autoreactive cd4- cd8- ajpt cells to vaccinate adjuvant arthritis

7
Immunology 1998 94 536-542 Autoreactive CD4 - CD8 - ajp T cells to vaccinate adjuvant arthritis M. A. HAQUE,* M. KIMOTO,t S. INADAt 0. TOKUNAGA§ & 0. KOHASHI¶ Departments of *Microbiology, tlmmunology and §Pathology, Saga Medical School, Nabeshima, Saga, Japan, ¶Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, USA, and tDepartment of Obstetrics and Pediatrics, Division of Human Genetics, University of Maryland, Baltimore, USA SUMMARY Studies suggested that experimental autoimmune diseases can effectively be prevented and treated by application of normal autoreactive T cells or autoreactive T cells in an attenuated form. In this study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolated for the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA). Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected rats from AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8- ap T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen- presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) and staphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacterium tuberculosis (Mt), or its 65000MW heat-shock protein (HSP). Rats protected from AA by inoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reaction compared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AA can be suppressed by autoreactive CD4- CD8- 4p T cells, and these cells may be used as therapeutic agents in experimental autoimmunity. INTRODUCTION The effectiveness of T-cell vaccination has been demonstrated in a variety of animal models of both induced and spontaneous autoimmune disease.1-4 T-cell vaccination can be highly specific and is likely to be non-toxic, because it exploits the immune system's own ability to control its autoimmune aber- ration.3'4 Organ-specific autoimmune diseases are caused by autoreactive T cells that attack the normal tissues of the individual.",2 Although activated, autoreactive T cells are potentially pathogenic, experimental autoimmune diseases can effectively be prevented and treated by application of autoreac- tive T cells in a normal or an attenuated form.3-6 Adjuvant arthritis (AA), a chronic inflammatory disease of the joints, is induced in rats by immunization with Mycobacterium tuberculosis (Mt).7'8 It has been demonstrated that clones of T lymphocytes reactive to Mt and 65 000 MW mycobacterial heat-shock protein (HSP) can not only induce but also pre- vent AA"',9"'0 Received 9 January 1998; revised 17 March 1998; accepted 6 April 1998. Abbreviations: AA, adjuvant arthritis; Mt, Mycobacterium tubercu- losis; HSP, heat-shock protein; DTH, delayed-type hypersensitivity; APC, antigen-presenting cells, TCR, T-cell receptor. Correspondence: Dr M. A. Haque, Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive (MS 255), Indianapolis, IN 46202, USA. Recently, CD4- CD8- oc4 T cells have been identified as rare subpopulations of mature T cells.`-3 The most striking characteristic of these novel CD4- CD8- oxI T cells are their highly restricted repertoire of VP gene usage, such that a single VP gene family (V138) is expressed on the majority of the cells.14" 5 However, the biology of these cells is still unknown. We have isolated several autoreactive CD4- CD8- acp T cell clones from the inguinal lymph nodes of Lewis rats with adjuvant arthritis, and an investigation was done to test whether these cells could be applied to vaccinate AA. MATERIALS AND METHODS Animals Inbred Lewis rats were obtained from Charles River Inc., Tokyo, Japan. Rats were housed at the Saga Medical School Animal Care facilities, fed a sterile commercial diet and water ad libitum, and maintained as described previously.'6 At the age of 3-4 weeks females and males were separated and only females (7-10 weeks) were used in the experiments. Antigens and adjuvant The 65 000 MW mycobacterial heat-shock protein (HSP) was a gift from Dr H. Nomaguchi.17 Mycobacterium tuberculosis (Mt) and incomplete Freund's adjuvant (IFA) were purchased from Difco Laboratories, Detroit, MI. Complete Freund's adjuvant (CFA) was prepared by suspending 10 mg Mt ground to a fine powder in 1 ml IFA. Concanavalin A (Con A) was 56 1998 Blackwell Science Ltd 536

