.8. Adoptive transfer CD8C SP thymocytes (5 ! 106), isolated from NOD FTOC at 24 h after treatment with anti-CD3 Ab (5 mg/ ml), and CD8C T cell-depleted splenocytes (5 ! 106), prepared from acutely diabetic NOD mice by negative selection using anti-CD8 labeled micromagnetic beads, were co-injected intravenously into 6e8-week-old NOD.scid mice. As controls, CD8C T cell-depleted splenocytes (5 ! 106) were injected into age-matched NOD.scid mice. The development of diabetes was determined by measuring urine and blood glucose every other day as previously described [19]. 2.9. Histological analysis Pancreatic sections of NOD.scid recipients were stained with hematoxylin and eosin [20]. The severity of insulitis was scored as follows: 0, normal islet; 1, mononuclear cell infiltration, largely in the periphery, in less than 25% of the islet; 2, 25e50% of the islet showing mononuclear cell infiltration; 3, 50e75% of the islet showing mononuclear cell infiltration; 4, over 75% of the islet showing mononuclear cell infiltration. 2.10. Statistical analysis The significance of differences between groups was analyzed by Student’s t test. A level of p ! 0.05 was accepted as significant.

13

3. Results 3.1. Efficient deletion of DP thymocytes after T cell activation in NOD mice To determine whether there is any difference in susceptibility of DP thymocytes to negative selection between diabetes-prone NOD mice and diabetes-resistant C57BL/6 mice, we examined the deletion profile of DP thymocytes in these mice at 24 and 48 h after injection with 10, 25, 100 or 200 mg/mouse of anti-CD3 Ab. We found that the proportion of DP thymocytes was significantly lower in NOD mice than in C57BL/6 mice (Fig. 1AeC), with the most significant differences being at doses of 25 and 100 mg of anti-CD3 Ab at 24 h after injection (Fig. 1B) and 10 and 25 mg of Ab at 48 h after injection (Fig. 1C). We then compared the sensitivity of DP and SP thymocytes from NOD and C57BL/6 mice to anti-CD3 Ab-induced deletion. The number of NOD DP thymocytes was reduced by 92%, and the numbers of NOD CD4CCD8ÿ and CD4ÿ CD8C SP thymocytes were slightly decreased at 24 h after anti-CD3 Ab injection. In contrast, the number of C57BL/6 DP thymocytes was only reduced by 39%, and the numbers of C57BL/6 SP thymocytes were unaffected by anti-CD3 Ab injection (Fig. 1D, E). These results indicate that DP thymocytes of NOD mice are more efficiently deleted than those of C57BL/6 mice and SP thymocytes from both strains of mice are less sensitive to anti-CD3 Ab-induced deletion as compared with DP thymocytes.

3.2. Deletion of DP thymocytes by Fas-independent apoptosis To determine whether the deletion of thymocytes is due to apoptosis, we examined chromosomal DNA fragmentation in NOD and C57BL/6 thymocytes at various times after anti-CD3 Ab injection. We found very mild fragmentation of chromosomal DNA in NOD thymocytes, but no fragmentation in C57BL/6 thymocytes at 12 h after anti-CD3 Ab injection (Fig. 2A). At 24 h after anti-CD3 antibody treatment, DNA fragmentation in NOD thymocytes was more severe than in C57 BL/6 thymocytes (Fig. 2A), indicating that thymocyte deletion is due to apoptosis and that NOD thymocytes are more susceptible to anti-CD3 antibody-induced apoptosis. We then determined which subpopulation of thymocytes are apoptosed by TUNEL staining and found that the majority of cells stained by TUNEL are DP thymocytes. The proportion of TUNEL-stained DP thymocytes was significantly higher in NOD mice than C57BL/6 mice (Fig. 2B), indicating that NOD DP thymocytes are more susceptible to apoptosis than

14

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

NOD isotype IgG 103

103

103

103

102

102

2

10

102

101

101

101

101

100

100 101 102

Proportion of DP cells ( )

103 104

100 100 101 102

0

10 4 103 10

4.3 77

10 65

2.3 17

4.6 40

3.4 15

3.7 22

B6 NOD

* **

40

**

20 0 10

25

100

200

80

B6 NOD

60 40

**

20

**

0 0

Anti-CD3 (µg/mouse)

D

Cell number (×106)

80 40

** 0

25

100

200

isotype IgG anti-CD3 30

DP

120

10

Anti-CD3 (µg/mouse)

E

isotype IgG anti-CD3

Cell number (×106)

4 103 10

20 35

C

0

100 100 101 102

5.7 75

80 60

4 103 10

100 101 102

anti-CD3

104

104

CD4

B

104

isotype IgG

104

Proportion of DP cells ( )

CD8

A

C57BL/6 anti-CD3

CD4+

CD8+

20

10

* 0

NOD

C57BL/6

NOD

C57BL/6

NOD

C57BL/6

Fig. 1. DP thymocytes of NOD mice are efficiently deleted after T cell activation. Thymocytes from NOD and C57BL/6 (B6) mice were isolated at different times after injection with various amounts of anti-CD3 Ab, stained with anti-CD4 and anti-CD8 Abs, and analyzed by FACS. (A) FACS profile of thymocytes at 24 h after treatment with 25 mg anti-CD3 Ab or isotype IgG. Representative result of five independent experiments is shown. Proportion of DP thymocytes in NOD and C57BL/6 mice at (B) 24 h or (C) 48 h after treatment with various amounts of anti-CD3 Ab. The number of (D) DP thymocytes and (E) CD4C and CD8C SP thymocytes of NOD and C57BL/6 mice at 24 h after treatment with 25 mg anti-CD3 Ab or isotype IgG. Numbers were calculated from the total number of thymocytes and the proportion of DP or SP thymocytes as determined by FACS. Values are means G SEM of more than three independent experiments (combined n R 4 for each data point). *p ! 0.03, **p ! 0.003, as compared to anti-CD3 Ab-treated C57BL/6 mice (B, C) or to isotype IgG treatment (D, E).

