Nagoya J. Med. Sci. 54.00 - 00, 1992

Invited Review Article

REGULATORY ROLE OF HEAT SHOCKPROTEIN-SPECIFIC T CELLSIN HOST DEFENSEYASUNOBU YOSHIKAI

Laboratory of Germfree Life, Research Institute for Disease Mechanism and Control,
Nagoya University School of Medicine, Nagoya 466, Japan.

ABSTRACTDuring infection with L.monocytogenes, a facultative intracellular bacteria, TcR y/b T cells specific for
65 kd hsp precede TcR a/ BT cells specific for the listerial antigens in appearance. The y/ b T cells provide
a first line of defense against the infection by recognizing exogenous and endogenous 65 kd hsp on infected
cells and producing cytokines such as yIFN. The hsp-specific T cells respond quickly to antigenically diverse
pathogens before antigen-specific T cells expand clonally, and they playa role in covering the gap between
the phagocytic system and highly evolved immune response. 65 kd hsp-specific T cells play important roles
not only in host defense mechanism against infection with various pathogens but also in induction of autoimmune disease. Both 65 kd hsp-specific yb T cells and 65 kd hsp-specific aB T cells abrogate the unresponsiveness of the self-reactive aB T cells and/or B cells by producing IL-2 and contribute to induction of
autoimmune disease.
Key Words: yb T cells, aB T cells, 65 kd HSP, Listeria monocytogenes, Autoimmune disease

INTRODUCTION
Heat shock proteins (hsp), polypeptides phylogenically conserved from prokaryotes to eukaryotes, are frequently the targets of humoral and cell-mediated immune responses during infection and autoimmune diseases. loS) Among the various kinds of hsp, the 65 kd (kilo dalton)
mycobacterial hsp has been identified as a target of T cells capable of causing autoimmune diseases in a rat model of adjuvant-induced arthritis,6) in the synovial infiltrates of rheumatoid arthritis patients,7) and in leprosy and tuberculosis. S ) Although most of the hsp-specific T cells express T cell receptor (TcR) a/~, there have been several lines of evidence that at least a substantial fraction of T cells bearing TcR y/ b are specialized to recognize mycobacterial antigens
including 65 kd hsp,912)
In the present study, we focus on the roles of hsp-specific T cells in host defense against infection with a facultative intracellular bacteria, Listeria monocytogenes, and in induction of
autoimmune diseases in mice. We have found that TcR y/b-bearing T cells capable of recognizing 65 kd hsp contribute to the first line of the host defense against primary infection withL.monocytogenes through production of y interferon (IFN) and that hsp-specific T cells, regardless of their TcR being a/~ or y/b, play an important role in the induction of autoimmune diseases in aged nude mice and newborn-thymectomized (NTX) mice.

11

12
Yasunobu Yoshikai

THE ROLE OF HSP-SPECIFIC y/O T CELLS IN HOST-DEFENSEAGAINST INFECTION WITH L.MONOCYTOGENES
Murine host response to L.monocytogenes has been a useful model for studying protective
immunity against facultative intracelluar pathogens. 13) The Listeria-immune T cells and their related cytokines represent the major mediators that confer protection against the disease. Recently, at least a substantial fraction of y / 6 T eels are reported to recognize common epitopes of
65 kd hsp derived from various bacteria and mammalian cells. 9 ,12) To elucidate a potential roleof the hsp-reactive y / 6 T cells in host defense against Listeria, we examined the kinetics, V
repertoire, specificity and functions of y / 6 T cells during an intraperitoneal infection with
L. monocytogenes.
The number of Listeria in the organs peaked on day 3 and gradually decreased by day S after
infection, CD3+CD4-CDS- cells bearing TcR y/6 in the peritoneal cavity, increased in proportion on day 3 and thereafter decreased by day S after infection, whereas CD3+CD4+CDS- cells
or CD3+CD4-CDS+ cells bearing TcR a/ B increased by day S after infection (Fig. 1). These results suggested that the y / 6 T cells precede the a/ BT cells in appearance during listerial infection, PCR analysis with TcR Vy and V6 primers revealed that the early-appearing y/6 T
Day 3

Day 1
(a)

••0 ".0 •••

J\.

.0'

'0 '

...

I
I
I
I

I •

Day 8
on.

