Clin. exp. Immunol. (1992) 89, 427-433

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-yb + lymphocytes E. HERMANN, A. W. LOHSE, W. J. MAYET, R. VAN DER ZEE*, W. VAN EDEN*, P. PROBST, T. PORALLA, K-H. MEYER ZUM BUSCHENFELDE & B. FLEISCHER First Department of Medicine, Johannes Gutenberg University, Mainz, Germany, and *Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands

(Acceptedfor publication 25 April 1992)

SUMMARY T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocolitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-yb+ lymphocytes in the 65-kD hsp-stimulated SF-derived lines. This expansion of TCR-yb+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-yb+ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-yb+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-yb+ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-af+ TLC responded vigorously to the human 65-kD hsp and in some cases also crossrecognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-yb+ cells in response to 65-kD hsp or live bacteria. Keywords TCR-yb+ lymphocytes 65-kD heat shock protein synovial fluid

INTRODUCTION The majority of T cells use as their receptors a heterodimer composed of an ax and a /3 chain. A minor subset has been identified which expresses TCRs consisting of y and 6 chains [1]. In the adult mouse, TCR-yb+ lymphocytes are detected in the thymus, in peripheral lymphoid organs and the peripheral blood (PB) in relatively low numbers while they predominate within epithelia, such as epidermis [2] and small intestine epithelium [3,4]. In man, TCR-yb+ cells are evenly distributed throughout the PB and thedifferentlymphoid tissues without asignificant t sgniicaingata and the in any enhichment thenriPB differentglymphoid Howi data single organ [5,6].tissues However, conflicting exist concerning the distribution of TCR-yb+ lymphocytes in the synovial fluid (SF) or synovial tissue of arthritic joints [7] Enrichment of TCR-y6+ cells in the SF as compared with PB Correspondence: Prof. Bernhard Fleischer, First Department of Medicine, Johannes Gutenberg University, Langenbeckstr.l1, D-6500 Mainz, Germnany.

427

could be observed in some rheumatoid arthritis (RA) patients [8,9] without reaching significance in a greater population [10]. T cell infiltrates of inflamed RA synovial membranes were also shown to be predominantly composed of TCR-afl-bearing lymphocytes [10]. Nonetheless, it has recently been discussed that TCR-yb+ cells may play a role in the pathogenesis of various diseases including RA [9]. In this context, the isolation of mycobacterial

65-kD heat shock protein (hsp)-specific TCR-yb+ lymphocyte clones from the SF of a patient with RA [11] has led to discussions concerning the relation between stress proteins and TRy el nifamtr FTcl ies.Mre Marked SF T cell TCR-y6+ cells in inflammatory joint disease.

proliferation in response to mycobacterial 65-kD hsp has been observed in patients with RA, reactive arthritis (ReA) and other forms of arthritis [12-14]. The fine specificities of several SFderived TCR-axf CD4+ clones suggest that both mycobacteriaspecific peptides [15] and conserved epitopes present on mycobacterial, enterobacterial and human 65-kD hsp [16] may be

E. Hermann et al.

428

recognized by these T cells. Similarly, at least in the mouse model a large fraction of the TCR-yb5+ cell repertoire seems to be directed against stress proteins of both bacterial and mammalian origin, suggesting an extensive cross antigenicity of hsp as defined by TCR-yb+ cell recognition [17]. Mouse tuberculinreactive TCR-yb+ lymphocyte hybridomas were shown to respond specifically to small synthetic peptides of heterologous and autologous stress proteins [18]. These data from TCR-y6+ cells in animal experiments, together with the findings that the human endogenous 65-kD hsp seems to be selectively expressed in the cartilage-pannus junction of arthritic joints [19] led to the assumption that human TCR-y6 + T cells in the SF might also be stimulated by self 65-kD hsp and/or cross-reacting epitopes on bacterial hsp. To test this hypothesis, we generated SF- and PB-derived T cell lines by incubating mononuclear cells with recombinant human or mycobacterial 65-kD hsp or control antigens and determined the distribution of TCR-a + and TCR-y3+ lymphocytes. Three lines of interest were cloned and further investigated for antigen-specific T cell responses. SUBJECTS AND METHODS

Subjects and preparation of mononuclear cells Peripheral blood of three healthy blood donors, one patient with RA and one patient with psoriatic arthritis (PsA) and SF of one patient with RA, three patients with ReA, two patients with PsA and one patient with ankylosing spondylitis (AS) were investigated. SF as well as PB specimens were collected into sterile heparinized tubes. SF mononuclear cells (SFMC) and PBMC were separated by Ficoll-Hypaque gradient centrifugation, washed three times and resuspended in RPMI 1640 (Biochrom, Berlin, Germany) containing 2 mm glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells were cryopreserved in 20% FCS and 10% dimethylsulphoxide (DMSO), and were stored in liquid nitrogen. Monocyte- and B cell-enriched PBMC (E-) were obtained by removing T cells by rosetting with neuraminidase-treated sheep erythrocytes and used as antigenpresenting cells (APC) in the proliferation assays. Antigens Yersinia enterocolitica 03 was cultured on Yersinia-selective agar (CIN agar medium, GIBCO, Karlsruhe, Germany), washed, resuspended in PBS, killed by heat and diluted to a protein concentration of 1 mg/ml. Live Y. enterocolitica 03 and Pseudomonas aeruginosa were counted and diluted to a final concentration of 2 x 107 cells/ml RPMI 1640 without antibiotics. Tetanus toxoid (TT) was kindly provided by Behringwerke, Marburg, Germany. Hsp from Mycobacterium bovis BCG was expressed from plasmid pRIB1300 and purified as described [20,21]. The human 65-kD hsp was expressed in Escherichia coli from plasmid pRH 710. This pEX2-derived plasmid was constructed from a phage lambda-gtl I clone containing the human 65-kD hsp gene that we obtained from Dr R. A. Young, Cambridge, MA. pEX2-derived proteins were isolated as the insoluble material after lysis of recombinant E. coli strains and purified as described [22]. The recombinant mycobacterial and the human 65-kD hsp fusion protein, as well as the pEX2-derived control protein fl-galactosidase were added at a final concentration of 5 Hig/ml.

