Eur. J. Immunol. 1990. 20: 1475-1482
Hiroyuki Yuuki, Yasunobu Yoshikai, Kenji Kishihara, Akinori Iwasaki, Goto Matsuzaki, Hiroaki Takimoto and Kikuo Nomoto Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka
Self-reactive T cells in aged nude mice
Clonal anergy in self-reactive a l p T cells is abrogated by heat-shock protein-reactive y/6 T cells in aged athymic nude mice* Although T cells proliferate and differentiate primarily in the thymus, athymic nude mice contain an appreciable level of T cell receptor a/fi and y/6 T cells, suggesting the existence of the extrathymic pathway in the development of bothT cells. Recent studies with nude mice indicate that clonal deletion of self-reactiveT cells does not occur extrathymically. In the present study, we have investigated the responsiveness of self-reactive T cells differentiating along an extrathymic pathway in aged BALB/c (H-2d, Mls-lb2", I-E+, 7-8 month old) nude mice. Consistent with recent reports,Tcells bearing Vp3 or Vpll, which are important for recognizing proteins encoded by the M I s - or ~ ~the I-E allele, respectively, are readily detected in age nude mice. The Vp3- or Vpll-bearing Tcells, however, do not proliferate in response to staphylococcal enterotoxin A which specifically stimulates Vp3- or Vpll-bearing T cells. When exogenous recombinant interleukin 2 was added to the culture, the Vp3-bearing T cells in aged nude mice significantly proliferated in response to staphylococcal enterotoxin A. Aged nude mice also contained a substantial level of y/6 Tcells which account for 15.6% of all Thy-1.2+ cells.The y/6 Tcells proliferated and produced a significant level of interleukin 2 in response to the 65-kDa mycobacterial heat-shock protein, which is highly homologous to its eukaryotic counterpart. These results suggest that unresponsiveness of self-reactive Tcells may be reversed by Tcells responding to stress proteins expressed by the invading microbes and/or the stressed autologous cells.
1 Introduction Tcell precursors derived from fetal liver or BM proliferate and differentiated in the thymus. During Tcell differentiation in the thymus, thymocytes with a broad range of affinities for self MHC are positively selected (positive selection) and thymocytes with high affinities for self antigens are rendered tolerant (negative selection) (reviewed in [l,21). The selection is carried out on the basis of expression of the TcR. Two types of TcR have been identified; aTcR a/fiheterodimer which recognizes nominal antigen in the context of self-MHC gene products and a TcR y/6 heterodimer which has been reported to recognize MHC molecules and/or mycobacterial antigens including the 65-kDa heat-shock protein (hsp) [3-81. Recently, it has been reported that Tcells bearing Vpll or Vpl7a capable of recognizing I-E molecules are eliminated from mature T cell repertoire of mice carrying I-E alleles
[I 83781
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This work was supported by grants to Y. Yoshikai from the Ministry of Education, Science and Culture (62480167, 01015081) and from Special Coordination Funds of the Science and Technology Agency of the Japanese Government.This work was also received financial support from the UNDPNorld B a n k N H O Special Programme for Research and Training in Tropical Disease (TDR).
Correspondence: Yasunobu Yoshikai, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812, Japan Abbreviations: hsp: Heat-schock protein SEA: Staphylococcal enterotoxin A
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
[9-111. TcR using Vp3 or Vp6/8.1 are also found to be specific for the products of MIS-2" or Mls-la and mice expressing Mls-la or Mls-2" eliminate Vp6/8.1- or Vp3bearing T cells in their pool of mature T cells, respectively [12-17].Thus, self tolerance is due mainly to the deletion of Tcells bearing TcR capable of recognizing the self antigens in the thymus. However, several lines of evidence suggest that in addition the self-reactive T cells are rendered tolerant peripherally by the induction of clonal anergy characterized by preservation of responsiveness to exogenous I L 2 but lack of IL2 production in response to self antigens [18-211. Although T cells proliferate and differentiate primarily in the thymus, several studies with athymic nude mice indicate that an extrathymic pathway exists in T cell development [22-261. Congenitally athymic nude mide, especially aged ones contained an appreciable level of a/F and y / 6 Tcells [27], suggesting that bothTcells do differentiate outside the thymus, albeit slowly. Recent studies with nude mice indicate that clonal deletion of self-reactive Tcells does not occur extrathymically [28,29]. However, whether the selfreactive T cells in nude mice respond normally to self antigens or are rendered tolerant by the induction of clonal anergy is not yet known. In the present study, we have investigated the fate of self-reactiveTcells and their responsiveness in aged athymic BALB/c (H-2d, M1s-lh2", I-E+) nude mice. Our results indicate that the self-reactive a/fi T cells differentiating outside the thymus are rendered tolerant by the induction of clonal anergy, whereas y/6 T cells in aged nude mice proliferate and produce a significant level of IL2 in response to the 65-kDa mycobacterial hsp. The implication of these findings for the development of autoimmune disease in aged nude mice is discussed. 0014-2980/90/0707-1475$3.50+ .25/0
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2 Materials and methods 2.1 Mice Congenitally athymic nude mice (ndnu, 5 weeks old) of a BALB/c background were obtained from Clea Japan Inc. (Tokyo) and were bred under specific pathogen-free conditions at the animal center of Kyushu University. Males (7-9 months old) were used for experiments.
Eur. J. Immunol. 1990. 20: 1475-1482 Con A (3 p.g/ml) (Sigma, St. Louis, MO) in the presence or not of 1 U/ml rIL2 (Takeda Chemical Industries, Osaka, Japan) at 37°C in a 5% COz incubator in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% FCS. Three days later, live cells were harvested and expanded for 3 days with 1U/ml rIL2. In some experiments, the blast cells were stained with each mAb without being cultured for 3 days with rIL2. Blasts were stained with FITCconjugated anti-hamster IgG or FITC-conjugated anti-rat IgG after treatment with KJ25,44-22-1, K T l l or 7D4 mAb and analyzed by FCM.
2.2 Antibodies The following antibodies were used for immunofluorescence staining: anti-CD3 E chain mAb (145-2C11; provided by Dr. J. A. Bluestone), anti-TcR a/@mAb (H57-597; provided by Dr. R. Kubo), anti-Vg3 mAb (KJ25; provided by Dr. J. W. Kappler), anti-Vg5 mAb (MR9-4; provided by Dr. 0. Kanagawa), anti-Vg6 mAb (44-22-1; provided by Dr. H. Hengartner), anti-Vg8 mAb (F23.1, KJ16 and F23.2; provided by Dr. J. W. Kappler), anti-Vp9 mAb (MR10-2; provided by Dr. 0. Kanagawa), anti-Vgll mAb (KT11; provided by Dr. K. Tomonari), anti-Vgl3 mAb (MR12-4; provided by Dr. 0. Kanagawa), anti-IL2R mAb (7D4), or biotin-conjugated anti-Thy-1.2 mAb (Becton Dickinson, Mountain View, CA), FITC-conjugated goat anti-hamster IgG, FITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rat IgG (Tago Inc., Burlingame, CA). 2.3 Staphylococcal enterotoxin A (SEA)
SEAwas isolated and prepared by the methods of Oda [30]. The culture SN of Staphylococcus aureus 13N-2909, provided by theTokyo Metropolitan Research of Public Health through the courtesy of Dr. H . Igarashi, was centrifuged and applied to a SP-Sephadex C-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) column chromatograph. The SEA was eluted with a pH gradient of pH 5 to pH 7.5 and the eluate was concentrated on an Amicon UM 10 membrane (Amicon Division, W. R. Grace and Co., MA). The concentrated SEA fraction was purified further by gel filtration on Sephadex G-75 (Pharmacia). The purified sample was shown to consist of a single band by SDSPAGE. SEA, stored as lyophilized powder at 4"C, was dissolved in saline.
2.4 FCM analysis Spleen and LN cells were taken from 7-9-month-old BALB/c nude mice. A single-cell suspension was stained with each antibody and was analyzed on a FACScan flow cytometer (Becton Dickinson). The percentage of positive cells was determined by integration from profiles based on 3 x lo4 cells.