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Page 1: Autoreactive CD4- CD8- ajpT cells to vaccinate adjuvant arthritis

Immunology 1998 94 536-542

Autoreactive CD4 - CD8 - ajp T cells to vaccinate adjuvant arthritis

M. A. HAQUE,* M. KIMOTO,t S. INADAt 0. TOKUNAGA§ & 0. KOHASHI¶ Departments of *Microbiology,

tlmmunology and §Pathology, Saga Medical School, Nabeshima, Saga, Japan, ¶Department ofMicrobiology and Immunology,Indiana University School ofMedicine, Indianapolis, USA, and tDepartment of Obstetrics and Pediatrics, Division ofHuman

Genetics, University ofMaryland, Baltimore, USA

SUMMARY

Studies suggested that experimental autoimmune diseases can effectively be prevented and treatedby application of normal autoreactive T cells or autoreactive T cells in an attenuated form. Inthis study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolatedfor the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA).Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected ratsfrom AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8-ap T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen-presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) andstaphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacteriumtuberculosis (Mt), or its 65000MW heat-shock protein (HSP). Rats protected from AA byinoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reactioncompared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AAcan be suppressed by autoreactive CD4- CD8- 4p T cells, and these cells may be used as

therapeutic agents in experimental autoimmunity.

INTRODUCTION

The effectiveness of T-cell vaccination has been demonstratedin a variety of animal models of both induced and spontaneousautoimmune disease.1-4 T-cell vaccination can be highlyspecific and is likely to be non-toxic, because it exploits theimmune system's own ability to control its autoimmune aber-ration.3'4 Organ-specific autoimmune diseases are caused byautoreactive T cells that attack the normal tissues of theindividual.",2 Although activated, autoreactive T cells arepotentially pathogenic, experimental autoimmune diseases caneffectively be prevented and treated by application of autoreac-tive T cells in a normal or an attenuated form.3-6 Adjuvantarthritis (AA), a chronic inflammatory disease of the joints,is induced in rats by immunization with Mycobacteriumtuberculosis (Mt).7'8 It has been demonstrated that clones ofT lymphocytes reactive to Mt and 65 000 MW mycobacterialheat-shock protein (HSP) can not only induce but also pre-vent AA"',9"'0

Received 9 January 1998; revised 17 March 1998; accepted6 April 1998.

Abbreviations: AA, adjuvant arthritis; Mt, Mycobacterium tubercu-losis; HSP, heat-shock protein; DTH, delayed-type hypersensitivity;APC, antigen-presenting cells, TCR, T-cell receptor.

Correspondence: Dr M. A. Haque, Department of Microbiologyand Immunology, Indiana University School of Medicine, 635 BarnhillDrive (MS 255), Indianapolis, IN 46202, USA.

Recently, CD4- CD8- oc4 T cells have been identified asrare subpopulations of mature T cells.`-3 The most strikingcharacteristic of these novel CD4- CD8- oxI T cells are theirhighly restricted repertoire of VP gene usage, such that a singleVP gene family (V138) is expressed on the majority of thecells.14" 5 However, the biology of these cells is still unknown.We have isolated several autoreactive CD4- CD8- acp T cellclones from the inguinal lymph nodes of Lewis rats withadjuvant arthritis, and an investigation was done to testwhether these cells could be applied to vaccinate AA.

MATERIALS AND METHODS

AnimalsInbred Lewis rats were obtained from Charles River Inc.,Tokyo, Japan. Rats were housed at the Saga Medical SchoolAnimal Care facilities, fed a sterile commercial diet and waterad libitum, and maintained as described previously.'6 At theage of 3-4 weeks females and males were separated and onlyfemales (7-10 weeks) were used in the experiments.