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41,6%

9,9%

43.0%

1
I

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1

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------------CD3 - - - - - - - - - - - (bl...J..'!~'~~l

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I, '.

r,

,

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,
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---,------------- CD8------------Fig. I.

,.'

Three-color FACS analysis of CD3, CD4 and CDS expression in the nonadherent population of PEC obtained from mice after i.p, inoculation with L.monocytogenes, Nonadherent population from the PEC on
day 1, 3 and S after i.p, injection of 1 X 10.1 L.monocytogenes was stained with F1TC-anti-CD3 and PEanti-CD4 mAb and biotin-CDS mAb, then with DuoCHROME-conjugated streptavidin,(a) Relative cell number of CD3+ was presented on day 1,3 and 8 after the infection.
(b) The profile of PE-CD4 and DuoCHROME-CDS was displayed after gating on the CD3+ cells using
forward light scalier to exclude dead cells and red blood cells,

13
HSP-SPECIFIC T CELLS IN HOST DEFENSE

V-gene segment Usage Analysis of 'YO--T Cellsv·o ,No..",.IPEC

..

v· ,
"

.)to

-at·

J,,2

.,

-

. ('..

·pl'
".

".

•

l~

"•.. l:W

Fig. 2.

V-gene segment usage analysis of yl>-T cells. RNA wcre prepared from peptone-induced PEC from normal mice or Listeria-infected mice. RNA were primed either with 10 pmol y chain C region primer or I>
chain C region primer in 20 ~d reaction mixture for reverse transcription. The PCR was performed on a
program temp. control system PC-500 (ASTEC, Japan). The 5' primers are as follows: Vyl/2
ACACAGCTATACATTGGTAC,
Vy2
CGGCAAAAAACAAATCAACAG,
Vy4
TGTCCTTGCAACCCCfACCC,
Vy5
TGTGCACfGGTACCAACTGA,
Vy6
GGAATTCAAAAGAAAACATTGTCT, Vy7 AAGCTAGAGGGGTCCfCTGC, Vol ATTCAGAAGGCAACAATGAAAG, V02 AGTTCCCTGCAGATCCAAGC, Vb3 TTCCTGGCfATTGCCfCfGAC,
VM CCGCTTCTCTGTGAACTTCC, Vb5 CAGATCCTTCCAGTTCATCC, V06 TCAAGTCCATCAGCCTTGTC, VI>7 CGCAGAGCfGCAGTGTAACf. Onc tenth of each y and O-PCR products
were electrophoresed on 1.5% agarose gel and transferred to Gene Screen Plus. The southern blots of yand b-PCR products were hybridized with np-labelJed y or b chain Jbl or J02 probe.

Medium

PPD

*

HSP
HKL

o

5000

10000

15000
CPM

Fig. 3.

Proliferation of yo·T cells stimulated with various antigens. In vitro stimulation of peritoneal yb-T cells in
Listeria-infected mice with mycobacterial antigens. PEC on day 6 were passed through nylon-wool andboth CD4 and CD8 cells were depleted by treatment with anti-CD4 mAb, anti-CD8 mAb and complement. Proliferative responses to PPD, HSP or HKL were measured by 12-h 'H-thymidine incorporation incells (3 x lOS/well) cultured for 72 h in the presence of irradiated (3000R) syngeneic spleen cells (3 x
1O'/well). The counts per minute (c.p.m.) for 'H-thymidinc incorporation were measured in triplicatedcultures. The results are mean ± SO.
*p < 0.005 when compared with the medium-alone control

14
Yasunobu Yoshikaicells preferentially used V yl/Vb6, which is reported to be often used by 65 kd hsp-specific y/
6 T cells (Fig.2).14) To determine the possible ligands for the early-appearing y/[) T cells duringlisteriosis, a y/ 6 T cell-enriched population in the PEC was cultured with heat-killed Listeria,
PPO or 65 kd hsp. A y/ [) T cell-enriched population was obtained by treatment of PEC passed
through nylon-wool, with anti-C04 mAb, anti-C08 mAb and complement. As shown in Fig.3,
the y/ 6 T cells significantly responded to PPO and 65 kd hsp in the presence of syngeneic irradiated spleen cells, while the y/ [) T cells did not proliferate in response to heat-killed Listeria.
The cytokine production was examined in the supernatants of 3-day culture of y/ [) T cells with
PPO/65 kd hsp. A significant level of yIFN was detected in the supernatants of the culture ofy/ 6 T cells with 65 kd hsp, whereas IL- 2 activity in the supernatants was hardly detected (Table1). These results suggest that the early-appearing y/6 T cells during Iisterial infection are specialized to recognize 65 kd hsp and produce yIFN.
Table J.