C57BL/6 DP thymocytes. In addition, we found that NOD CD8C SP thymocytes are more susceptible to apoptosis than C57BL/6 CD8C SP thymocytes (Fig. 2B). To determine whether Fas is involved in the apoptosis of thymocytes, we injected anti-CD3 Ab into Fas-deficient NODlpr/lpr, NODC/lpr and NODC/C mice and examined the proportion of DP thymocytes at 24 h after Ab treatment. We found there was no difference in the deletion of thymocytes from NODlpr/lpr, NODC/lpr and NODC/C mice (data not shown), confirming the Fas-independent deletion of DP thymocytes, as previously reported [21,22].

3.3. Efficient deletion of immature DP thymocytes after T cell activation in NOD FTOC It has been reported that TCR-specific peptide or Abmediated peripheral T cell activation affects DP thymocyte deletion in vivo, due to bystander cell death by proinflammatory cytokines released from stimulated peripheral T cells [23e25]. To exclude this possibility, we used an in vitro system, FTOC. We performed NOD and C57BL/6 FTOC in the presence of anti-CD3 Ab or isotype IgG as a control and examined the proportion of DP thymocytes 24 h later. There was no difference in the proportion of DP thymocytes in isotype IgG-treated

15

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

A

B C57BL/6 0h

12h

24h

104

NOD 0h

12h

C57BL/6 10 60

103

24h

90

4.0 26

2

10

10

CD4

CD8

120 60

1

15

98.9

10

0

30

0 100 101 102 103 104

100 0 10 101 102 103 104

DP 300

20

5

200

95.3 2.9

0 100 101 102 103 104

100

91.6 5.2

0 100 101 102 103 104

NOD 104 10

CD8

102 101 0

10 0 10 101 102 103 104

CD4

17 33 4.0 46

80 40

100

15

120

#Cells

3

10

98.3 0

100 101 102 103 104

5

80

85.5 11

100 101 102 103 104

60 40 20

74.8 20.1

0 100 101 102 103 104

TUNEL

Fig. 2. DP thymocytes of NOD mice are deleted through Fas-independent apoptosis. NOD and C57BL/6 mice were injected with 25 mg of anti-CD3 Ab and thymocytes were isolated at different times thereafter. (A) DNA fragmentation analysis of thymocytes at various times after anti-CD3 Ab administration. (B) TUNEL staining of SP and DP thymocytes. Thymocytes isolated at 24 h after anti-CD3 Ab treatment were analyzed by FACS (left panel) and each CD4C SP, CD8C SP and DP subset was further analyzed by TUNEL staining (right panel).

FTOC between NOD and C57BL/6 mice, whereas the proportion of DP thymocytes in NOD FTOC was significantly lower than that in C57BL/6 FTOC at 24 h after anti-CD3 Ab (5 mg/ml) treatment (Fig. 3A). In addition, we found that treatment with zVAD-fmk, a caspase inhibitor, inhibited anti-CD3 Ab-induced DP thymocyte deletion in both NOD and C57BL/6 mice (Fig. 3A). Taken together, these in vitro results confirm that DP thymocyte of NOD mice are more efficiently deleted after T cell activation and the deletion of DP thymocytes is due to activation-induced apoptosis by anti-CD3 Ab, consistent with the results obtained in vivo. We then determined the dose-dependent effect of anti-CD3 Ab treatment on DP thymocyte deletion, and we found that the proportion of NOD DP thymocytes was significantly lower than those of C57BL/6 and Balb/c DP thymocytes at the lowest dose of 5 mg/ml (Fig. 3B). In contrast, there was no significant difference in the proportion of DP thymocytes between NOD and C57BL/6 and Balb/c mice at the high dose of anti-CD3 Ab (40e100 mg/ml) for 24 h (Fig. 3B). We also found that the proportion of DP thymocytes from NOD mice was significantly decreased compared to C57BL/6 and Balb/c mice in FTOC at 48 h after a low dose of antiCD3 Ab treatment (Fig. 3C).

To determine whether such signaling molecules are more readily activated due to a lower activation threshold in NOD DP thymocytes as compared to C57BL/6 DP thymocytes, we examined the phosphorylation of ERK and JNK in both total thymocytes, 80e85% of which are DP thymocytes, and purified thymocytes from NOD or C57BL/6 mice after activation with anti-CD3 Ab. We found that the phosphorylation of ERK was significantly increased at 2 and 4 h after activation in NOD thymocytes as compared with C57BL/6 thymocytes (Fig. 4A, B). Similarly, the phosphorylation of JNK was increased at 8 and 12 h after activation as compared to C57BL/6 thymocytes (Fig. 4C, D). Consistent with the results obtained using total thymocytes, we found that phosphorylation of ERK and JNK in purified NOD DP thymocytes was significantly higher than that of C57BL/ 6 DP thymocytes after activation (Fig. 4EeH). These results indicate that DP thymocytes of NOD mice have a lower activation threshold than those of C57BL/6 mice, resulting in more efficient phosphorylation of ERK and JNK in NOD thymocytes as compared to C57BL/6 DP thymocytes after activation.