2.5 In vitro culture of T cells with Con A or SEA Spleen cells were stimulated with SEA or Con A as described by Takimoto et al. [31]. Briefly, spleen cells (1 x 106/ml) were incubated with SEA (0.1 pg/ml) or
2.6 In vitro culture of T cells with PPD or hsp
The LN cells from aged nude mice were cultured with 30 pg/ml PPD derived from Mycobacterium tuberculosis (H37Ra; Kainosu Inc., Tokyo, Japan), 25 pg/ml recombinant 65-kDa hsp derived from Mycobacterium bovis (kindly provided by Drs. R.Van der Zee and J. D. A.Van Embden; [32]), or 25 pg/ml trypsin-digested hsp [digested with L- 1-tosylamido-2-pheny lethyl-chloromethyl ketonetreated immobilized trypsin (Sigma)] at 37°C in a 5% C02 incubator for 72 h in RPMI 1640 (Gibco) supplemented with 10% FCS. 2.7 IL 2 production of nude mice T cells in response to hSP
The ILZdependent CTLL-2 cells (1 x 104) were incubated with a dilution of SN from cultures in which aged nude mice Tcells were stimulated with PPD or hsp in 0.2 ml medium at 37°C for 48 h, and then pulsed with 1 pCi = 37 kBq [3H]thymidine. After another 6 h of incubation the cells were harvested. 2.8 Titer of anti-DNA antibody in serum
Anti-DNA titer in the serum from aged nude mice was determined by ELISA. Briefly, calf thymus DNA (type I, Sigma) diluted to a concentration of 10 pg/ml with coating buffer (15 mM Na2C03, 34 mM NaHC03, 0.2 mM NaN3, pH 9.2) was added to each well of flat-bottom microtiter plates (Sanko Pure Chemical Co., Tokyo) and kept overnight at 4°C. Nonspecific binding sites were saturated by addition of 200 pl of PBS containing 1% BSA (fraction V; Boehringer Mannheim, Mannheim, FRG). After a 1-h incubation at room temperature, the plate was washed three times with PBS containing 0.05% Tween 20 (PBSTween). For each group of mice, sera were pooled in equal volume and serially diluted with PBS-Tween.Then 100 p1 of diluted serum was added into each well of antigen-coated plates and incubated at 37°C for 1h. After washing with PBS-Tween, 100 p1 of alkaline phosphatase-conjugated goat anti-mouse IgG (Tago) diluted to 1/1OOO with PBSTween was added to each well and incubated at 37°C for 1 h. After washing, 1 mg of p-nitrophenyl phosphate (Wako, Osaka, Japan), solubilized in 10% diethanolamine solution (pH 9.8) per 1 ml, was added to the well as a substrate and incubated for 5 min at room temperature. After stopping the enzyme reaction with 3 N NaOH, the absorbance at 405 nm was determined with a ELISA spectrophotometer.
Self-reactive T cells in aged nude mice
Eur. J. Immunol. 1990. 20: 1475-1482
3 Results
3.2 TcR Vp repertoire of T cells differentiating along an extrathymic pathway
3.1 Development of both TcRdp and y/6 T cells along an extrathymic pathway To confirm that T cells develop along an extrathymic pathway, we determined Tcell surface markers on the LN cells of young (8- to 10-weeks old) and aged (8- to 10-months old) BALB/c (H-2d, M l ~ l ~ nude 2 ~ ) mice by FCM analysis. In the LN of young nude mice, few CD3+CD4+CD8- or CD3+CD4-CD8+ cells were detected, whereas a substantial number of CD3+CD4-CD8cells (data not shown) and of CD4 or CD8 single-positive T cells were detected in those aged nude mice in which CD8+ cells predominated over CD4+ cells (Fig. 1). Thus, a substantial number of phenotypically mature T cells can develop along an extrathymic pathway. Expression of TcR on the T cells in aged nude mice was further analyzed by double staining with anti-Thy-1.2 mAb and anti-TcR a$ mAb (H57-597) or anti-TcRy/6 mAb (UC7-13D5). As shown in Fig. 2b, 78.5% of Thy-l+ cells were TcRa/b+ in the L N of aged nude mice, whereas y/6 Tcells account for 15.6% of Thy-l+ cells in these mice.Thus, both a@and y/8 T cells can develop outside the thymus, albeit slowly. Consistent with report by Lawetzky and Hunig [24], the intensity of TcR/CD3 expression on T cells in aged nude mice was a little lower than that in euthymic mice (Fig. 2a) but higher than that in CD4+CD8+ thymocytes (data not shown).
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To examine the TcR repertoire of the a / p T cells in aged nude mice, the LN cells were double-stained with antiThy-1 mAb and various anti-Vp mAb including anti-Vp3 mAb (KJ25), anti-Vp5 mAb (MR9-4), anti-Vg6 mAb (44-22-1), anti-Vp8 mAb (F23.1, KJ16 and F23.2), anti-Vp9
(a)
Ib)
CD3
BALB/c
aged nulnu
I
CDS
Figure 1. Two-color FCM analysis of CD4 vs. CD8 expression on LN cells from aged (28 week) nude mice. (a) nu/+, 8 weeks; (b) nu/nu, 8 weeks: (c) nulnu, 28 weeks. LN cells were stained with PE-anti-CD4 mAb and FITC-anti-CD8 mAb.
Figure 2. Expression of CD3, TcRyl6, TcR a.@ or Vp on LN cells from aged nude mice (7-9 months old) and normal euthymic mice (2-3 months old). (a) Density of CD3 expression on total LN cells and density of V136expression on Thy-l.2+ LN cells from an aged nude mice (solid line) and normal euthymic mice (dotted line); (b) quantitation of TcR ylb-. TcR dp- or Vg-positive cells in Thy-1.2+ LN cells. LN cells were stained with each mAb and simultaneously with anti-Thy-1.2 mAb.Thy-1.2- cells are gated out and shown are histograms of TcRyl6,TcR a/p or Vg expression of Thy-1.2+ cells. The frequency of positive cells is calculated as the percentage of cells staining with each mAb followed by FITC-conjugated goat anti-hamster, anti-rat or anti-mouse IgG as the secondary reagent, minus the percentage staining with second reagent alone. Thirty thousand cells per sample were analyzed on a FACScan flow cytometer.
Fluorescence
intensity
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Table 1. Quantitation of CD3-,TcR a@- and TcR Vp-positive cells in LN from BALB/c nude mice at 7-9 months of age and from normal BALB/c micea) Nude mouse no.
YO CD3+
dB
vp3
v!35
Fraction of Thy-1.2+ cells (YO) Vp6 Vp8.1 Vg8.2 Vp8.3
Vp9
Vpll
ND 5.1 7.1 2.6 14.0 11.3 ND 7.8 1 21.6 74.0 3.4 ND 5.2 8.1 13.0 ND 13.9 78.5 10.5 2 30.3 6.3 ND 8.7 8.2 15.6 13.2 8.1 ND 3 20.7 73.5 2.5 8.7 2.0 3.8 19.0 1.6 9.9 5.4 4 22.6 85.5 4.3 10.1 2.6 5.6 14.2 2.4 10.8 8.4 5 20.3 69.1 MeanfSDb) 23.1f4.1 76.1k6.2 8.0f1.8 2.0k0.6 11.8k1.7 5.7k2.5 15.222.3 8.0f1.9 2.3f0.4 4.3f1.5 BALB/cC)
76.3f2.9 98.5f0.2 0.3f0.2 0.2kO.l 9 . 8 f 0 . 7 6.7k0.4 13.1k0.410.2+1.1 0 . 4 f 0 . 1 O.lkO.1
Vp13
ND ND ND 1.1 2.5 1.8f1.0
O.lfO.l
a) LN cells from five individual aged BALB/c nude mice (7-9 months of age) were stained and analyzed as described in legends of Fig. 1. The frequency of Tcells expressing the CD3 E chain is calculated as a percentage of total LN cells and the frequency of cells expressing other markers is calculated as a perccntage of Thy-1.2+ cells. The typical data from one experiment are shown in Fig. 1. b) Percentages represent the mean value (kSD) of five individual animals. c) Percentage represent the mean value (k SD) of three individual animals.
mAb (MR10-2), anti-Vgll mAb (KT11) or anti-Vgl3 mAb (MR12-4). A typical fluorescence histogram is shown in Fig. 2 and the results of five individual mice are summarized in Table 1. A direct link has been found between the reactivity to the products of the M l ~ - or 2 ~the I-E allele and TcR Vg3 or VgSNg11 on T cells, respectively. Consistent with the earlier findings, BALB/c euthymic mice have deleted Vp3-,VgS- and Vgll-bearingTcells in their matureT cell pool as a result of clonal deletion. By contrast, an appreciable number of Vg3-,Vg-5- or Vgll-bearing T cells were detected in the LN of aged nude mice. These results confirm that T cells differentiating along an extrathymic pathway have not undergone negative selection.
3.3 Functional analysis of self-reactive T cells in nude mice
To investigate the functional aspect of the self-reactive Tcells differentiating in aged nude mice, we tried to activate the Vp3- or Vpll-bearing T cells by SEA, a "superantigen" which specifically stimulates Tcells bearing Vg3 or Vpll irrespective of the a chain expressed by theseT cells. Stimulation of Tcells with Con A served as positive control to demonstrate general responsiveness of the population. As shown in Fig. 3, Vg3:bearing T cells and Vpll-bearing T cells significantly proliferated in response to SEA in C57BL/6 mice carrying M l ~ - 2and ~ I-E-.