Antigens and adjuvantThe 65 000 MW mycobacterial heat-shock protein (HSP) wasa gift from Dr H. Nomaguchi.17 Mycobacterium tuberculosis(Mt) and incomplete Freund's adjuvant (IFA) were purchasedfrom Difco Laboratories, Detroit, MI. Complete Freund'sadjuvant (CFA) was prepared by suspending 10 mg Mt groundto a fine powder in 1 ml IFA. Concanavalin A (Con A) was

56 1998 Blackwell Science Ltd536

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Vaccination against AA with CD4- CD8- 43# T cells

purchased from Sigma (St Louis, MO), and staphylococcalenterotoxin B (SEB) was a gift from Dr T. Uchiyama(Department of Microbiology and Immunology, TokyoWomen's Medical College, Tokyo, Japan).

Induction ofarthritisAdjuvant arthritis was induced by intradermal injection at thebase of the tail with 0-1 ml CFA containing 10 mg/ml Mt.

Clinical evaluation ofarthritisThe development of arthritis was assessed by standard method-ology.18 Rats were observed daily for the presence of clinicalsigns of AA. All clinical evaluations were performed in ablinded fashion. Clinical observations were then confirmed byhistological analysis.

Cloning ofT cellsRats (7-8 weeks) were immunized with Mt (10 mg/ml in IFA)by an intradermal injection into the base of the tail. Elevendays after immunization, inguinal lymph nodes were separatedand a single cell suspension was prepared. These lymph nodecells (LNC; 6 x I06/ml) were cultured in RPMI-1640 (FlowLaboratories, Paisley, UK) containing 100 U/ml penicillin,100 jg/ml streptomycin, 5 x 10- M 2-mercaptoethanol, 1%non-essential amino acid, and 10% heat-inactivated fetal calfserum (FCS) (Intergen, Purchase, NY) in 24-well plate(Falcon, 3047; Becton-Dickinson, Franklin Lakes, NJ) in thepresence of 5 jg/ml Mt. A line reactive to Mt was first isolatedby repeated stimulation with Mt, and then it was cloned bylimiting dilution in the presence of irradiated (3000 rad)syngeneic spleen cells as antigen-presenting cells (APC), andCon A-stimulated spleen cell supernatant (CAS). Mixtures of0-1-10 cells, 1 x 106 APC, and 20% (v/v) CAS were culturedin 200 gl of complete medium in 96-well plates (Falcon, 3072).Positive wells were transferred into 24-well plates which con-tained 1 x I07 APC, and 10% CAS in 1-5 ml complete medium.Cloned cells were maintained by resting culture and restimula-tion cycle as described previously.'9

Proliferation assayT-cell clones (5 x 104) were cultured with 5 x 106 irradiatedAPC (3000 rad) in 96-well flat bottomed plates containing200 jil complete medium in the presence of Mt (5 jg/ml), andmycobacterial 65000 MW HSP (5 jg/ml). After 48 hr ofculture, 1 gCi of ['HI thymidine (ICN Biomedicals Inc., CostaMesa, CA, USA) was added to each well and incubation wascontinued for 18 hr. Cells were harvested using a cell harvester,and radioactivity was determined by liquid scintillationcounting.

Fluorescence-activated cell sorting (FACS) analysisT-cell clones were cultured for 7 days and viable cells wereseparated from cellular debris by centrifugation on a Ficollgradient. The viable cells (5 x 105) were washed in stainingbuffer (0-1% FCS and 0 1% NaN3 in phosphate-bufferedsaline; PBS), and stained with appropriately diluted anti-CD3(Seikagaku Corp., Tokyo, Japan), CD4, CD8, ar4 T-cell recep-tor (TCR; Serotec, Oxford, UK), y6 TCR (PharMingen, SanDiego, CA), and IL-2R (Serotec) for 20 min on ice. Afterwashing with cold staining buffer, cells were further incubatedwith protein A-fluorescein isothiocyanate (FITC; Zymed, San

Francisco, CA). For staining of CD3, cells were incubatedwith appropriately diluted anti-CD3 (mouse immunoglobulinM; IgM). The second antibody was biotinylated goat anti-mouse IgM followed by streptavidin-FITC. Twenty microlitresof 10 gg/ml propidium iodide (Sigma) was added for the last10 min. Cells were washed twice with staining buffer andanalysed by FACScan (Becton Dickinson ImmunocytometrySystems, Mountain View, CA). Dead cells were gated outfrom the analysis.