3.4. Efficient activation of ERK and JNK in NOD DP thymocytes

Since NOD DP thymocytes are efficiently deleted by activation-induced apoptosis after anti-CD3 Ab treatment both in vivo and in vitro, we hypothesized that immature DP thymocytes may have a lower activation threshold in NOD mice as compared to C57BL/6 mice, resulting in efficient activation of DP thymocytes. We tested this hypothesis by measuring the expression of the

Mitogen-activated protein kinases (MAPKs) such as ERK and JNK are known to be phosphorylated after TCR-mediated T cell activation and involved in the positive and negative selection of DP thymocytes [2,3].

3.5. Generation of SP thymocytes after T cell activation by efficient positive selection of immature DP thymocytes in NOD FTOC

16

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

C57BL/6

A isotype IgG (7.9×105)

104

anti-CD3 (4.5×105)

104

zVAD-fmk anti-CD3

zVAD-fmk 104

(7.4×105)

103

103

103

103

102

102

102

102

101

1

1

101

10

100 100 101 102

103 104

100

10 100 101 102

103 104

100

(7.2×105)

104

100 100 101 102

103 104

100 101 102

4 103 10

11 66

15 55

4.4 83

5.5 81

7.4 16

9.0 21

2.6 10

1.5 12

NOD (7.5×105)

104 103 102 1

CD8

10

0

10

100 101 102

103 104

(5.2×105)

(8.2×105)

104

104

103

103

103

2

10

2

10

102

101

101

101

0

0

10

100 101 102

103 104

10

100 101 102

(6.8×105)

104

103 104

100

100 101 102

103 104

11 61

25 41

6.3 76

8.5 71

7.5 19

11 23

6.0 12

2.8 17

CD4

B

C 60

60

40

40

*** B6

20

*** B6

20

NOD

NOD

Balb/c

Balb/c

*

0

0 0

5

10

40

100

Anti-CD3 (µg/ml)

0

24

48

Time(h)

Fig. 3. DP thymocytes of NOD mice are efficiently deleted through apoptosis after T cell activation in vitro. Fetal thymic lobes were isolated from NOD, Balb/c and C57BL/6 (B6) mice, incubated for 4 days in FTOC and then treated with anti-CD3 Ab or isotype IgG as indicated. Thymocytes were stained and analyzed by FACS. (A) FACS profiles of thymocytes in NOD and C57BL/6 FTOC after 24 h of incubation with 5 mg/ml of isotype IgG or anti-CD3 Ab with or without the caspase inhibitor, zVAD-fmk (50 mm). Representative results from five independent experiments are shown; total cell numbers are shown in parentheses. The proportion of DP thymocytes in NOD, C57BL/6 and Balb/c FTOC after 24 h of incubation with (B) various doses of anti-CD3 Ab or (C) 5 mg/ml of anti-CD3 Ab for various times. Values are means G SEM of four independent experiments. *p ! 0.03, ***p ! 0.0002 as compared to anti-CD3 Ab-treated C57BL/6 FTOC.

activation marker, CD69, on DP thymocytes at 24 h after anti-CD3 Ab treatment in NOD and C57BL/6 FTOC. We found that the proportion and number of CD69C DP thymocytes in NOD FTOC was significantly higher than that in C57BL/6 FTOC (Fig. 5A, B). This result indicates that immature DP thymocytes in NOD FTOC are more efficiently activated and differentiated into CD69C DP thymocytes after anti-CD3 Ab treatment than those in C57BL/6 FTOC.

We quantified each thymocyte subset in FTOC after anti-CD3 Ab treatment and found that there was no difference in the total number of thymocytes between C57BL/6 and NOD mice (Fig. 5C). In contrast, the number of DP thymocytes in NOD FTOC was significantly lower than in C57BL/6 FTOC (Fig. 5D). In addition, we found that the numbers of both CD4C and CD8C SP thymocytes, particularly CD8C SP thymocytes, in NOD FTOC were significantly higher

17

A

B NOD

C57BL/6 2

0

8

4

2

4

8

p44 p42 1.0 1.1 2.2 2.1

C

0

p54 P-JNK

4

8

0

8

4

12

D

p54 JNK p46

**

1.0 1.3 2.7 2.3

B6 0

1 2

0

p42 p44 p42 1.0

** **

2

1 4

2.1

8

0

8

p54 P-JNK

p46 p54

JNK p46

1.0

1.5

1.0

3.5

Ratio(P-JNK/JNK)

0

H

NOD

B6

8

12

3 0h 2

*

2h

1

B6

G

8

2

F

0.9

6

Time (hour)

p44

1.0

4

B6 NOD

3

NOD 2

*

2

0

E

ERK

3

0

p46

P-ERK

B6 NOD

1.0 2.7 3.6 1.7

12

1.0 1.4 1.7 1.4

4

Ratio(P-ERK/ERK)

ERK

Ratio(P-JNK/JNK)

0 p44 P-ERK p42

Ratio(P-ERK/ERK)