Fluorescence
intensity
Figure 3. FCM analysisof Con A (a) or SEA (b)-activated blast cells. Spleen cells (1 X 106/ml)were incubated in 10 ml medium with SEA (0.1 kg/ml) or Con A (3 pglml) in a humidified atmosphere with 5% CO2 at 37"C.Three days later, live cells were harvested and expanded for 3 days in the presence of 1 U/ml rIL2 to increase the intensity of TcR Vp expression on the blasts. Blasts were stained with FITC-conjugated anti-hamster IgG or FITC-conjugated anti-rat IgG after treatment with KJ25,44-22-1or K T l l mAb and analyzed on a FACScan .
Self-reactive T cells in aged nude mice
Eur. J. Immunol. 1990. 20: 1475-1482 after before
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Similarly, the number of Vp3-bearing T cells significantly increased after stimulation with SEA in AKR/J mice carrying Mls-2" as compared with stimulation with Con A [31]. In euthymic BALB/c mice, however, noVp3-bearingT cells proliferated in response to SEA because of toleranceinduced deletion of self antigens. The low but significant levels of proliferation observed inVgll-bearingTcells in the BALB/c mice may suggest that not all Vgll-bearing Tcells recognize I-E molecules. On the other hand, in spite of the presence of Vg3- and Vgll-bearing T cells in aged nude mice, these T cells showed little, if any, proliferation in response to SEA. Thus, the self-reactive T cells maturing extrathymically may be rendered tolerant to self antigens. To elucidate whether the unresponsiveness of the Tcells in aged nude mice is due to a defect in the function of the TcR/CD3 complex in theTcells or to an induction of clonal anergy,we investigated the responsiveness of Vp3-bearingT cells to SEA in the presence of exogenous rIL2. Approximately 45% of nude spleen cells expressed IL2R after stimulation into SEA and I L 2 (Fig. 4). The intensity of Vp3-expression was relatively low because the blasts were obtained after stimulation with SEA and IL2 but without an additional culture with IL 2. However, theVg3-bearingT cells in aged nude mice significantly proliferated in response to SEA in the presence of exogenous rIL2 as compared with those in euthymic BALBk mice (Fig. 4).
3.4 Increased number of TcR $6 T cells in aged nude mice bred under conventional conditions To investigate whether $6 Tcells in LN cells in nude mice are activated in vivo by environmental antigens, we examined the number of y/6 Tcells in aged nude mice bred under conventional conditions for several weeks instead of pathogen-free conditions. As shown in Fig. 5, the percentage of y/6 T cells in the LN cells significantly increased from 15.6% in nude mice bred under pathogen-free conditions to 38.4% in those bred under conventional conditions.
Figure 4. FCM analysis of normal BALBlc spleen cells (a) or spleen cells from 7-9-monthold nude mice (b). For stimulation spleen cells (1 x 10h/ml) were incubated in 10ml medium with SEA (0.1 pglml) plus IL2 (1 Ulml) in a humidified atmosphere with 5% CO2 at 37°C for 3 days.Then, live cells were harvested. Blasts were stained with KJ25, 44-22-1 or 7D4 mAb and simultaneous1 with anti-Thy-1.2 mAb and analyzed on a FACScan. Thy1.2- cells were gated out. Ibl I
Fluorescence
intensity
Figure 5. Expression of TcR y16 on LN cells from aged nude mice bred under pathogen-free (a) or conventional conditions (b). The LN cells from aged nude mice bred under conventional conditions for 2 or 3 weeks instead of pathogen-free conditions or from mice bred continuously under pathogen-free conditions were stained with anti-TcRy/6 mAb and anti-Thy-1.2 mAb. Thy-1.2- cells are gated out and shown are histograms of TcRy16 expression of Thy-1.2+ cells. The frequency of positive cells is calculated as the percentage of cells staining with the anti-TcRy16 mAb followed by FITC-conjugated goat anti-hamster IgG as the secondary reagent, minus the percentage staining with secondary reagent alone.Thirty thousand cells per sample were analyzed on a FACScan.
3.5 Responsiveness of TcR $6 T cells in nude mice to mycobacterial hsp Recently, a significant fraction of $6 T cells has been shown to be specialized to recognize mycobacterial antigens including phylogenically conserved stress and heat-shock proteins [5-81. To determine whether the T cells differentiating in aged nude mice respond to mycobacterial hsp, we tested the reactivity of T cells to PPD and to t h e recombinant 65-kDa hsp derived from Mycobacterium bovis. As shown in Fig. 6, the proportion of y/6 T cells significantly increased (from 34% to approximately 55%) after stimulation with hsp and trypsin-digested hsp in absence of exogenous IL 2. Furthermore, the LN cells in aged nude mice produced a significant level of IL 2 in the response to the mycobacterial hsp (Fig. 7).These results suggested that
Eur. J. Immunol. 1990. 20: 1475-1482
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"1
Before c u l t u r e
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la'
After culture PPD
55.406 Tryp-hsp
Figure 6. Responsiveness of normal BALBk (a) or aged nude mice (b) y/6 T cells to PPD or mycobacterial hsp. LN cells from aged nude mice were cultured with PPD (30 pg/ml), recombinant 65kDa of M. bovis (25 pg/ml) or trypsindigested (Try-hsp) hsp (25 pg/ml) for 3 days. Cultured cells were stained with anti-TcRyy16 mAb and with anti-Thy-1.2. Thy-1.2- cells are gated out and shown are histograms of TcR y16 expression of Thy-1.2+ cells.
at least a significant fraction of y / 6 T cells differentiating outside thymus may be activated to produce IL 2 by the 65 -kDa mycobacterial hsp.
11 tl12
:1/4
'118
x1
X1116
XI12
XlIU
xll8
XI116
Dilution
Figure 7. I L 2 production of LN cells from aged nude mice stimulated with PPD (a) or hsp (b). CTLL-2 cells (2 X 10") were incubated with diluted SN from cultures in which nude rniceTcells were stimulated with PPD or hsp (described in Fig. 5) in 0.2 ml medium at 37°C for 48 h, and then pulsed with 1 pCi [3H]dThd. Culture were incubated for another 6 h and cells were harvested.
3.6 Detection of high titer of anti-DNA antibodies in aged nude mice bred under conventional conditions It is known that nude mice spontaneously develop autoimmunity [33].To confirm that the a@ Tcells in nude mice are activated in vivo and contribute to the development of autoimmunity in vivo, we measured the titer of anti-DNA antibodies in the serum in aged nude mice bred under conventional conditions for several weeks. As shown in Fig. 8, the titer of anti-DNA antibodies significantly increased in aged nude mice bred under conventional conditions in correlation with the increased percentage of y/6 T cells. These results suggested that environmental antigens may activate the hsp-reactive y/6 Tcells to produce IL 2, and consequently reverse the unresponsiveness of self-reactive a/p Tcells differentiating outside the thymus, resulting in the development of autoimmunity. Our hypothesis is shown in Fig. 9.
4 Discussion Our data indicate that self-reactive T cells bearing Vg3 or Vpll are not clonally deleted during maturation along an
Self-reactive Tcells in aged nude mice
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n u i n u [Sw)
P-
P+ 10000
lOO00
30000
Reciprocal tiler
Figure 8. Reciprocal titer of anti-DNA antibody in serum from aged nude mice. Diluted serum was added to each well of plates coated with calf thymus DNA and incubated at 37 "Cfor 1 h. After washing three times, 100 pl of alkaline phosphatase-conjugated goat anti-mouse IgG was added to each well and incubated at 37°C for 1 h . After washing, the substrate p-nitrophenyl phosphate was added to the well and incubated for 5 min at room temperature. After stopping the enzyme reaction with 3 N NaOH, the absorbance at 405 nm was determined with a spectrophotometer.
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self-reactive T cells are made tolerant by clonal anergy [34, 351. The clonal anergy is also induced in mature Vp6-bearing Mls-la-reactive T cells upon immunization Mls-lb mice with Mls-la-expressing cells [36]. Several lines of evidence from studies with Tcell clones in vitro reveal that TcR occupancy in the absence of a co-stimulatory signal coming from the APC such as M@ and dendritic cells, induces a state of proliferative unresponsiveness in theTcells [37].The tolerance induction in the self-reactiveT cells maturing extrathymically may be due to an inadequate signaling from the TcR/CD3 complex which is occupied by self antigens present continuously on non-lymphoid cells in the absence of a co-stimulatory signal coming from the APC. Further biochemical analysis of the TcR molecules in self-reactive T cells of aged nude mice is required to elucidate the extrathymic tolerance mechanism.