Cytokine Production of Listeria-primed y&-T Cells Stimulated with
Various Antigens

Antigen

y-INF

IL-2

PPD

1680 Vlml

undetectable"

HSP

940

undetectable

HKL

33

undetectable

Medium66

undetectableTo further investigate the protective role of y/ 6 T cells in listerial infection, TcR a/ ~ T celldepleted mice or TcR y/ 6 T cell-depleted mice were prepared by in vivo treatment with antiTcR a/~ mAb or anti-TcR y/6 mAb, respectively. We confirmed with FCM analysis that the
number of TcR a/ ~ T cells or TcR y/ [) T cells remained at an undetectable level in the peripheral lymphoid tissues by day 8 after an intraperitoneal injection of 100 ~g purified anti-TcR a/~mAb or anti-TcR y/6 mAb. A sublethal dose of viable Listeria was injected into the peritoneal
cavity of mice three days after treatment with anti-TcR a/~ mAb or anti-TcR y/[) mAb, andthe kinetics of bacterial growth were examined at various intervals after infection. As shown in
FigAa, the number of Listeria in the spleen of TcR a/~ T cell-depleted mice on day 3 after infection was much the same as that of untreated mice at this stage, while a significantly increased
number of Listeria were detected in TcR y/ [) T cell-depleted mice as compared with those in
TcR a/~ T cell-depleted mice and untreated mice at this stage after infection. The number ofbacteria in the untreated mice decreased to an undetectable level by day 8 after infection. On
the other hand, an appreciable number of bacteria remained in TcR a/~ mAb-treated mice onday 8 after infection in spite of the presence of TcR y/ 6 T cells. On the other hand, Listeria
were completely eliminated in TcR y/6 T cell-depleted mice at this stage (FigAb). These results
suggest that the early- appearing y/ 6 T cells may participate in host defense at the early stage ofIisterial infection. The notable findings in the present study are as follows:
1) The y / [) T cells precede the a/ ~ T cells in appearance in the peritoneal cavity during an intraperitoneal infection with Listeria.
2) The early-appearing y/[) T cells display a limited Vy/V[) gene usage such as Vyl/Vb6.
3) The y/ [) T cells are specialized to recognize 65 kd hsp but not heat-killed Listeria.
4) The early-appearing y/6 T cells produce yIFN in response to 65 kd hsp.
5) The y/ 6 T cell-depleted mice show impaired protection at the early stage after listerial infection.

15
HSP-SPECIFIC T CELLS IN HOST DEFENSE

A)
lOR

10

7

--0--

Control·····0····

H57-597(anti-TeR a~ )r::treatedQ)~6
0.' 10

en

--

~

U.

U

10

5

/

..

l'O

.;:

Q)

f Il

'£" '.

i'

10

4

10

3

'.

·····£··············f.................:

j

-I

10

2

0

10

5

15

20

25

Days after infection

B)
10

r::

10

8

...................

7

en

--

UC7-13D5(anti-TcR yo)

Q)
Q)

0.

Control
treated

10 6

~

u.

U

10

5

l'O

.;:

Q)

fIl

:.:i

10

4

J03

10

2

0

5

10

15

Days after infection

Fig. 4.

Effects of in vivo administration of anti-TCR a~ (H57-597) (A) or anti-TCR yO (B) on recovery of bacteria from spleens. Female C3H mice were inoculated intraperitoneally with 3 x 10 3 of L.monocytogenes
on day 0 and with 200 lAg of anti-TCR a~ mAb or 200 lAg of anti-TCR yO mAb on day 3. The number of
Listeria recovered from spleens of infected mice on the indicated days were determined by colony formation assay on TSA. Values are means ± SD for a group of five mice.