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

NOD

5 0h 3

*

8h

1 B6

NOD

Fig. 4. Efficient activation of ERK and JNK in immature DP thymocytes of NOD mice after T cell activation. NOD and C57BL/6 (B6) mice were injected with 25 mg anti-CD3 Ab. Total (AeD) or sorted (EeH) DP thymocytes were isolated at the indicated times (hours) thereafter. Western blots were performed using phospho-specific Abs against ERK (P-ERK) (A, E) or JNK (P-JNK) (C, G) and the ratio of phosphorylated ERK to total ERK (B, F) or phosphorylated JNK to total JNK (D, H) was determined. p42/44 bands are ERK and p46/54 bands are JNK. Values are means G SEM (n Z 3/data point). *p ! 0.03, **p ! 0.02 as compared to C57BL/6 thymocytes.

than those in C57BL/6 FTOC (Fig. 5E, F). These results suggest that in NOD FTOC, immature DP thymocytes are more efficiently deleted by negative selection and both CD4C and CD8C SP thymocytes are more efficiently generated by positive selection after T cell activation, as compared to C57BL/6 FTOC. 3.6. Preferential differentiation of DP thymocytes of NOD mice into SP thymocytes after T cell activation To determine whether immature DP thymocytes of NOD mice differentiate more efficiently into SP thymocytes than those of C57BL/6 mice, we performed reaggregate thymic organ culture (RTOC) in the

presence of anti-CD3 Ab (0.1 mg/ml) and examined the proportion of CD4CCD8ÿ and CD4ÿCD8C SP thymocytes. At 2 days after RTOC, the proportion of CD4CCD8ÿ and CD4ÿCD8C SP thymocytes was significantly higher in NOD RTOC as compared with C57BL/6 RTOC (Fig. 6B, D). The proportion of SP thymocytes, especially CD4ÿCD8C SP thymocytes, was also significantly higher in isotype IgG control Abtreated NOD RTOC than that of C57BL/6 RTOC (Fig. 6A, C). At 4 days after RTOC, most DP thymocytes from both NOD and C57BL/6 mice had differentiated into SP thymocytes (Fig. 6EeH). These results indicate that DP thymocytes of NOD mice differentiate more efficiently into SP thymocytes after anti-CD3 Ab-induced activation than those of C57BL/6 mice.

18

A

B

#Cells

B6/isotype IgG NOD/isotypeIgG B6/anti-CD3 NOD/anti-CD3

100

101

102

103

104

Number of CD69+DP(×104)

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

B6 NOD

6

**

4

2 0

isotype

anti-DC3

CD69 80

Cell number (×104)

C

Total

60

D

B6 NOD

40

Cell number (×104)

B6 NOD

40

* 20

20 0

E

DP

0 CD4+

16

B6 NOD

F *

12

CD8+ 12

B6 NOD

*

8

8 4

4 0

isotype

anti-DC3

0

isotype

anti-DC3

Fig. 5. DP thymocytes of NOD mice efficiently differentiate into SP thymocytes by efficient positive selection after T cell activation in vitro. Fetal thymic lobes were isolated from NOD, Balb/c and C57BL/6 (B6) mice, incubated for 4 days in FTOC and then treated with anti-CD3 Ab or isotype IgG as indicated. Thymocytes were stained and analyzed by FACS. (A) Expression of CD69 on DP thymocytes after 24 h of incubation with 5 mg/ml anti-CD3 Ab or isotype IgG. DP thymocytes were gated and further analyzed for the expression of CD69. Representative results from four independent experiments are shown. The number of (B) CD69CCD4CCD8C DP, (C) total, (D) DP, (E) CD4C SP, and (F) CD8C SP thymocytes in NOD and C57BL/6 FTOC after 24 h of incubation with 5 mg/ml anti-CD3 Ab or isotype IgG. The number of thymocytes in each subset was calculated from the total thymocyte number and the proportion of thymocytes in each subset determined by FACS. Values are means G SEM of four independent experiments. *p ! 0.03, **p ! 0.001 as compared to anti-CD3 Ab-treated C57BL/6 FTOC.

3.7. Induction of diabetes in NOD.scid mice by SP thymocytes generated in NOD FTOC To determine whether SP thymocytes generated in NOD FTOC are diabetogenic in NOD mice, we isolated CD4ÿCD8C SP thymocytes from NOD FTOC at 24 h after anti-CD3 Ab (5 mg/ml) treatment and adoptively transferred them into NOD.scid mice with CD8C T celldepleted diabetogenic splenocytes prepared from acutely diabetic NOD mice. We found that 5/6 (83%) of NOD.scid mice receiving both CD4ÿCD8C SP thymocytes and CD8C T cell-depleted splenocytes became diabetic within 8 weeks of the transfusion, whereas 1/8 (12%) of the NOD.scid control mice receiving CD8C T cell-depleted diabetogenic splenocytes alone became diabetic (Fig. 7A). Most pancreatic islets from diabetic NOD.scid mice that received both CD4ÿCD8C thymocytes and CD8C T cell-depleted diabetogenic splenocytes showed severe insulitis (Fig. 7B, C). In contrast,

most pancreatic islets from NOD.scid mice that received CD8C T cell-depleted diabetogenic splenocytes alone showed mild to moderate insulitis (Fig. 7B, D). This result indicates that SP thymocytes generated by positive selection of DP thymocytes in NOD FTOC are capable of killing b cells, leading to the development of type 1 diabetes.