TcR y/6 represents the first CD3-associated TcR in ontogeny and displays a more limited diversity thanTcR alp. y/6 T cells are distributed in different anatomical sites such as the epidermis [38, 391, intestine [40, 411 and reproductive organs [42]. Recently, there has been convincing evidence that some of Tcells are specialized to recognize epitopes on mycobacterial antigens. Holoshitz et al. [7] cloned human $6 Tcells reactive to mycobacterial antigens from synovial fluid of patients with rheumatoid arthritis. Modlin et al. [43] found that $6 T cells accumulated in the reactive granulomatous lesions of leprosy. Haregewoin et al. [44] showed that y/6 T cells in a BCG-immune individual expanded in response to PPD. Janis et al. [6] showed that T cells in mice immunized with M . tuberculosis were activated to produce I L 2 by soluble extract with M . tuberculosis [6]. Similarly,antigen-unselected y/6 Tcell hybridomas from murine newborn thymus have been reported to respond to PPD [5]. Among the many potential mycobacterial antigens, hsp have been implicated as immunodominant antigens. In fact, a significant fraction of y/6 T cells reactive to mycobacterial antigens can also respond to the recombinant 65-kDa hsp [ 5 , 7, 441. hsp are polypeptides phylogenically conserved between eukaryotes and prokaryotes. Koga et al. [45] have shown that molecules similar Figure 9. Model of development of autoimmunity in aged nude to bacterial 65 hsp were targets of mycobacterial hspmice. specific T cell clones. Taken together, y/6 T cells may represent the first line of defense against the invasion of extrathymic pathway in aged BALB/c nude mice. Instead, various pathogens by elimination of stressed autologous the self-reactive T cells are in a state of clonal anergy. The cells such as infected or transformed cells [46]. In our density of TcR/CD3 on their surface was a little lower than results, y/6 T cells differentiating in aged nude mice that of normal control Tcells, suggesting that the unre- proliferated and produced a significant level of IL2 in sponsiveness of these Tcells may be due to down-regulation response to 65-kDa hsp. Our results also revealed that the of the receptor molecules. However, in the presence of self-reactive a@ T cells in nude mice could respond exogenous rIL 2, these T cells significantly proliferate in normally to signals through the TcR in the presence of IL 2. response to SEA.Therefore, the self-reactiveTcells may be Therefore, it is possible that mycobacterial infection may rendered tolerant by clonal anergy which is characterized induce autoimmune diseases through production of IL2 in by preservation of responsiveness to I L 2 but lack of IL2 the response to hsp expressed by mycobacterium and/or production. Recently, Hodes et al. [28] have reported that stressed autologous cells. We have previously reported that the self-reactive Tcells are detected in athymic nude mice. environmental antigens play an important role in the Furthermore, Fry et al. [29] have shown that the self- age-associated increase of the number of $6 Tcells in both reactive T cells bearing Vg3 or Vpll can significantly euthymic and athymic mice [47,48]. Consistent with these respond to SEA in the presence of exogenous IL2. Our data, aged nude mice bred under conventional conditions contained a larger number of y/6 Tcells than that found in data are consistent with their results. mice bred under specific pathogen-free conditions; such The mechanism of tolerance outside the thymus is quite mice appear to be likely to develop autoimmunity. Recentdifferent from that in the thymus.The recent description of ly, Fujihashi et al. [49] have reported that y/6 T cells from tolerance in transgenic mice expressing MHC class I or aged nude mice contribute to abrogate oral tolerance. At class I1 molecules in pancreatic cells indicate that the least some of y/6 Tcells would participate in the develop-
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H.Yuuki,YYoshikai, K. Kishihara et al.
ment autoimmunity by reversing the unresponsiveness of the self-reactive T cells. We have previously reported that young nude mice expressed functional messages of y chain genes composed of Vy2-J,2-q2 or V y 4 - J y 1 - ~and 1 6 chain genes composed of V&Dsl-Dg2-J~l-C~ [23]. Recently, Happ et al. [50] have determined that most of PPD-reactive T cells expressed V,1-Jyl-C,4 and Vy6-Dyl-Jyl-C[50]. Further analysis on the y/S Tcells in aged nude mice are required to elucidate the nature of theTcR repertoire of y/6 Tcells differentiating along an extrathymic pathway. We thank Dr. J. A . Bluestone (Chicago University) forproviding the mAb 145-2Cll and UC7-13D5, Dr. J. 1.1.1 Kappler and i? Marrack (National Jewish Center for Immunology and Respiratory Medicine) for providing the mAb KJ25, n 3 . 1 , KJ16 and F23.2, Dr. H. Hengartner (University Hospital Zurich) for providing the mAb 44-22-1, Dr. K. Tomonari (MRC Clinical Research Center) for providing the mAb KTII, Dr. 0. Kanagawa (Lilly Research Laboratories) for providing the mAb MR9-4, MR10-2 and MR12-4, Dr. R. Kubo (National Jewish Center Immunology and Respiratory Medicine) for providing the mAb H57-597, Drs. R. Van der Zee and J. D. A . Van Embden (National Institute of Public Health and Environmental Protection, bacteriology, Netherland) for providing recombinant M. bovis hsp 65, and 7: Otani (Research Institute, Daiichi Seiyaku Co., Ltd., Tokyo) for providing SEA. Received March 1, 1990.
5 References 1 Zinkernagel, R. M., Immunol. Rev. 1987. 42: 224. 2 Marrack, P. and Kappler, J., Immunol. Today 1988. 9: 308. 3 Fowlkes, B. J. and Pardoll, D. M., Adv. Immunol. 1989. 44: 207. 4 Brenner, M. B., Strominger, J. L. and Krangel, M. S., Adv. Immunol. 1988. 43: 133. 5 O'Brien, R. L., Happ, M. I?, Dallas, A., Palmer, E., Kubo, R. and Born, W. K., Cell 1989. 57: 667. 6 Janis, E. M., Kaufmann, S. H. E., Schwartz, R. H. and Pardoll, D. M., Science 1989. 224: 713. 7 Holoshitz, H., Konning, F.. Colligan, J. E., Bruyn, J. D. and Strober, S., Nature 1989. 339: 226. 8 Raulet, D. H., Nature 1989. 339: 342. 9 Kappler, J. W., Roehm, N. and Marrack, P., Cell 1987. 49: 273. 10 Tomonari, K. and Lovering, E., Immunogenetics 1988. 28: 445. 11 Bill, J., Kanagawa, O.,Woodland, D. L. and Palmer, E. D., J. Exp. Med. 1989. 169: 1405. 12 Kappler, J. W., Staerz, U.,White, J. and Marrack, P. C., Nature 1988. 332: 35. 13 MacDonald, H. R.,Schneider, R., Lees, R. K., Howe, R. C., Acha-Orbea, H., Festenstein, H., Zinkernagel, R. M. and Hengartner, H., Nature 1988. 332: 40. 14 Pullen, A.M., Marrack, I? and Kappler, J.W., Nature 1988.335: 796. 15 Abe, R. ,Vacchio, M. S., Fox, B. and Hodes, R. J., Nature 1988. 335: 827. 16 Fry, A. M. and Matis, L. A., Nature 1988. 335: 830. 17 Hengartner, H., Odermatt, B., Schneider, R., Schreyer, M., Walle, G.. MacDonald, H. R. and Zinkernagel, R. M., Nature 1988. 336: 338. 18 Lamb, J. R., Skidmore, B. J., Green, N., Chiller, J. M. and Feldmann, M.. J. Exp. Med. 1983. 157: 1434.