T-cell vaccinationTo investigate the role of autoreactive T cells in the suppressionof AA, A2, A6, Al 3 cells and spleen cells (SPC) were activatedby culture with Con A for 72 hr. Then the cells were washedand suspended in PBS, and either A2, A6, A13, T-cell blasts(3 x 107 cells/rat), or PBS was injected intraperitoneally intonaive Lewis rats (9-10 rats/group). Three weeks later, ratswere re-inoculated with A2, A6, A13, T-cell blasts (2 x 107cells/rat) and PBS, respectively. In another experiment, rats(six rats/group) were injected once with 1 x 107 cells (A2, A6,A13, T-cell blasts) and PBS. Four weeks after the first inocu-lation ofT cells, all rats were immunized with Mt to induce AA.

Delayed-type hypersensitivity testsA2, A6, A13 cells and T-cell blasts were stimulated with ConA for 72 hr. Naive female Lewis rats were intraperitoneallyinoculated with either A2, A6, A13 cells, T-cell blasts (3 x 107cells/rat) or PBS. Three weeks later all rats were re-inoculatedwith the same cells (3 x 107 cells/rat) or PBS, as describedabove. Four weeks after the first inoculation of T cells, ratswere immunized with Mt to induce AA. Seven days afterimmunization, activated irradiated (2500 rad) T cells (2 x 10'cells in 50 il PBS) were subcutaneously injected into the leftear, following respective groups. Ear thickness was measuredin a blinded fashion before and 48 hr after challenge, using a

micrometer caliper (Ozaki MFG. Co., Tokyo, Japan). Changesin thickness before and after challenge were recorded for eachanimal and results expressed as the mean for each experimentalgroup± SEM; each group consisted of six rats.

HistologyTo confirm the suppression of AA by Al 3 cells as observedin clinical evaluation, naive rats were intraperitoneally inocu-lated with either PBS or A13 cells (only one injection with1 x 107 cells/rat). Four weeks later, rats were immunized withMt to induce AA. Twenty-one days after immunization, ratswere killed and the tissue specimens were fixed in 10% formalin,decalcified in formic acid, and stained with haematoxylin andeosin as described previously.16'20 Each group consisted ofsix rats.

RESULTS

Autoreactivity and the proliferative responses to variousstimulants

At first we isolated seven T-cell clones from the Mt-reactiveT-cell line and selected three clones (A2, A6 and A13 cells)based on similar proliferative responses to various antigens,and phenotypic markers, and investigated the effects of thesecells on AA. As observed in the proliferation assay, A2, A6and A13 cells showed marked proliferative responses to

C 1998 Blackwell Science Ltd, Immunology, 94, 536-542

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M. Azizul Haque et al.

syngeneic spleen cells demonstrating that the cells were auto-reactive. The cells did not proliferate against Mt or its65 000 MW HSP although the original line was raised againstMt. However, the cells showed high proliferative responses toCon A and SEB in the presence of APC (Table 1).

Phenotypic analysis of autoreactive T cells

Flow cytometric analysis of A13 cells demonstrated that thecells expressed CD3, oc4 TCR and interleukin-2R (IL-2R), butdid not express CD4, CD8, and y6 TCR (Fig. 1). A2 and A6cells also showed similar phenotypic markers (data not shown).

Suppression of AA by inoculation with A13 cells

To test the ability of autoreactive CD4- CD8- c43 T cells tosuppress AA, rats were inoculated with either A2, A6, or Al 3cells before induction of the disease. Rats received either PBS,

A2, A6 or T-cell blasts developed severe arthritis that persistedfor 45 days. Surprisingly, rats inoculated with A13 cells (atfirst 3 x IO' and then 2 x IO' cells) were markedly protected,although some rats (three out of nine) developed mild arthritis(Table 2). Intraperitoneal inoculation of A13 cells also inducedearly remission of AA, as well as resistance to subsequentdisease. Only one injection of as low as 1 x IO' cells alsoresulted in marked suppression of AA (Fig. 2).