4. Discussion Defects in negative selection in the thymus are thought to result in the failure to delete potential b cell-reactive T cells, contributing to the development of autoimmune diabetes in the NOD mouse. We investigated this possibility by comparing the deletion profile of DP thymocytes in NOD mice with that of diabetes-resistant C57BL/6 and Balb/c mice after

19

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

A

104

2-day

E 104

103 102

isotype IgG

101 100 0 10 101 102 103 104

B anti-CD3

103 102

0.9 81

101

12 5.6

100 0 10 101 102 103 104

104

F 104

103

103

102

102

9.9 36

101 100 0 10 101 102 103 104

C

101

46 7.8

100 0 10 101 102 103 104

G

104 103 102

isotype IgG

101 100 100 101 102 103 104

102

CD8

101 100 100 101 102 103 104

74

15

4.9 0.4 78

16

104

102

7.0 67

4.5 1.3

101

20 6.0

100 0 10 101 102 103 104

H

103

anti-CD3

4.1 7.0

103

104

D

4-day

70

24

104 103

20

26

40

14

102 101 100 100 101 102 103 104

7.0 0.6 72

20

CD4 Fig. 6. SP thymocytes are efficiently generated from DP thymocytes of NOD mice after T cell activation. Thymocytes were isolated from 1- to 2week-old C57BL/6 mice (A, B, E, F) and NOD mice (C, D, G, H), sorted, and the purity was examined by FACS. The purified DP thymocytes were used for RTOC. At 2 days (AeD) and 4 days (EeH) after RTOC, the proportion of DP and SP thymocytes was analyzed by FACS. Representative results from three independent experiments are shown.

anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway [26e29]. It is known that high and low doses of a single peptide can mimic the physiological high and low affinity/avidity interactions between TCR and peptide/ MHC complex [30]. Although anti-CD3 antibody does not exactly mimic physiological conditions, we used various doses of this antibody in vivo and in vitro to mimic different strengths of affinity/avidity interactions between the TCR and peptide/MHC complex (Figs. 1 and 3). We found that a very low dose of anti-CD3 antibody did not result in significant activation or deletion of DP thymocytes in C57BL/6 and NOD mice, whereas a high dose of anti-CD3 antibody induced strong activation and deletion of DP thymocytes in both strains of mice. In contrast, an optimized low dose of anti-CD3 antibody induced mild activation of DP thymocytes and resulted in differential activation and deletion of DP thymocytes between C57BL/6 and NOD

mice. We found that DP thymocytes of NOD mice were more efficiently deleted than those of C57BL/6 and Balb/c mice when injected with the optimized low dose of anti-CD3 antibody (25 mg). Supporting this result, it was recently reported that DP thymocytes of NOD mice are more susceptible to apoptosis than those of C57BL/6 mice in single-cell suspension culture [31]. As well, the poor proliferation of NOD thymocytes after activation with anti-CD3 Ab as compared with C57BL/6 and Balb/c thymocytes [17] was probably due to the efficient anti-CD3 Ab-induced apoptosis of NOD DP thymocytes. NOR mice are a diabetes-resistant strain of mice, although their genome is 89% homologous with NOD mice, including critical idd loci such as idd1. We examined anti-CD3 antibody-induced activation and deletion of DP thymocytes in NOR mice and found that there were no differences in anti-CD3 antibody-induced activation and deletion of DP thymocytes between NOR and NOD mice (data not shown). Therefore, the

20

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

Incidence of diabetes ( )

A

C

100 CD8+-thymocytes 80

Control

60 40 20 0 1

2

3

4

5

6

7

8

Time (week)

Insulitis ( )

B

D

100 80

4

60

3 40

2 1

20

0 0

CD8+-Thymocytes

Control

Fig. 7. CD8 SP thymocytes generated in NOD FTOC after T cell activation induce insulitis and diabetes in NOD.scid mice. (A) CD8C SP thymocytes (5 ! 106 cells) sorted from NOD FTOC were transferred into NOD.scid mice (n Z 6) along with CD8C T cell-depleted diabetogenic splenocytes (5 ! 106 cells) or CD8C T cell-depleted diabetogenic splenocytes alone were transferred into NOD.scid mice as a control (n Z 8). The cumulative incidence of diabetes in these mice was determined. (B) The pancreas was removed from NOD.scid recipients at the onset of diabetes or 8 weeks after adoptive transfer, sectioned and histologically examined. The degree of insulitis was analyzed as described in Section 2. Representative islets from NOD.scid recipients of (C) both CD8C SP thymocytes and CD8C T cell-depleted diabetogenic splenocytes or (D) CD8C T cell-depleted diabetogenic splenocytes alone (!200). C

development of b cell-specific autoreactive T cells in NOR mice might be the same as in NOD mice, but the induction of peripheral tolerance might be different between NOR and NOD mice. It has been suggested that TCR-specific peptide- or anti-CD3 Ab-induced thymocyte deletion is affected by activated peripheral T cells [23e25,32]. To exclude any effects of activated peripheral T cells on the deletion of DP thymocytes, we used FTOC, which maintains sites for positive and negative selection. Consistent with our in vivo results, we found that DP thymocytes of NOD mice were more efficiently deleted than those of C57BL/6 mice at 24 h after a low dose of anti-CD3 Ab (5 mg/ml) treatment in FTOC. Since peripheral CD4C T cells of NOD mice are less sensitive to antiCD3 Ab-induced activation and proliferation as compared with those of C57BL/6 mice [33,34] and DP thymocytes of NOD mice are more resistant to glucocorticoid-induced apoptosis than those of C57BL/6 and Balb/c mice [35], glucocorticoids induced by peripheral T cell activation may not be a factor for the efficient deletion of NOD DP thymocytes. Supporting this, treatment with a glucocorticoid antagonist, RU486, could not completely recover the number of DP thymocytes deleted by anti-CD3 Ab or TCR-specific