Eur. J. Irnmunol. 1990. 20: 1475-1482 19 Lamb, J. R. and Feldmann, M., Nature 1984.308: 72. 20 Quill, H. and Schwartz, R. H., J. Immunol. 1987. 138: 3704. 21 Jenkins, M. K., Pardoll. D. M.. Mizuguchi. J.. Chused.T. M. and Schwartz, R. H., Proc. Natl. A A d . Sci. USA 1987. 84: 5409. 22 Hunig, T., Immunol. Today 1983. 4: 84. 23 Kishihara, K., Yoshikai, Y., Matsuzaki, G., Mak, T. W. and Nomoto, K., Eur. J. Immunol. 1987. 17: 477. 24 Lawetzky, A. and Hiinig, T., Eur. J. Immunol. 1988. 18: 409. 25 Yoshikai, Y., Reis, M. D. and Mak, T. W., Nature 1986. 324: 482. 26 Yoshikai,Y., Matsuzaki, G., Takeda, Y., Kishihara, K., Yuuki, H. and Nomoto, K., Eur. J. Immunol. 1988. 18: 1039. 27 Pardoll, D. M., Fowlkes, B. J., Lew, A. M., Maloy, W. L., Weston, M. A., Bluestone, J. A., Schwartz, R. H., Coligan, J. E. and Kruisbeek, A. M., J. Immunol. 1988. 146: 4091. 28 Hode, R. J., Sharrow, S. 0.and Solomon, A., Science 1989.246: 1041. 29 Fry, A. M., Jones, L. A., Kruisbeek, A. M. and Matis, L. A., Science 1989. 246: 1044. 30 Oda, T., Jpn. J. Bacteriol. 1978. 33: 743. 31 Takimoto, H., Yoshikai, Y., Kishihara, K.. Matsuzaki, G., Kuga, H., Otani,T. and Nomoto, K., Eur. J. Immunol. 1990. 20: 617. 32 Thole, J. E . R., Dauwerse, H. G., Das, P. K., Groothuis, D. G., Schouls, L. M. and Van Embden, J. D. A., Infect. Immun. 1985. 50: 800. 33 Monier, J. C. and Sepetjian, M., Ann. Irnmunol. (Inst. Pasteur) 1975. 126C: 63. 34 Morahan, G., Allison, J. and Miller, J. F. A. I?, Nature 1989. 339: 622. 35 Burkly, L. C., Lo, D., Kanagawa, O., Brinster, R. L. and Flavel, R. A., Nature 1989. 342: 564. 36 Rammensee, H.-G., Kroschewski, R. and Frangoulius, B., Nature 1989. 339: 541. 37 Schwartz, R. H., Cell 1989. 57: 1073. 38 Konning, F., Stingl, S., Yokoyama, W. M., Tschachler, E., Shevach, E. M., Coligan, J. E. and Maloy,W. L., Science 1987. 236: 834. 39 Stingl, G., Konning, F., Yamada, H., Yokoyama, W. M., Tschachler, E., Bluestone, J. A., Steiner, G., Samelson, L. E., Lew, A. M., Coligan, J. E. and Shevach, E. M., Proc. Natl. Acad. Sci. USA 1987. 84: 4586. 40 Goodman, T. and Lefraniois, L., Nature 1988. 333: 855. 41 Bonneville, M., Janeway, Jr., C. A., Ito, K., Haser,W.. Ishida, I., Nakanishi, N. and Tonegawa, S., Nature 1988. 336: 479. 42 Lafaille, J. J., DeCloux. A., Bonneville, M.,Takagaki,Y. and Tonegawa, S., Cell 1989. 59: 859. 43 Modlin, R. L., Pirmetz, C., Hofman, F. M., Torigian, V., Uyemura, K., Rea,T. H., Bloon, B. R. and Brenner. M. B., Nature 1989. 339: 544. 44 Haregewoin, A.,Soman, G., Hom, R. C. and Finberg, R. W.. Nature 1989. 340: 309. 45 Koga,T.,Wand-Wurttenberger,A., DeBruyn, J.,Munk, M. E., Schoel, B. and Kaufmann, S. H. E., Science 1989. 245: 1112. 46 Young, R. A. and Elliott,T. J., Cell 1989. 59: 5. 47 Matsuzaki, G., Yoshikai, Y., Kishihara, K., Nomoto, K., Yokokura, T. and Nomoto, K., Eur. J. Immunol. 1988. 18: 1779. 48 Yoshikai, Y.,Matsuzaki, G., Kishihara, K., Nomoto, K., Yokokura, T. and Nomoto, K., Infect. Immun. 1988. 56: 2069. 49 Fujihashi, K., Kiyono, H., Aicher,W. K., Green, D. R., Singh, B., Eldridge, J. H. and McGhee, J. R., J. Immunol. 1989.143: 3415. 50 Happ, M. I?, Kubo, R.T., Palmar, E., Born,W. K. and OBrien, R. L.. Nature 1989. 342: 696.
Hiroyuki Yuuki, Yasunobu Yoshikai, Kenji Kishihara, Akinori Iwasaki, Goto Matsuzaki, Hiroaki Takimoto and Kikuo Nomoto Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka
Self-reactive T cells in aged nude mice
Clonal anergy in self-reactive a l p T cells is abrogated by heat-shock protein-reactive y/6 T cells in aged athymic nude mice* Although T cells proliferate and differentiate primarily in the thymus, athymic nude mice contain an appreciable level of T cell receptor a/fi and y/6 T cells, suggesting the existence of the extrathymic pathway in the development of bothT cells. Recent studies with nude mice indicate that clonal deletion of self-reactiveT cells does not occur extrathymically. In the present study, we have investigated the responsiveness of self-reactive T cells differentiating along an extrathymic pathway in aged BALB/c (H-2d, Mls-lb2", I-E+, 7-8 month old) nude mice. Consistent with recent reports,Tcells bearing Vp3 or Vpll, which are important for recognizing proteins encoded by the M I s - or ~ ~the I-E allele, respectively, are readily detected in age nude mice. The Vp3- or Vpll-bearing Tcells, however, do not proliferate in response to staphylococcal enterotoxin A which specifically stimulates Vp3- or Vpll-bearing T cells. When exogenous recombinant interleukin 2 was added to the culture, the Vp3-bearing T cells in aged nude mice significantly proliferated in response to staphylococcal enterotoxin A. Aged nude mice also contained a substantial level of y/6 Tcells which account for 15.6% of all Thy-1.2+ cells.The y/6 Tcells proliferated and produced a significant level of interleukin 2 in response to the 65-kDa mycobacterial heat-shock protein, which is highly homologous to its eukaryotic counterpart. These results suggest that unresponsiveness of self-reactive Tcells may be reversed by Tcells responding to stress proteins expressed by the invading microbes and/or the stressed autologous cells.
1 Introduction Tcell precursors derived from fetal liver or BM proliferate and differentiated in the thymus. During Tcell differentiation in the thymus, thymocytes with a broad range of affinities for self MHC are positively selected (positive selection) and thymocytes with high affinities for self antigens are rendered tolerant (negative selection) (reviewed in [l,21). The selection is carried out on the basis of expression of the TcR. Two types of TcR have been identified; aTcR a/fiheterodimer which recognizes nominal antigen in the context of self-MHC gene products and a TcR y/6 heterodimer which has been reported to recognize MHC molecules and/or mycobacterial antigens including the 65-kDa heat-shock protein (hsp) [3-81. Recently, it has been reported that Tcells bearing Vpll or Vpl7a capable of recognizing I-E molecules are eliminated from mature T cell repertoire of mice carrying I-E alleles
[I 83781
*
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This work was supported by grants to Y. Yoshikai from the Ministry of Education, Science and Culture (62480167, 01015081) and from Special Coordination Funds of the Science and Technology Agency of the Japanese Government.This work was also received financial support from the UNDPNorld B a n k N H O Special Programme for Research and Training in Tropical Disease (TDR).
Correspondence: Yasunobu Yoshikai, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812, Japan Abbreviations: hsp: Heat-schock protein SEA: Staphylococcal enterotoxin A
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
[9-111. TcR using Vp3 or Vp6/8.1 are also found to be specific for the products of MIS-2" or Mls-la and mice expressing Mls-la or Mls-2" eliminate Vp6/8.1- or Vp3bearing T cells in their pool of mature T cells, respectively [12-17].Thus, self tolerance is due mainly to the deletion of Tcells bearing TcR capable of recognizing the self antigens in the thymus. However, several lines of evidence suggest that in addition the self-reactive T cells are rendered tolerant peripherally by the induction of clonal anergy characterized by preservation of responsiveness to exogenous I L 2 but lack of IL2 production in response to self antigens [18-211. Although T cells proliferate and differentiate primarily in the thymus, several studies with athymic nude mice indicate that an extrathymic pathway exists in T cell development [22-261. Congenitally athymic nude mide, especially aged ones contained an appreciable level of a/F and y / 6 Tcells [27], suggesting that bothTcells do differentiate outside the thymus, albeit slowly. Recent studies with nude mice indicate that clonal deletion of self-reactive Tcells does not occur extrathymically [28,29]. However, whether the selfreactive T cells in nude mice respond normally to self antigens or are rendered tolerant by the induction of clonal anergy is not yet known. In the present study, we have investigated the fate of self-reactiveTcells and their responsiveness in aged athymic BALB/c (H-2d, M1s-lh2", I-E+) nude mice. Our results indicate that the self-reactive a/fi T cells differentiating outside the thymus are rendered tolerant by the induction of clonal anergy, whereas y/6 T cells in aged nude mice proliferate and produce a significant level of IL2 in response to the 65-kDa mycobacterial hsp. The implication of these findings for the development of autoimmune disease in aged nude mice is discussed. 0014-2980/90/0707-1475$3.50+ .25/0
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2 Materials and methods 2.1 Mice Congenitally athymic nude mice (ndnu, 5 weeks old) of a BALB/c background were obtained from Clea Japan Inc. (Tokyo) and were bred under specific pathogen-free conditions at the animal center of Kyushu University. Males (7-9 months old) were used for experiments.