16
Yasunobu Yoshikai

Listeriosis is caused by the gram-positive rod, L.monocytogenes, which is one of the intracelluar bacteria as well as Mycobacterium tuberculosis. The protective mechanisms against listeriosis are largely divided into two phases. 15-17) The early response, which occurs during the first
48 h, is attributed to resident macrophages and early influx of bone marrow-derived phagocytes
in the liver and spleen. The late response, beginning at about four days, is characterized by the
proliferation of Iisterial antigen-dependent T cells which further enhance bacterial killing invivo. The early-appearing y/6 T cells may playa role in covering the gap between the phagocytic systme and the highly evolved type of immune responses mediated by a/0 T cells in hostdefense against listerial infection.
Hsp are polypeptides phylogenically highly conserved between eukaryotes and prokaryotes. I ,2) Under a variety of stress conditions such as heat shock, nutrient deprivation, and oxigen radicals, eukaryotic cells have been shown to produce stress proteins to preserve cellularfunctions.;;) Recently, Rajasekar et al. have reported that a subset of murine y/6 T cells canreact to antigens on self cells in which a heat-shock response is induced. 12 ) Born et al. haveshown that the PPO-specific y/ 6 T cell hybridomas derived from murine newborn thymus can
respond to the equivalent portion of the autologous homolog to 65 kd mycobacterial hSp.~·lX)
Thus, it is possible that the early-appearing y/ 6 T cells during listeriosis may recognize endogenous hsp of autologous cells infected with Listeria as well as exogenous hsp derived fromviable Listeria.
Our results with y/ () T cells during listeriosis indicate that at least a subset of y/ () T cells provides a first line of defense against infection, by recognizing 65 kd hsp and producing cytokines
such as yIFN. Recently, we have found that the y/6 T cells precede a/0 T cells in appearance
during infection with Bacilllus Calmette Guerin (BCG).l~) It may be generalized that y/6 T cells
may appear first in the infected site and the early-appearing y/ 6 T cells may serve as a first lineof defense against at least some infectious agents. Our finding is an important clue not only in
elucidating the possible ligands for y/ () T cells but also in understanding the functional role ofy/ 6 T cells in host defense mechanisms.

THE ROLE OF HSP-SPECIFIC y/6 T CELLS AND a/~ T CELLSIN INDUCTION OF AUTOIMMUNE DISEASES
Although T cells proliferate and differentiate primarily in the thymus, several studies with athymic nude mice and neonatally thymectomized (NTX) mice have revealed that an extrathymic
pathway exists in T cell development. 20-2;;) Aged nude mice and NTX mice are reported to develop autoimmune diseases often. 26 .27 ) To elucidate the mechanism of induction of the autoimune diseases in these mice, we have investigated the fate of self-reactive T cells and the
responsiveness of T cells differentiating outside the thymus to 65 kd hsp. 2X.2~)
To confirm that T cells develop along an extrathymic pathway, we conducted FCM analysis
for TcR/C03 on LN cells of aged BALB/c nude mice and BALB/c NTX mice. An appreciable number of C03+ cells were detected in the LN cells of both aged nude mice and aged NTX
mice (6 to 9 months old). Expression of TcR on the T cells in these mice was analyzed bydouble staining with anti-Thy1.2 mAb and anti-TcR a/0 mAb or anti-TcR y/b mAb. As
shown in Fig.5a, and appreciable nubmer of TcR y/ () T cells were detected in the aged nude
mice, while most of the T cells in the NTX mice expressed TcR a/0 on their surface (Fig.5b).
To examine the TcR repertoire of the TcR a/0 T cells differentiating outside the thymus innude mice and NTX mice, the LN cells were double stained with anti-Thy1.2 mAb and various
anti-V0 mAbs including anti-V03, anti-V06, anti-V08 and anti-V011 mAb. Consistent with

17

HSP-SPECIFIC T CELLS IN HOST DEFENSEBALB I c

0.7%

/f

rio

- - , .. ,Aged nu I nu

\11 ,,,,,"-(~(-f11"'T-_or" '''''1-,,'11,1\,

98.5%

0.2%

13.9%

.J

. Llf,""aFluorescence intensity

18
Yasunobu Yoshikai

BALB I c

NTX

96.6%

98.9%

ull

0.2%

,.t.----r-rn~"Tlll

10.5%

11.8%

20.7% 14.1 %b

Fig. 5.