peptide [23e25]. In addition, TCR-specific peptide induced the deletion of DP thymocytes without peripheral T cell activation [36]. Therefore, the efficient deletion of DP thymocytes in NOD mice may not be due to the effects of peripheral T cell activation, but due to intrinsic characteristics of NOD thymocytes. We then hypothesized that immature CD69ÿCD3low DP thymocytes of NOD mice may have a lower activation threshold and be more susceptible to TCRinduced activation than those of C57BL/6 mice. This may result in both faster deletion of some cells by negative selection and more efficient differentiation of others within the NOD DP thymocyte population by positive selection, depending on the strength of the activation signal. We examined this possibility by measuring the expression of the activation marker, CD69, on DP thymocytes in NOD and C57BL/6 FTOC after anti-CD3 Ab treatment. We found that the proportion and number of CD69C DP thymocytes was significantly higher in NOD FTOC as compared with C57BL/6 FTOC. It has been shown that CD69C DP thymocytes are generated during positive selection of immature DP thymocytes [37e40] and that overexpression of CD69 in the thymus results in a dramatic increase in CD4C and CD8C SP thymocytes, due to an

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

increase in the efficiency of positive selection [39]. Consistent with this, we found that SP thymocytes, especially CD4ÿCD8C SP thymocytes, were more efficiently differentiated from DP thymocytes in NOD RTOC as compared to those of C57BL/6 RTOC. Taken together, our results suggest that the DP thymocytes of NOD mice are more sensitive to positive selection, resulting in efficient generation of CD69C DP thymocytes, which then can further differentiate into CD4C and CD8C SP thymocytes. It is known that mitogen activated protein kinases (MAPKs) such as ERK, JNK, and p38 MAPK are activated after TCR-mediated T cell activation [2,3]. ERK activation is required for differentiation of DN thymocytes into DP thymocytes [41], positive selection of DP thymocytes [41e45], and negative selection of DP thymocytes [46,47]. JNK and p38 MAPK activation is involved in the negative selection of DP thymocytes [48,49]. We found that phosphorylation of ERK and JNK in NOD DP thymocytes occurred sooner and at a higher level than in C57BL/6 DP thymocytes after T cell activation. The phosphorylation of ERK occurred sooner than the phosphorylation of JNK, probably because JNK requires costimulatory signals for activation [48]. It appears that the lower activation threshold in immature DP thymocytes of NOD mice, as compared with C57BL/6 DP thymocytes, results in more efficient phosphorylation of JNK and ERK after activation, which would promote both negative and positive selection, leading to efficient differentiation into CD69CCD3high DP thymocytes. In our experimental results, the number of CD4CCD8ÿ and CD4ÿCD8C SP thymocytes in NOD FTOC was significantly higher than in C57BL/6 FTOC, in contrast with our in vivo results, which showed no significant differences in the number of SP thymocytes between NOD and C57BL/6 mice after anti-CD3 Ab injection. This difference between in vivo and in vitro results is probably due to the continuous release of CD4C and CD8C SP thymocytes into the periphery in vivo, whereas these cells accumulate in vitro. These results clearly support our hypothesis that SP thymocytes are more efficiently differentiated from DP thymocytes in NOD mice as compared with C57BL/6 mice. We found that these differentiated SP thymocytes, particularly CD4ÿCD8C thymocytes, have diabetogenic potential. It was recently shown that semi-mature HSACCD4CCD8ÿ SP thymocytes of NOD mice are resistant to negative selection in the medulla of the thymus as compared to those of C57BL/6 mice after anti-TCRb monoclonal Ab treatment [50]. We examined the proportion of HSACCD4CCD8ÿ SP thymocytes in NOD and C57BL/6 mice at different times after the administration of anti-CD3 Ab (25 mg). We found that the proportion of semi-mature HSACCD4C SP thymocytes of NOD mice was higher than C57BL/6 mice at