Eur. J. Immunol. 1990. 20: 1475-1482 Con A (3 p.g/ml) (Sigma, St. Louis, MO) in the presence or not of 1 U/ml rIL2 (Takeda Chemical Industries, Osaka, Japan) at 37°C in a 5% COz incubator in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% FCS. Three days later, live cells were harvested and expanded for 3 days with 1U/ml rIL2. In some experiments, the blast cells were stained with each mAb without being cultured for 3 days with rIL2. Blasts were stained with FITCconjugated anti-hamster IgG or FITC-conjugated anti-rat IgG after treatment with KJ25,44-22-1, K T l l or 7D4 mAb and analyzed by FCM.
2.2 Antibodies The following antibodies were used for immunofluorescence staining: anti-CD3 E chain mAb (145-2C11; provided by Dr. J. A. Bluestone), anti-TcR a/@mAb (H57-597; provided by Dr. R. Kubo), anti-Vg3 mAb (KJ25; provided by Dr. J. W. Kappler), anti-Vg5 mAb (MR9-4; provided by Dr. 0. Kanagawa), anti-Vg6 mAb (44-22-1; provided by Dr. H. Hengartner), anti-Vg8 mAb (F23.1, KJ16 and F23.2; provided by Dr. J. W. Kappler), anti-Vp9 mAb (MR10-2; provided by Dr. 0. Kanagawa), anti-Vgll mAb (KT11; provided by Dr. K. Tomonari), anti-Vgl3 mAb (MR12-4; provided by Dr. 0. Kanagawa), anti-IL2R mAb (7D4), or biotin-conjugated anti-Thy-1.2 mAb (Becton Dickinson, Mountain View, CA), FITC-conjugated goat anti-hamster IgG, FITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rat IgG (Tago Inc., Burlingame, CA). 2.3 Staphylococcal enterotoxin A (SEA)
SEAwas isolated and prepared by the methods of Oda [30]. The culture SN of Staphylococcus aureus 13N-2909, provided by theTokyo Metropolitan Research of Public Health through the courtesy of Dr. H . Igarashi, was centrifuged and applied to a SP-Sephadex C-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) column chromatograph. The SEA was eluted with a pH gradient of pH 5 to pH 7.5 and the eluate was concentrated on an Amicon UM 10 membrane (Amicon Division, W. R. Grace and Co., MA). The concentrated SEA fraction was purified further by gel filtration on Sephadex G-75 (Pharmacia). The purified sample was shown to consist of a single band by SDSPAGE. SEA, stored as lyophilized powder at 4"C, was dissolved in saline.
2.4 FCM analysis Spleen and LN cells were taken from 7-9-month-old BALB/c nude mice. A single-cell suspension was stained with each antibody and was analyzed on a FACScan flow cytometer (Becton Dickinson). The percentage of positive cells was determined by integration from profiles based on 3 x lo4 cells.
2.5 In vitro culture of T cells with Con A or SEA Spleen cells were stimulated with SEA or Con A as described by Takimoto et al. [31]. Briefly, spleen cells (1 x 106/ml) were incubated with SEA (0.1 pg/ml) or
2.6 In vitro culture of T cells with PPD or hsp
The LN cells from aged nude mice were cultured with 30 pg/ml PPD derived from Mycobacterium tuberculosis (H37Ra; Kainosu Inc., Tokyo, Japan), 25 pg/ml recombinant 65-kDa hsp derived from Mycobacterium bovis (kindly provided by Drs. R.Van der Zee and J. D. A.Van Embden; [32]), or 25 pg/ml trypsin-digested hsp [digested with L- 1-tosylamido-2-pheny lethyl-chloromethyl ketonetreated immobilized trypsin (Sigma)] at 37°C in a 5% C02 incubator for 72 h in RPMI 1640 (Gibco) supplemented with 10% FCS. 2.7 IL 2 production of nude mice T cells in response to hSP
The ILZdependent CTLL-2 cells (1 x 104) were incubated with a dilution of SN from cultures in which aged nude mice Tcells were stimulated with PPD or hsp in 0.2 ml medium at 37°C for 48 h, and then pulsed with 1 pCi = 37 kBq [3H]thymidine. After another 6 h of incubation the cells were harvested. 2.8 Titer of anti-DNA antibody in serum
Anti-DNA titer in the serum from aged nude mice was determined by ELISA. Briefly, calf thymus DNA (type I, Sigma) diluted to a concentration of 10 pg/ml with coating buffer (15 mM Na2C03, 34 mM NaHC03, 0.2 mM NaN3, pH 9.2) was added to each well of flat-bottom microtiter plates (Sanko Pure Chemical Co., Tokyo) and kept overnight at 4°C. Nonspecific binding sites were saturated by addition of 200 pl of PBS containing 1% BSA (fraction V; Boehringer Mannheim, Mannheim, FRG). After a 1-h incubation at room temperature, the plate was washed three times with PBS containing 0.05% Tween 20 (PBSTween). For each group of mice, sera were pooled in equal volume and serially diluted with PBS-Tween.Then 100 p1 of diluted serum was added into each well of antigen-coated plates and incubated at 37°C for 1h. After washing with PBS-Tween, 100 p1 of alkaline phosphatase-conjugated goat anti-mouse IgG (Tago) diluted to 1/1OOO with PBSTween was added to each well and incubated at 37°C for 1 h. After washing, 1 mg of p-nitrophenyl phosphate (Wako, Osaka, Japan), solubilized in 10% diethanolamine solution (pH 9.8) per 1 ml, was added to the well as a substrate and incubated for 5 min at room temperature. After stopping the enzyme reaction with 3 N NaOH, the absorbance at 405 nm was determined with a ELISA spectrophotometer.
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Eur. J. Immunol. 1990. 20: 1475-1482
3 Results
3.2 TcR Vp repertoire of T cells differentiating along an extrathymic pathway
3.1 Development of both TcRdp and y/6 T cells along an extrathymic pathway To confirm that T cells develop along an extrathymic pathway, we determined Tcell surface markers on the LN cells of young (8- to 10-weeks old) and aged (8- to 10-months old) BALB/c (H-2d, M l ~ l ~ nude 2 ~ ) mice by FCM analysis. In the LN of young nude mice, few CD3+CD4+CD8- or CD3+CD4-CD8+ cells were detected, whereas a substantial number of CD3+CD4-CD8cells (data not shown) and of CD4 or CD8 single-positive T cells were detected in those aged nude mice in which CD8+ cells predominated over CD4+ cells (Fig. 1). Thus, a substantial number of phenotypically mature T cells can develop along an extrathymic pathway. Expression of TcR on the T cells in aged nude mice was further analyzed by double staining with anti-Thy-1.2 mAb and anti-TcR a$ mAb (H57-597) or anti-TcRy/6 mAb (UC7-13D5). As shown in Fig. 2b, 78.5% of Thy-l+ cells were TcRa/b+ in the L N of aged nude mice, whereas y/6 Tcells account for 15.6% of Thy-l+ cells in these mice.Thus, both a@and y/8 T cells can develop outside the thymus, albeit slowly. Consistent with report by Lawetzky and Hunig [24], the intensity of TcR/CD3 expression on T cells in aged nude mice was a little lower than that in euthymic mice (Fig. 2a) but higher than that in CD4+CD8+ thymocytes (data not shown).
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To examine the TcR repertoire of the a / p T cells in aged nude mice, the LN cells were double-stained with antiThy-1 mAb and various anti-Vp mAb including anti-Vp3 mAb (KJ25), anti-Vp5 mAb (MR9-4), anti-Vg6 mAb (44-22-1), anti-Vp8 mAb (F23.1, KJ16 and F23.2), anti-Vp9
(a)
Ib)
CD3
BALB/c
aged nulnu
I
CDS
Figure 1. Two-color FCM analysis of CD4 vs. CD8 expression on LN cells from aged (28 week) nude mice. (a) nu/+, 8 weeks; (b) nu/nu, 8 weeks: (c) nulnu, 28 weeks. LN cells were stained with PE-anti-CD4 mAb and FITC-anti-CD8 mAb.
Figure 2. Expression of CD3, TcRyl6, TcR a.@ or Vp on LN cells from aged nude mice (7-9 months old) and normal euthymic mice (2-3 months old). (a) Density of CD3 expression on total LN cells and density of V136expression on Thy-l.2+ LN cells from an aged nude mice (solid line) and normal euthymic mice (dotted line); (b) quantitation of TcR ylb-. TcR dp- or Vg-positive cells in Thy-1.2+ LN cells. LN cells were stained with each mAb and simultaneously with anti-Thy-1.2 mAb.Thy-1.2- cells are gated out and shown are histograms of TcRyl6,TcR a/p or Vg expression of Thy-1.2+ cells. The frequency of positive cells is calculated as the percentage of cells staining with each mAb followed by FITC-conjugated goat anti-hamster, anti-rat or anti-mouse IgG as the secondary reagent, minus the percentage staining with second reagent alone. Thirty thousand cells per sample were analyzed on a FACScan flow cytometer.