Histological analysis

Intraperitoneal inoculation of as low as 1 x 107 A13 cells (byonly one injection with 1 x 107 cells) suppressed the develop-ment of AA which was confirmed by histological examination(at day 21 after immunization) (Fig. 3). Acute fibrinous exu-date and marked synovial thickening in the affected joint spaceof control rats were extensive (Fig. 3a), but no fibrinousexudate and slight or no synovial thickening were observed in

Table 1. Proliferative responses of T cells isolated from Lewis rats with AA

Proliferative responses(mean c.p.m. x 103)

Cells Med APC Mt HSP SEB Con A

A2 047+003 5535+27 5988+35 5958+34 21967+158 261 12+233A6 046+002 5831 +30 61 43 +34 5922+24 251 75+203 24406+21 1A13 045 +008 5861 +1 6 61 16+34 6035+20 214 19+12-0 255-46+128

A2, A6 or A13 cells (5 x 104) were cultured with either Mt (5 jg/ml), 65 000 MW HSP(5 ptg/ml), SEB (1 pig/ml) or Con A (1 ptg/ml) in the presence of APC (5 x 106) in completeRPMI for 48 hr as described in Materials and Methods. Proliferative responses weremeasured by the incorporation in 1 pCi of [3H ]thymidine pulsed for the last 18 hr of culture.

see

CD4

11 'a! I.,I, ,2,l a Is s2 to

Log fluorescence intensity

Figure 1. Phenotypic analysis of A13 cells. Cells obtained more than 1 week after restimulation were purified on Ficoll-Paque toremove feeder cells and cell debris. The viable cells (5 x 105) were then stained with appropriately diluted anti-CD3, -CD4, -CD8,y6 TCR, c,4 TCR, and IL-2R followed by protein A-FITC. For staining of CD3, cells were incubated with appropriately dilutedanti-CD3 (mouse IgM) for 20 min. The second antibody was biotinylated goat antimouse IgM followed by streptavidin-FITC.T cells were also directly stained with FITC-labelled anti-y6 TCR. Twenty microlitres of 10 ptg/ml propidium iodide (Sigma) wasadded for the last O min, and cells were analysed by FACScan. Dead cells were gated out from the analysis. The dotted linerepresents control.

c 1998 Blackwell Science Ltd, Immunology, 94, 536-542

538

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Vaccination against AA with CD4- CD8- 4x# T cells

Table 2. Inoculation of A13 cells protects rats from AA*

Clinical evaluation of AA

Maximum% protection Mean day Duration clinical

Cells Stimulation (no. rats) of onset (days) score

A2 Con A 0 (10) 12-5 30-45 11-3A6 ConA 0(9) 12-0 30-45 11-0A13 Con A 66 (9) 19-0 10-20 48SPC Con A 0 (10) 12-8 30-45 117

*A2, A6, A13 cells or SPC were stimulated in vitro with Con A for 72 hr, and washedtwice with phosphate-buffered saline. The cells were injected intraperitoneally (twice) intonaive rats as described in Materials and Methods. One week after the last injection, ratswere tested for susceptibility to induction of AA by immunization with Mt. Rats werechecked and clinical score was given to the four paws as described.

-0-

-0-

.. . . 15, . . . . . . .

5 10 15 20 25

DTH reactions to A13 cells (Fig. 4). Control rats which wereinoculated with either PBS or T-cell blasts did not show anti-idiotypic DTH reactions. A13-cell inoculated rats, whichshowed a marked DTH reaction to A13 cells, were resistantto AA. In contrast, rats inoculated with either A2 or A6 cells,which did not show any significant anti-idiotypic DTH reac-tions were susceptible to AA. These findings indicated a goodrelationship between anti-idiotypic DTH responses and thesuppression of AA. To find out the specificity, DTH reactionsto A2 or A6 cells were also measured in rats inoculated withA13 cells, but no significant reactions were observed (datanot shown).