21

72 h after treatment (data not shown), consistent with the previous finding [50]. Therefore, it may be possible that a portion of the positively selected SP thymocytes that developed from the efficiently activated CD69CCD3high DP thymocytes, possibly in the corticomedullary junction, may escape deletion due to defects in negative selection in the medulla of thymus in NOD mice. The synergistic effect of efficient positive selection of immature DP thymocytes in the cortex and defects in negative selection of semi-mature SP thymocytes in the medulla may augment potentially autoreactive diabetogenic T cells in NOD mice. Our in vivo and in vitro data consistently show that there are anomalies in the negative and positive selection of immature CD4CCD8C DP thymocytes in NOD mice. Affinity and avidity interactions between the TCR and peptide/MHC complex are believed to determine the fate of CD4CCD8C DP thymocytes through positive and negative selection in the thymus [30,46,51]. Strongly activated immature DP thymocytes are rapidly deleted by negative selection, whereas weakly activated DP thymocytes differentiate into SP thymocytes by positive selection. Thymocytes with extremely weak affinity between the TCR and peptide/MHC complex undergo programmed cell death. We suggest that the activation threshold for DP thymocytes in NOD mice is much lower than that of C57BL/6 mice, allowing a very weak TCRmediated signal to trigger positive selection in NOD DP thymocytes. For example, a very weak TCR-mediated signal may induce immature DP thymocytes that express b cell autoantigen-specific TCRs to efficiently differentiate into SP thymocytes in NOD mice. In contrast, the same weak TCR-mediated signal may not result in positive selection of immature DP thymocytes that express b cell autoantigen-specific TCRs in C57BL/6 mice, due to their higher activation threshold as compared to NOD mice, and thus these potentially autoreactive cells may be deleted through programmed cell death. In conclusion, immature NOD CD4CCD8C DP thymocytes have a low activation threshold, resulting in efficient phosphorylation of ERK and JNK and differentiation into CD69CCD3high DP thymocytes. Activated DP thymocytes expressing b cell autoantigen-specific TCRs efficiently differentiate into potential diabetogenic CD4C and CD8C SP thymocytes through inordinate positive selection in the corticomedullary junction. Positively selected SP thymocytes may escape deletion due to defects in negative selection in the medulla of the thymus, resulting in the augmentation of autoreactive diabetogenic T cells in the thymus of NOD mice.

Acknowledgements We are grateful to Laurie Robertson for assistance with flow cytometry and Dr. Ann L. Kyle for editorial

22

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

assistance. This work was supported by a grant from the Canadian Institutes of Health Research (MA 9584). J.W.Y. holds a Canada Research Chair in Diabetes.

References [1] Goldrath AW, Bevan MJ. Selecting and maintaining a diverse T-cell repertoire. Nature 1999;402:255–62. [2] Sebzda E, Mariathasan S, Ohteki T, Jones R, Bachmann MF, Ohashi PS. Selection of the T cell repertoire. Annu Rev Immunol 1999;17:829–74. [3] Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu Rev Immunol 2003;21:139–76. [4] Abai AM, Hobart PM, Barnhart KM. Insulin delivery with plasmid DNA. Hum Gene Ther 1999;10:2637–49. [5] Yoon JW, Jun HS. Insulin-dependent diabetes mellitus. In: Roitt IM, Delves PJ editors. Encyclopedia of immunology. London: Academic Press; 1998, p. 1390–8. [6] Tisch R, McDevitt H. Insulin-dependent diabetes mellitus. Cell 1996;85:291–7. [7] Benoist C, Mathis D. Cell death mediators in autoimmune diabetesdno shortage of suspects. Cell 1997;89:1–3. [8] Jun HS, Yoon CS, Zbytnuik L, van Rooijen N, Yoon JW. The role of macrophages in T cell-mediated autoimmune diabetes in nonobese diabetic mice. J Exp Med 1999;189:347–58. [9] Yoon JW, Yoon CS, Lim HW, Huang QQ, Kang Y, Pyun KH, et al. Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells. Science 1999;284:1183–7. [10] Cameron MJ, Arreaza GA, Zucker P, Chensue SW, Strieter RM, Chakrabarti S, et al. IL-4 prevents insulitis and insulin-dependent diabetes mellitus in nonobese diabetic mice by potentiation of regulatory T helper-2 cell function. J Immunol 1997;159:4686–92. [11] Falcone M, Yeung B, Tucker L, Rodriguez E, Sarvetnick N. A defect in interleukin 12-induced activation and interferon gamma secretion of peripheral natural killer T cells in nonobese diabetic mice suggests new pathogenic mechanisms for insulindependent diabetes mellitus. J Exp Med 1999;190:963–72. [12] Piganelli JD, Martin T, Haskins K. Splenic macrophages from the NOD mouse are defective in the ability to present antigen. Diabetes 1998;47:1212–8. [13] Serreze DV, Gaskins HR, Leiter EH. Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J Immunol 1993;150:2534–43. [14] Colucci F, Bergman ML, Penha-Goncalves C, Cilio CM, Holmberg D. Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region. Proc Natl Acad Sci U S A 1997;94:8670–4. [15] Wu AJ, Hua H, Munson SH, McDevitt HO. Tumor necrosis factor-alpha regulation of CD4CCD25C T cell levels in NOD mice. Proc Natl Acad Sci U S A 2002;99:12287–92. [16] Kreuwel HT, Biggs JA, Pilip IM, Pamer EG, Lo D, Sherman LA. Defective CD8C T cell peripheral tolerance in nonobese diabetic mice. J Immunol 2001;167:1112–7. [17] Zipris D, Lazarus AH, Crow AR, Hadzija M, Delovitch TL. Defective thymic T cell activation by concanavalin A and antiCD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis. J Immunol 1991;146:3763–71. [18] Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 1994;22:5506–7. [19] Lee HC, Kim SJ, Kim KS, Shin HC, Yoon JW. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue. Nature 2000;408:483–8.