Fluorescence
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H.Yuuki,Y. Yoshikai, K. Kishihara et al.
Table 1. Quantitation of CD3-,TcR a@- and TcR Vp-positive cells in LN from BALB/c nude mice at 7-9 months of age and from normal BALB/c micea) Nude mouse no.
YO CD3+
dB
vp3
v!35
Fraction of Thy-1.2+ cells (YO) Vp6 Vp8.1 Vg8.2 Vp8.3
Vp9
Vpll
ND 5.1 7.1 2.6 14.0 11.3 ND 7.8 1 21.6 74.0 3.4 ND 5.2 8.1 13.0 ND 13.9 78.5 10.5 2 30.3 6.3 ND 8.7 8.2 15.6 13.2 8.1 ND 3 20.7 73.5 2.5 8.7 2.0 3.8 19.0 1.6 9.9 5.4 4 22.6 85.5 4.3 10.1 2.6 5.6 14.2 2.4 10.8 8.4 5 20.3 69.1 MeanfSDb) 23.1f4.1 76.1k6.2 8.0f1.8 2.0k0.6 11.8k1.7 5.7k2.5 15.222.3 8.0f1.9 2.3f0.4 4.3f1.5 BALB/cC)
76.3f2.9 98.5f0.2 0.3f0.2 0.2kO.l 9 . 8 f 0 . 7 6.7k0.4 13.1k0.410.2+1.1 0 . 4 f 0 . 1 O.lkO.1
Vp13
ND ND ND 1.1 2.5 1.8f1.0
O.lfO.l
a) LN cells from five individual aged BALB/c nude mice (7-9 months of age) were stained and analyzed as described in legends of Fig. 1. The frequency of Tcells expressing the CD3 E chain is calculated as a percentage of total LN cells and the frequency of cells expressing other markers is calculated as a perccntage of Thy-1.2+ cells. The typical data from one experiment are shown in Fig. 1. b) Percentages represent the mean value (kSD) of five individual animals. c) Percentage represent the mean value (k SD) of three individual animals.
mAb (MR10-2), anti-Vgll mAb (KT11) or anti-Vgl3 mAb (MR12-4). A typical fluorescence histogram is shown in Fig. 2 and the results of five individual mice are summarized in Table 1. A direct link has been found between the reactivity to the products of the M l ~ - or 2 ~the I-E allele and TcR Vg3 or VgSNg11 on T cells, respectively. Consistent with the earlier findings, BALB/c euthymic mice have deleted Vp3-,VgS- and Vgll-bearingTcells in their matureT cell pool as a result of clonal deletion. By contrast, an appreciable number of Vg3-,Vg-5- or Vgll-bearing T cells were detected in the LN of aged nude mice. These results confirm that T cells differentiating along an extrathymic pathway have not undergone negative selection.
3.3 Functional analysis of self-reactive T cells in nude mice
To investigate the functional aspect of the self-reactive Tcells differentiating in aged nude mice, we tried to activate the Vp3- or Vpll-bearing T cells by SEA, a "superantigen" which specifically stimulates Tcells bearing Vg3 or Vpll irrespective of the a chain expressed by theseT cells. Stimulation of Tcells with Con A served as positive control to demonstrate general responsiveness of the population. As shown in Fig. 3, Vg3:bearing T cells and Vpll-bearing T cells significantly proliferated in response to SEA in C57BL/6 mice carrying M l ~ - 2and ~ I-E-.
Fluorescence
intensity
Figure 3. FCM analysisof Con A (a) or SEA (b)-activated blast cells. Spleen cells (1 X 106/ml)were incubated in 10 ml medium with SEA (0.1 kg/ml) or Con A (3 pglml) in a humidified atmosphere with 5% CO2 at 37"C.Three days later, live cells were harvested and expanded for 3 days in the presence of 1 U/ml rIL2 to increase the intensity of TcR Vp expression on the blasts. Blasts were stained with FITC-conjugated anti-hamster IgG or FITC-conjugated anti-rat IgG after treatment with KJ25,44-22-1or K T l l mAb and analyzed on a FACScan .
Self-reactive T cells in aged nude mice
Eur. J. Immunol. 1990. 20: 1475-1482 after before
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1ti.
a%
i
5 2 . 9%
Fluorescence
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Similarly, the number of Vp3-bearing T cells significantly increased after stimulation with SEA in AKR/J mice carrying Mls-2" as compared with stimulation with Con A [31]. In euthymic BALB/c mice, however, noVp3-bearingT cells proliferated in response to SEA because of toleranceinduced deletion of self antigens. The low but significant levels of proliferation observed inVgll-bearingTcells in the BALB/c mice may suggest that not all Vgll-bearing Tcells recognize I-E molecules. On the other hand, in spite of the presence of Vg3- and Vgll-bearing T cells in aged nude mice, these T cells showed little, if any, proliferation in response to SEA. Thus, the self-reactive T cells maturing extrathymically may be rendered tolerant to self antigens. To elucidate whether the unresponsiveness of the Tcells in aged nude mice is due to a defect in the function of the TcR/CD3 complex in theTcells or to an induction of clonal anergy,we investigated the responsiveness of Vp3-bearingT cells to SEA in the presence of exogenous rIL2. Approximately 45% of nude spleen cells expressed IL2R after stimulation into SEA and I L 2 (Fig. 4). The intensity of Vp3-expression was relatively low because the blasts were obtained after stimulation with SEA and IL2 but without an additional culture with IL 2. However, theVg3-bearingT cells in aged nude mice significantly proliferated in response to SEA in the presence of exogenous rIL2 as compared with those in euthymic BALBk mice (Fig. 4).
3.4 Increased number of TcR $6 T cells in aged nude mice bred under conventional conditions To investigate whether $6 Tcells in LN cells in nude mice are activated in vivo by environmental antigens, we examined the number of y/6 Tcells in aged nude mice bred under conventional conditions for several weeks instead of pathogen-free conditions. As shown in Fig. 5, the percentage of y/6 T cells in the LN cells significantly increased from 15.6% in nude mice bred under pathogen-free conditions to 38.4% in those bred under conventional conditions.
Figure 4. FCM analysis of normal BALBlc spleen cells (a) or spleen cells from 7-9-monthold nude mice (b). For stimulation spleen cells (1 x 10h/ml) were incubated in 10ml medium with SEA (0.1 pglml) plus IL2 (1 Ulml) in a humidified atmosphere with 5% CO2 at 37°C for 3 days.Then, live cells were harvested. Blasts were stained with KJ25, 44-22-1 or 7D4 mAb and simultaneous1 with anti-Thy-1.2 mAb and analyzed on a FACScan. Thy1.2- cells were gated out. Ibl I
Fluorescence
intensity
Figure 5. Expression of TcR y16 on LN cells from aged nude mice bred under pathogen-free (a) or conventional conditions (b). The LN cells from aged nude mice bred under conventional conditions for 2 or 3 weeks instead of pathogen-free conditions or from mice bred continuously under pathogen-free conditions were stained with anti-TcRy/6 mAb and anti-Thy-1.2 mAb. Thy-1.2- cells are gated out and shown are histograms of TcRy16 expression of Thy-1.2+ cells. The frequency of positive cells is calculated as the percentage of cells staining with the anti-TcRy16 mAb followed by FITC-conjugated goat anti-hamster IgG as the secondary reagent, minus the percentage staining with secondary reagent alone.Thirty thousand cells per sample were analyzed on a FACScan.
3.5 Responsiveness of TcR $6 T cells in nude mice to mycobacterial hsp Recently, a significant fraction of $6 T cells has been shown to be specialized to recognize mycobacterial antigens including phylogenically conserved stress and heat-shock proteins [5-81. To determine whether the T cells differentiating in aged nude mice respond to mycobacterial hsp, we tested the reactivity of T cells to PPD and to t h e recombinant 65-kDa hsp derived from Mycobacterium bovis. As shown in Fig. 6, the proportion of y/6 T cells significantly increased (from 34% to approximately 55%) after stimulation with hsp and trypsin-digested hsp in absence of exogenous IL 2. Furthermore, the LN cells in aged nude mice produced a significant level of IL 2 in the response to the mycobacterial hsp (Fig. 7).These results suggested that
Eur. J. Immunol. 1990. 20: 1475-1482
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"1
Before c u l t u r e
0.8%
la'
After culture PPD
55.406 Tryp-hsp
Figure 6. Responsiveness of normal BALBk (a) or aged nude mice (b) y/6 T cells to PPD or mycobacterial hsp. LN cells from aged nude mice were cultured with PPD (30 pg/ml), recombinant 65kDa of M. bovis (25 pg/ml) or trypsindigested (Try-hsp) hsp (25 pg/ml) for 3 days. Cultured cells were stained with anti-TcRyy16 mAb and with anti-Thy-1.2. Thy-1.2- cells are gated out and shown are histograms of TcR y16 expression of Thy-1.2+ cells.
at least a significant fraction of y / 6 T cells differentiating outside thymus may be activated to produce IL 2 by the 65 -kDa mycobacterial hsp.