Cell surface expression of TcR a/~, TcR y/6 or V~s on Thyl + cells in the LN cells from euthymic control
BALB/c mice, aged nude mice (A) or NTX mice (B). The whole LN cell was stained with FITC-anti
V~3, anti- VfJ6, anti- V08.1 +8.2, anti- V~ II mAb or anti-TCR a~ mAb, anti-TCR y6 mAb plus FITCami-hamster IgG and PE-anti-Thy1.2 mAb.

19

HSP-SPECIFIC T CELLS IN HOST DEFENSE

the earlier findings,311.31) euthymic BALB/c control mice deleted V~3- or V~lI-bearing T cells
capable of recognizing Mls2 a plus MHC class 2IA/lE, or MTV(mammary tumor virus)-related
antigens plus IE, respectively, in a mature T cell pool as a result of intrathymic clonal deletion. 32 .33 ) On the other hand, an appreciable number of V~ II-bearing T cells were present in the
LN of both the nude mice and NTX mice. Although the V~3-bearing T cells were eliminated in
the LN of NTX mice, V~3-bearing T cells were present in the LN of nude mice (Fig.5a,b).
These results suggest that T cells differentiating along an extrathymic pathway have not undergone negative selection and that timing of clonal deletion may be different in each self-reactive
T cell population. 34 )To investigate the functional aspect of the self-reactive T cells in nude mice and NTX mice,
we tried to activate the V~3- or V~ II-bearing T cells by SEA, a superantigen that specificallystimulates T cells bearing V~3 or V~ 11 irrespective of the a chain expressed by these T cells. 3:;)V~3-and V~ II-bearing T cells in the nude mice and V~ II-bearing T cells in the NTX miceproliferated significantly in response to SEA in the presence of exogenous IL- 2 (data notshown). No V~3-bearing T cells proliferated in response to SEA because of tolerance-induced
deletion of self-reactive T cells. Thus, the self-reactive T cells in the nude mice and NTX mice
can normally respond to stimulation via their TcR and be functional.
It is well known that NTX mice develop multiple organ-localized autoimmune diseases. 27 )Nude mice are also known often to develop autoimmune diseases. 27 ) To confirm whether aged
nude mice and NTX mice spontaneously develop autoimmune diseases, we investigated theTiter of anti-DNA antibody in serum

BALB Ie
NTX

a

1000

2000

3000

10 000

20 00030 000nu 1+ (8w)
nu I nu (8w)

nu I nlj ( 28-36w )b

Reciprocal tiler
Fig. 6.

Titers of anti-DNA antibody in the serum of BALB/c control mice, NTX mice (A) or aged nude mice
(B). Anti-DNA titers in the serum were measured by ELISA. Each column and horizontal bar represent
the mean of three individual mice ± SD.

20
Yasunobu Yoshikai

level of anti-DNA antibody in the serum and the stomach histology of these mice. As shown in
Fig.6a, anti-DNA titers in sera of NTX mice were much higher than those in the control euthymic mice. Histological examination revealed that the NTX mice had gastritis characterized by
infiltration of many mononuclear cells into the mucosa and submucosa of the stomach (data notshown). Although aged nude mice bred under SPF condition did not develop autoimmune disease, a substantially higher titer of anti- DNA antibody was detected in the sera of the aged nude
mice bred under conventional conditions (Fig.6b).
To determine whether T cells capable of recognizing 65 kd hsp are involved in teh development of autoimmune diseases in aged nude mice and NTX mice, we tested the reactivity of LN
cells to recombinant 65 kd hsp derived from Mycobacterium bovis in aged nude mice and NTX
mice. The LN cells of the nude mice and NTX mice proliferated notably and produced a higher
level of IL-2 in response to the 65 kd hsp as compared with those in normal mice (Fig.7a,b).
These results suggest that aged nude mice and NTX mice may contain an increased number of
65 kd hsp-specific T cells in their mature T cell pool. To investigate which population of T cells
proliferate in response to 65 kd hsp in these mice, we examined by FCM the responder cells
after culture with 65 kd hsp. As shown in Fig.8a, the proportion of y/ 6 T cells in aged nude
mice bred under conventional conditions substantially increased after stimulation with PPD, recombinant 65 kd hsp, or trypsin-treated hsp. PCR analysis with Vy and V6 primers revealed
that the 65 kd hsp-specific y/6 T cells preferentially expressed Vyl/VM (data not shown). In
case of NTX mice, hsp-responding cells were mainly TcR a/ ~ T cells rather than TcR y/ 6 T
cells. Among TcR a/ ~ T cells, V~ ll-bearing T cells proliferated greatly after culture with the
65 kd hsp, whereas the proportions of other T cells bearing V~6 or V~8.1+8.2 did not increase(Fig.8b). To further determine whether V~ ll-bearing T cells enriched after culture with the 65
cpm