[20] Yoon JW, Rodrigues MM, Currier C, Notkins AL. Long-term complications of virus-induced diabetes mellitus in mice. Nature 1982;296:566–9. [21] Sidman CL, Marshall JD, Von Boehmer H. Transgenic T cell receptor interactions in the lymphoproliferative and autoimmune syndromes of lpr and gld mutant mice. Eur J Immunol 1992;22:499–504. [22] Singer GG, Abbas AK. The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1994;1:365–71. [23] Martin S, Bevan MJ. Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection. Eur J Immunol 1997;27:2726–36. [24] Jondal M, Okret S, McConkey D. Killing of immature CD4C CD8C thymocytes in vivo by anti-CD3 or 5#-(N-ethyl)-carboxamide adenosine is blocked by glucocorticoid receptor antagonist RU-486. Eur J Immunol 1993;23:1246–50. [25] Xue Y, Murdjeva M, Okret S, McConkey D, Kiuossis D, Jondal M. Inhibition of I-Ad-, but not Db-restricted peptideinduced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice. Eur J Immunol 1996;26:428–34. [26] Shi YF, Bissonnette RP, Parfrey N, Szalay M, Kubo RT, Green DR. In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes. J Immunol 1991;146:3340–6. [27] Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JJ. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 1989;337:181–4. [28] Jenkins MK, Schwartz RH, Pardoll DM. Effects of cyclosporine A on T cell development and clonal deletion. Science 1988;241:1655–8. [29] Migita K, Eguchi K, Kawabe Y, Mizokami A, Tsukada T, Nagataki S. Prevention of anti-CD3 monoclonal antibodyinduced thymic apoptosis by protein tyrosine kinase inhibitors. J Immunol 1994;153:3457–65. [30] Sebzda E, Wallace VA, Mayer J, Yeung RS, Mak TW, Ohashi PS. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science 1994;263:1615–8. [31] Villunger A, Marsden VS, Strasser A. Efficient T cell receptormediated apoptosis in nonobese diabetic mouse thymocytes. Nat Immunol 2003;4:717. author reply 718. [32] Ashwell JD, Lu FW, Vacchio MS. Glucocorticoids in T cell development and function. Annu Rev Immunol 2000;18:309–45. [33] Dahlen E, Hedlund G, Dawe K. Low CD86 expression in the nonobese diabetic mouse results in the impairment of both T cell activation and CTLA-4 up-regulation. J Immunol 2000;164:2444– 56. [34] Noorchashm H, Moore DJ, Noto LE, Noorchashm N, Reed AJ, Reed AL, et al. Impaired CD4 T cell activation due to reliance upon B cell-mediated costimulation in nonobese diabetic (NOD) mice. J Immunol 2000;165:4685–96. [35] Martins TC, Aguas AP. Involvement of c-myc in the resistance of non-obese diabetic mice to glucocorticoid-induced apoptosis. Immunology 1998;95:377–82. [36] Zhan Y, Purton JF, Godfrey DI, Cole TJ, Heath WR, Lew AM. Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation. Proc Natl Acad Sci U S A 2003;100:1197–202. [37] Swat W, Dessing M, von Boehmer H, Kisielow P. CD69 expression during selection and maturation of CD4C 8C thymocytes. Eur J Immunol 1993;23:739–46. [38] Hare KJ, Jenkinson EJ, Anderson G. CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection. J Immunol 1999;162:3978–83. [39] Nakayama T, Kasprowicz DJ, Yamashita M, Schubert LA, Gillard G, Kimura M, et al. The generation of mature,

H. Kwon et al. / Journal of Autoimmunity 24 (2005) 11e23

[40]

[41]

[42] [43]

[44]

[45]

single-positive thymocytes in vivo is dysregulated by CD69 blockade or overexpression. J Immunol 2002;168:87–94. Ohashi PS, Pircher H, Burki K, Zinkernagel RM, Hengartner H. Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities. Nature 1990;346:861–3. Alberola-Ila J, Forbush KA, Seger R, Krebs EG, Perlmutter RM. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 1995;373:620–3. O’Shea CC, Crompton T, Rosewell IR, Hayday AC, Owen MJ. Raf regulates positive selection. Eur J Immunol 1996;26:2350–5. Alberola-Ila J, Hogquist KA, Swan KA, Bevan MJ, Perlmutter RM. Positive and negative selection invoke distinct signaling pathways. J Exp Med 1996;184:9–18. Swat W, Shinkai Y, Cheng HL, Davidson L, Alt FW. Activated Ras signals differentiation and expansion of CD4C 8C thymocytes. Proc Natl Acad Sci U S A 1996;93:4683–7. Werlen G, Hausmann B, Palmer E. A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity. Nature 2000;406:422–6.

23

[46] Mariathasan S, Zakarian A, Bouchard D, Michie AM, ZunigaPflucker JC, Ohashi PS. Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection. J Immunol 2001;167:4966–73. [47] Bommhardt U, Scheuring Y, Bickel C, Zamoyska R, Hunig T. MEK activity regulates negative selection of immature CD4CCD8C thymocytes. J Immunol 2000;164:2326–37. [48] Sugawara T, Moriguchi T, Nishida E, Takahama Y. Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 1998;9:565–74. [49] Rincon M, Whitmarsh A, Yang DD, Weiss L, Derijard B, Jayaraj P, et al. The JNK pathway regulates the in vivo deletion of immature CD4(C)CD8(C) thymocytes. J Exp Med 1998;188:1817–30. [50] Kishimoto H, Sprent J. A defect in central tolerance in NOD mice. Nat Immunol 2001;2:1025–31. [51] Stefanski HE, Mayerova D, Jameson SC, Hogquist KA. A low affinity TCR ligand restores positive selection of CD8C T cells in vivo. J Immunol 2001;166:6602–7.