PBSA2A6A13Blast

0. . 5.. 0. .

30 35 40 45

Days after immunization

Figure 2. Suppression of AA by inoculation with autoreactiveCD4- CD8 - o43 T cells (A13 cells). Naive rats were intraperitoneallyinoculated with either A2, A6 or A13 cells after stimulation with ConA. As control either PBS or Con A-activated spleen cells (T-cellblasts) were used. Four weeks later, AA was induced by an intradermalinjection of CFA. Rats were checked and the arthritis was scored as

described in Materials and Methods. The results were expressed as

the mean for each experimental group + SEM.

the joint space of the rats treated with A13 cells (Fig. 3b).Marked and diffused granulation tissue was observed in thePBS group (Fig. 3c), and focal granulation tissue was observedin the test group (Fig. 3d). Marked infiltration of inflammatorycells (Fig. 3e) was detected in the control rats, whereas slightinfiltration of inflammatory cells (Fig. 3f ) was detected in therats treated with A13 cells, suggesting that A13 cells reallysuppressed the inflammatory reactions around the joint.

Anti-idiotypic delayed-type hypersensitivity (DTH) reactionsinduced by A13 cells

To determine whether the suppression ofAA by Al 3 cells wascaused by an anti-idiotypic DTH response, rats inoculatedwith either A2, A6, or T-cell blasts were compared with rats

inoculated with A13 cells. Rats inoculated with either A2 or

A6 did not develop significant anti-idiotypic DTH reactions,whereas rats inoculated with A13 cells showed an increased

1998 Blackwell Science Ltd, Immunology, 94, 536-542

DISCUSSION

The results of this study demonstrated that autoreactive T-cellclones designated as A2, A6 and A13 could be isolated fromrats with AA. These clones were CD4- CD8- ap T cellswhich were confirmed by flow cytometric analysis.Interestingly, intraperitoneal inoculation with A13 cells butnot A2 or A6 cells protected rats from AA although some

rats developed mild diseases with delayed onset. Vaccinationwith A13 cells induced lasting remission of the disease andprevented the development of subsequent induction of AA.Histological examination revealed an acute fibrinous exu-

dation, synovial thickening, granulation tissue, and inflamma-tory cell infiltration in all the control rats inoculated with PBS,and no such inflammatory reactions were observed in ratsintraperitoneally inoculated with only one injection of 1 x 107A13 cells. The presence or absence of clinical signs of thedisease corresponded with the histological evaluation ofthese animals.

Flow cytometric analysis revealed that A13 cells expressedsurface markers such as CD3 and IL-2R characteristic ofmature peripheral blood T cells, but did not express eitherCD4 or CD8 molecules. It was observed that A13 cells didnot show any proliferative responses to Mt or 65000MWHSP, although the original line was raised against Mt.However, mycobacterial antigen-specific human CD4 - CD8 -T-cell clones have been reported.2' A13 cell showed highproliferative responses to Con A, but did not respond toanti-CD3 (data not shown) which correlated with the previousreport.22 Because the relevant auto-antigens are as yet

539

14 -

12 -

a) 10 -10-o0

_ 8-.aC. 6-cas2 4 -

2-

0

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M. Azizul Haque et al.