1000

cpm

.--

-

CI>
:,t;

<1J

50000

a.
:>
Q>

c

'0

'E

>-

.<:

f-

I

10000

c::o...

5000~L~lJI •• ••8

Fig. 7.

xl

x 1/2 x 1/4 x 1/8 x 1/16 b

b 1/2

1 /4

1 /8

1 /16

1/32

1/64

IL-2 production of the LN cells from aged nude mice (A) or NTX mice (B) stimulated with 65 kd hsp.
CfLL-2 cells (2 x 104 ) were incubated with diluted supernatants from the cultures in which LN cells were
stimulated with 65 kd hsp (25 f.lg/ml) in 0.2 ml medium at 37C for 48 h, and then pulsed with If.lCi [.1H]dThd. Cultures werc incubated for another 6 h and cells were harvested.

21
HSP-SPECIFIC T CELLS IN HOST DEFENSE

Aged nu / nu

BALB / c

1,

Before culture

0.0%~W~~"-""--I"""'--~"""6""'" ~~After culture PPD

!fi

;
2.8%

~

~
t

•

I

I

m
n

...... .OJ

'I ;\

hsp

42.5%

16'

..

14.1 %:~ ,,~.. ~7.3%

Tryp-hsp

Ii.

'1/\

(. )

a

Fig. 8.

I•

0.1 %:~\-'.-.,.~-(A) Responsiveness of normal BALB/c (a) or aged nude mice (b) y/b T cells to PPD or mycobacterial
hsp. LN cells from aged nude mice were cultured with PPD (30 ~g/ml), recombinant 65 kd of M.bovis(25~g/ml) or trypsin-digested (Try-hsp) hsp (25~g/ml) for 3 days. Cultured cells were stained with antiTcR y/6 mAb and with anti-Thy-1.2. Thy-1.2- cells were gated out and shown are histograms ofTcR y/
6 expression of Thy-1.2+ cells.

22
Yasunobu Yoshikai

Before culture

1.P

After culture with hsp

3.4

12.6

16.3

24.4

b

Fig. 8.

(8) Activation of VIHI-bearing T cells in NTX mice by 65 kd hsp. The LN cells (l x IOt'/ml) from
NTX mice were cultured with the 65 kd hsp at 25 ~lg/ml for three days. Cultured cells were stained with
anti- Vp mAb, anti-VB6 mAb, anti- VB8.1 +8.2 mAb, or anti- VI)11 mAb plus FlTC-anti-hamster or antirat IgG and PE-anti-Thy-1.2 mAb, FITC-anti-CD8 mAb, PE-anti-CD4 mAb.