11 tl12
:1/4
'118
x1
X1116
XI12
XlIU
xll8
XI116
Dilution
Figure 7. I L 2 production of LN cells from aged nude mice stimulated with PPD (a) or hsp (b). CTLL-2 cells (2 X 10") were incubated with diluted SN from cultures in which nude rniceTcells were stimulated with PPD or hsp (described in Fig. 5) in 0.2 ml medium at 37°C for 48 h, and then pulsed with 1 pCi [3H]dThd. Culture were incubated for another 6 h and cells were harvested.
3.6 Detection of high titer of anti-DNA antibodies in aged nude mice bred under conventional conditions It is known that nude mice spontaneously develop autoimmunity [33].To confirm that the a@ Tcells in nude mice are activated in vivo and contribute to the development of autoimmunity in vivo, we measured the titer of anti-DNA antibodies in the serum in aged nude mice bred under conventional conditions for several weeks. As shown in Fig. 8, the titer of anti-DNA antibodies significantly increased in aged nude mice bred under conventional conditions in correlation with the increased percentage of y/6 T cells. These results suggested that environmental antigens may activate the hsp-reactive y/6 Tcells to produce IL 2, and consequently reverse the unresponsiveness of self-reactive a/p Tcells differentiating outside the thymus, resulting in the development of autoimmunity. Our hypothesis is shown in Fig. 9.
4 Discussion Our data indicate that self-reactive T cells bearing Vg3 or Vpll are not clonally deleted during maturation along an
Self-reactive Tcells in aged nude mice
Eur. J. Immunol. 19Y0. 20: 1475-1482 n u / + l8w)
n u i n u [Sw)
P-
P+ 10000
lOO00
30000
Reciprocal tiler
Figure 8. Reciprocal titer of anti-DNA antibody in serum from aged nude mice. Diluted serum was added to each well of plates coated with calf thymus DNA and incubated at 37 "Cfor 1 h. After washing three times, 100 pl of alkaline phosphatase-conjugated goat anti-mouse IgG was added to each well and incubated at 37°C for 1 h . After washing, the substrate p-nitrophenyl phosphate was added to the well and incubated for 5 min at room temperature. After stopping the enzyme reaction with 3 N NaOH, the absorbance at 405 nm was determined with a spectrophotometer.
1481
self-reactive T cells are made tolerant by clonal anergy [34, 351. The clonal anergy is also induced in mature Vp6-bearing Mls-la-reactive T cells upon immunization Mls-lb mice with Mls-la-expressing cells [36]. Several lines of evidence from studies with Tcell clones in vitro reveal that TcR occupancy in the absence of a co-stimulatory signal coming from the APC such as M@ and dendritic cells, induces a state of proliferative unresponsiveness in theTcells [37].The tolerance induction in the self-reactiveT cells maturing extrathymically may be due to an inadequate signaling from the TcR/CD3 complex which is occupied by self antigens present continuously on non-lymphoid cells in the absence of a co-stimulatory signal coming from the APC. Further biochemical analysis of the TcR molecules in self-reactive T cells of aged nude mice is required to elucidate the extrathymic tolerance mechanism.
TcR y/6 represents the first CD3-associated TcR in ontogeny and displays a more limited diversity thanTcR alp. y/6 T cells are distributed in different anatomical sites such as the epidermis [38, 391, intestine [40, 411 and reproductive organs [42]. Recently, there has been convincing evidence that some of Tcells are specialized to recognize epitopes on mycobacterial antigens. Holoshitz et al. [7] cloned human $6 Tcells reactive to mycobacterial antigens from synovial fluid of patients with rheumatoid arthritis. Modlin et al. [43] found that $6 T cells accumulated in the reactive granulomatous lesions of leprosy. Haregewoin et al. [44] showed that y/6 T cells in a BCG-immune individual expanded in response to PPD. Janis et al. [6] showed that T cells in mice immunized with M . tuberculosis were activated to produce I L 2 by soluble extract with M . tuberculosis [6]. Similarly,antigen-unselected y/6 Tcell hybridomas from murine newborn thymus have been reported to respond to PPD [5]. Among the many potential mycobacterial antigens, hsp have been implicated as immunodominant antigens. In fact, a significant fraction of y/6 T cells reactive to mycobacterial antigens can also respond to the recombinant 65-kDa hsp [ 5 , 7, 441. hsp are polypeptides phylogenically conserved between eukaryotes and prokaryotes. Koga et al. [45] have shown that molecules similar Figure 9. Model of development of autoimmunity in aged nude to bacterial 65 hsp were targets of mycobacterial hspmice. specific T cell clones. Taken together, y/6 T cells may represent the first line of defense against the invasion of extrathymic pathway in aged BALB/c nude mice. Instead, various pathogens by elimination of stressed autologous the self-reactive T cells are in a state of clonal anergy. The cells such as infected or transformed cells [46]. In our density of TcR/CD3 on their surface was a little lower than results, y/6 T cells differentiating in aged nude mice that of normal control Tcells, suggesting that the unre- proliferated and produced a significant level of IL2 in sponsiveness of these Tcells may be due to down-regulation response to 65-kDa hsp. Our results also revealed that the of the receptor molecules. However, in the presence of self-reactive a@ T cells in nude mice could respond exogenous rIL 2, these T cells significantly proliferate in normally to signals through the TcR in the presence of IL 2. response to SEA.Therefore, the self-reactiveTcells may be Therefore, it is possible that mycobacterial infection may rendered tolerant by clonal anergy which is characterized induce autoimmune diseases through production of IL2 in by preservation of responsiveness to I L 2 but lack of IL2 the response to hsp expressed by mycobacterium and/or production. Recently, Hodes et al. [28] have reported that stressed autologous cells. We have previously reported that the self-reactive Tcells are detected in athymic nude mice. environmental antigens play an important role in the Furthermore, Fry et al. [29] have shown that the self- age-associated increase of the number of $6 Tcells in both reactive T cells bearing Vg3 or Vpll can significantly euthymic and athymic mice [47,48]. Consistent with these respond to SEA in the presence of exogenous IL2. Our data, aged nude mice bred under conventional conditions contained a larger number of y/6 Tcells than that found in data are consistent with their results. mice bred under specific pathogen-free conditions; such The mechanism of tolerance outside the thymus is quite mice appear to be likely to develop autoimmunity. Recentdifferent from that in the thymus.The recent description of ly, Fujihashi et al. [49] have reported that y/6 T cells from tolerance in transgenic mice expressing MHC class I or aged nude mice contribute to abrogate oral tolerance. At class I1 molecules in pancreatic cells indicate that the least some of y/6 Tcells would participate in the develop-
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H.Yuuki,YYoshikai, K. Kishihara et al.
ment autoimmunity by reversing the unresponsiveness of the self-reactive T cells. We have previously reported that young nude mice expressed functional messages of y chain genes composed of Vy2-J,2-q2 or V y 4 - J y 1 - ~and 1 6 chain genes composed of V&Dsl-Dg2-J~l-C~ [23]. Recently, Happ et al. [50] have determined that most of PPD-reactive T cells expressed V,1-Jyl-C,4 and Vy6-Dyl-Jyl-C[50]. Further analysis on the y/S Tcells in aged nude mice are required to elucidate the nature of theTcR repertoire of y/6 Tcells differentiating along an extrathymic pathway. We thank Dr. J. A . Bluestone (Chicago University) forproviding the mAb 145-2Cll and UC7-13D5, Dr. J. 1.1.1 Kappler and i? Marrack (National Jewish Center for Immunology and Respiratory Medicine) for providing the mAb KJ25, n 3 . 1 , KJ16 and F23.2, Dr. H. Hengartner (University Hospital Zurich) for providing the mAb 44-22-1, Dr. K. Tomonari (MRC Clinical Research Center) for providing the mAb KTII, Dr. 0. Kanagawa (Lilly Research Laboratories) for providing the mAb MR9-4, MR10-2 and MR12-4, Dr. R. Kubo (National Jewish Center Immunology and Respiratory Medicine) for providing the mAb H57-597, Drs. R. Van der Zee and J. D. A . Van Embden (National Institute of Public Health and Environmental Protection, bacteriology, Netherland) for providing recombinant M. bovis hsp 65, and 7: Otani (Research Institute, Daiichi Seiyaku Co., Ltd., Tokyo) for providing SEA. Received March 1, 1990.
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