Figure 3. Histological analysis of joints (day 21 after immunization) of rats intraperitoneally inoculated with PBS (a, c, e,respectively) and A13 cells (b, d, f, respectively). (a) acute fibrinous exudate (arrowheads) in the joint space, and synovial thickening(b) no evidence of either fibrinous exudate in the joint space or synovial thickening; (c) marked granulation tissue is observed;(d) granulation tissue (arrowhead) is focally observed; (e) marked infiltration of inflammatory cells; (f) slight infiltration ofinflammatory cells (arrowhead).

unidentified; A2, A6 and Al 3 cells were stimulated non-

specifically by culture with Con A. In a number of studies,stimulation with mitogen, although non-specific, has beenfound to give an advantage to antigen-specific primed T cellsin experimental animals.10'23'24 Moreover, our data demon-strated that A13 cells responded well to superantigen SEB. Ithas been reported that superantigens stimulate a high pro-

portion of T cells bearing specific VP3 regions of the TCR, inthe presence of major histocompatibility complex (MHC)class II molecules.25'26 We therefore suggest that CD4 CD8-

ap T cells are mature end-stage cells as demonstrated pre-

viously,27 and the cells may be generated and expanded by thereactivity with still undetermined superantigens, autoantigens,or other foreign viral antigens.

Furthermore, rats protected from AA by inoculation withA13 cells showed a specific anti-idiotypic DTH responses.Inoculation with A2 or A6 cells neither suppressed the develop-ment of AA nor induced anti-idiotypic DTH responses. Thesedata have provided an indirect evidence that the protection isdirected against certain idiotype of the A13 cells, but not ofA2 or A6 cells. It is possible that the anti-idiotypic CD4' andCD8+ T cells might in vivo aid resistance by inducing DTH

© 1998 Blackwell Science Ltd, Immunology, 94, 536-542

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1

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Vaccination against AA with CD4- CD8- 43# T cells 541

PBS

A2

AlS

A13 _

T-cell blast

0 20 40 60 80

Swelling (x0001 mm)

Figure 4. Induction of anti-idiotypic delayed type hypersensitivityreactions by A13 cells. Naive female Lewis rats were intraperitoneallyinoculated (twice) with either A2, A6, Al3 cells, T-cell blasts or PBSas described in Materials and Methods. Four weeks after the firstinoculation of T cells, rats were immunized with Mt to induce AA.Seven days after immunization, irradiated (2500 rad) T cells (2 x iO0in 50 jld PBS) were subcutaneously injected into the left ear. Earthickness was measured, and the changes in thickness before and afterchallenge were recorded for each animal. The results were expressedas the mean for each experimental group ± SEM.

reaction to the autoimmune effector cells or by driving theidiotype-anti-idiotype network to enhance suppression as pro-posed previously.28,29 The immunoregulatory activity demon-strated here of a defined region of the TCR expressed by Al 3cells is an important step forward in understanding anti-idiotypic regulation, and provides a clear explanation for theprotective effects of the vaccination approach. Our studysuggest that the vaccination with A13 cells appears to inducean anti-idiotypic response to the cells that includes both CD4+and CD8+ T cells, as demonstrated in human anti-idiotypicCD8+ T-cell responses specific for receptors that recognizeviral antigens30 or alloantigen.3132 It has been demonstratedthat not all autoimmune T cells are able to induce an anti-idiotypic response or resistance to disease.27 Attempts weremade to study the cytokines involved. Unfortunately, somemajor cytokines, and antibodies are still not available in ratmodel, and it made difficult to see insight. However, the resultsof our study demonstrate for the first time that AA can besuppressed by autoreactive CD4- CD8 - a3 T cells, and thesecells may be used as therapeutic agents in experimental autoim-munity. Furthermore these new therapeutic approachesderived from animal models may offer a hope of more selectiveinterventions for the treatment ofhuman autoimmune diseases.Certainly, the existence of these unusual cells warrants furtherinvestigation of the exact mechanism responsible for thepotential role of these cells in the pathogenesis and vaccinationof AA.

ACKNOWLEDGMENTS

We thank Dr T. Uchiyama for providing SEB, and Dr Yamashita(Department of Immunology, Saga Medical School) for biotinylatedgoat antimouse IgM and streptavidin-FITC. This study was supportedby a Grant-in-Aid for Scientific Research from the Ministry ofEducation, Science and Culture of Japan.

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