23
HSP-SPECIFIC T CELLS IN HOST DEFENSE

kd hsp were really in an activated state, the size of the V[311-bearing T cells was assessed bytheir forward light scatter profile. Approximately half of the VB II-bearing T cells were blastoid
cells, whereas VB8.1 +8. 2-bearing T cells were small. (data not shown). Thus, VB II-bearing T
cel1s may proliferate substantially after culture with 65 kd hsp.
A notable finding in the present sutdy was that the aged nude mice and NTX mice that developed autoimmune diseases contained a large number of 65 kd hsp-specific T cells in their
peripheral lymphoid tissues. In aged nude mice, the hsp-responding T cells are mainly Vyll
VM-bearing T cells, whereas self-reactive VBll-bearing T cells in NTX mice increased in number after culture with the 65 kd hsp. Both T cel1s seem to produce a high level of IL- 2 in response to 65 kd hsp. Hsp are polypeptides phylogenetical1y conserved between prokaryotes andeukaryotes. 2) We speculate that the 65 kd hsp-specific T cells may be activated by hsp expressedby bacteria and autologous cells stressed with various pathogens, resulting in the development of
autoimmune diseases in such animals.
Several lines of evidence have shown that tolerance to self-antigens is mainly due to intrathymic clonal deletion of self-reactive T cells. 1IU1 ) Our results reveal that a substantial number ofself-reactive T cells such as VB II-bearing T cells were present in the LN of nude mice and NTX
mice. These self-reactive T cells can respond normally to signals via the TcR in the presence ofexogenous IL- 2. Our results also revealed that the self-reactive VB 11-T cells in NTX mice proliferate substantially after culture with 65 kd hsp. These T cells expressed a high level of IL-2Ra
even before stimulation. 28 ,29) Therefore it is possible that hsp-reactive T cells other than VB 11bearing T cells may respond to the 65 kd hsp and subsequently produce a stimulus such as IL-2
for proliferation of the self-reactive VB II-bearing T cells. In case of aged nude mice, hspresponding cells are found mainly in y/b T cells expressing VyllVM. At present, we can notdetect any increase in the number of particular T cells including y I 6 T cells other than VB 11bearing T cells in the NTX mice. Recently we have found that an autoreactive T cel1line specificfor self MHC class II recognized 65 kg hsp.3h) Therefore, it is alternatively possible that VB 11bearing T cells in NTX mice can directly recognize the 65 kd hsp in the context of a self MHCmolecule. The reactivity of VB II-bearing T cel1s to the 65 kd hsp should be determined at aclonal level to clarity this possibility.
The self-reactive T cel1s are rendered tolerant mainly by clonal energy and clonal suppression
via suppressor cells. 37 ,38) although recent studies reveal that clonal deletion occurs extrathymically.39.41) It has been reported that autoimmune diseases in NTX mice were clearly prevented
by the reconstitution of these mice with T cells from syngeneic, normal animals. A deficit ofsuppressor T cells may be a cause of autoimmune disease in NTX mice. 42 ) The high reactivity ofT cells to the 65 kd hsp in NTX mice may result from depletion of a specific T cell subpopulation responsible for checking and controlling the hsp-specific T cells which is normally presnet
in the mature T cell pool in euthymic mice. An increase in the number of 65 kd hsp-specific T
cel1s was evident in athymic nude mice bred under conventional conditions. Environmental antigens such as those of intestinal microflora may also play an important role in the expansion of
the hsp-specific T cells in aged nude and NTX mice.

CONCLUDING REMARKS
Our results presented here suggest that 65 kd hsp-specific T cel1s play an importnat role not
only in induction of autoimmune diseases but also in the host defense mechanism against infection by various pathogens. During infection with L.monocytogenes, TcR y/6 T cells specific for
65 kd hsp precede TcR alB T cells specific for the listerial antigen in appearance. The y/6 T

24
Yasunobu Yoshikai~\.icytokines

IL-2------~~~Ag unresponsivenessFig. 9.

autoimmunity

Ag responsiveness

Model of roles of hsp-specific T cells in host defense against bacteiral infection and induction of autoimmune disease.

cells seem to provide a first line of defense against the infection by recognizing exogenous andendogenous 65 kd hsp on infected cells and producing cytokines such as yIFN. Thus, the hspspecfic T cells may respond quickly to antigenically diverse pathogens before antigen-specific T
cells expand clonally, and they may playa role in covering the gap between the phagocytic system and the highly evolved type of immune responses (Fig.9).4J.4.l)Athymic mice such as nude mice and NTX mice contain an appreciable number of self-reactive T cells that have not undergone inrathymic clonal deletion. The self-reactive T cells express
IL2 receptor and respond normally to signals delivered through the TcR in the presence ofexogenous IL2. Notably, a substantially higher level of hsp reactivity was observed in the LN
cells in such animals, especially those bred under conventional conditions. In aged nude mice,y/ b T cells greatly proliferated and produced IL2 in response to 65 kd hsp. On the other hand,the self-reactive V~ll-bearing T cells notably proliferated and a high level of IL-2 was detected
after culture with 65 kd hsp in the NTX mice. Both groups of animals spontaneously developed
autoimmune diseases. Taking all the previous results into consideration, we speculate that 65 kd
hsp-specific T cells may abrogate the unresponsiveness of the self-reactive T cells and/ or B cells
by producting IL-2 and that they may play an important role in the induction to autoimmune
diseases (Fig.9).

25
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