Establishment of SF and PB-derived T cell lines T cell lines were established by incubating PBMC and SFMC (1 5 x 106 ml) with either human 65-kD hsp (5 ,g/ml), myc 65kD hsp (5 ug/ml), TT (10 Mg/ml) or heat killed Y. enterocolitica 03 (50 yg/ml) in supplemented RPMI-1640 containing 10% heat-inactivated human AB serum (HUS) (i.e. culture medium) in 24-well plates (Costar, Cambridge, MA). The concentrations used had previously been found to be optimal in T cell proliferation assays. SFMC from one patient (patient 4) and PBMC from one healthy donor (donor 3) were stimulated with 5 ug/ml of the control antigen pEX2/fl-galactosidase. In some experiments live Y. enterocolitica 03 were used to establish T cell lines, in one patient, live Ps. aeruginosa were also used as a control. SFMC (I x 106) were incubated with 1 x 107 Y. enterocolitica 03 in 1 ml of RPMI-1640/ 10% HUS without antibiotics. After 2 h gentamycin (Serva Feinbiochemica, Heidelberg, Germany) was added to the cultures at a final concentration of 100 pg/ml in order to kill extracellular bacteria. The SFMC culture containing Ps. aeruginosa was supplemented with ciprofloxacin 1:500 (kindly provided by Bayer AG, Hamburg, Germany) after 2 h of culture. In all T cell lines, 0-5 ml of culture medium supplemented with 20 U/ml recombinant IL-2 (rIL-2; Boehringer Mannheim, Germany) were added to each well after 4 days of cultures. rIL2-supplemented culture medium was replaced on day 8. On day 12 the cultures were split with new antigen- and rIL-2supplemented culture medium and with 1 5 x 106 irradiated (40 Gy) autologous PBMC per well. Subsequent splits were made approximately every 4-6 days with rIL-2 supplemented medium. Cell sorter analysis Cell sorter analysis of the T cell lines as well as of unstimulated PBMC and SFMC was performed on day 15 of culture. Cells (3 x 105 per test) were labelled first with the unconjugated MoAbs anti-CD3, anti-CD4, anti-CD8 (hybridoma supernatants), BMA.03 1 (specific for a framework determinant on the TCR-af3, kind gift of Dr R. Kurrle, Behringwerke), TCR-61 (specific for a framework determinant on the 5 chain of the TCR-y6, T-Cell Sciences, Cambridge, MA) and TiyA (specific for the Vy9 encoded epitope, kind gift of Dr T. Hercend, Villejuif, France [23]) and then, with FITC-conjugated polyclonal goat anti-mouse immunoglobulin antibody (Medac, Hamburg, Germany) in RPMI-1640/10% FCS. Samples were washed twice between each step. Cytofluorometric analysis, counting 6000 cells per sample, was performed on a fluorescence-activated cell sorter (FACStar, Becton Dickinson).

Cloning of T cell lines From two different patients (patients 2 and 5), SF-derived T cell lines stimulated with the human 65-kD hsp and one line stimulated with heat-killed Yersinia were cloned. One of these lines (JPl, stimulated with the human 65-kD hsp) contained predominantly y6+ T cells, the other lines (JP2, stimulated with dead Yersinia, and HSI, stimulated with the human 65-kD hsp) contained predominantly TCR-a4+ cells. Cloning of the T cell lines was performed in Terasaki microtest plates (Nunc, Roskilde, Denmark) by limiting dilution using phytohaemagglutinin (PHA-P, GIBCO) at 1 ,ug/ml and rIL-2 (20 U/ml) supplemented medium and irradiated feeder cells (1 ~2 x 104 cells/well). On day 10 of culture, growing T cell clones were expanded in

Synovial TCR-y5 + T cells and heat shock protein larger plates (96-well microtitre plates (Nunc) and 12-well plates) with rIL-2-supplemented culture medium and were then tested after another week of culture. All clones were phenotyped using a beta-galactosidase linked single cell immunoassay [24]. Assay for proliferative activity of T cell clones Proliferative responses were determined in triplicates of 150 pI culture medium (RPMI-1640/10% HUS) containing 1 x 104 cloned T cells as responders and I x 104 autologous E- as APC in U-shaped, 96-well microtitre plates (Nunc). The appropriate antigens (human or mycobacterial 65-kD hsp, heat-killed Yersinia) were added to the cultures at the final protein concentrations described above. After 24 h of incubation the cultures were pulsed with 3H-thymidine (Amersham International, Amersham, UK) at 0-25 pCi per well, harvested on glass fibre filters after another 24 h and counted in a fl-scintillation counter (Betaplate, Pharmacia, Sweden). The results were expressed as mean ct/min incorporation of triplicates. RESULTS Phenotypes of antigen-stimulated SF and PB T cells Consistent with published data [9,24] we found that the percentage of TCR-yb+ T cells in the unstimulated PBMC and SFMC ranged between 3-2% and 9-6% (Table 1). There was no difference between the numbers of TCR-yb+ cells in the SFMC and PBMC. Analysis of cell surface markers after in vitro stimulation with different soluble antigens (human 65-kD hsp, mycobacterial 65-kD hsp, TT) for 15 days revealed a marked expansion of TCR-yb+ cells only in the 65-kD hsp-stimulated

429

lines (Table 1). Flow-cytometry profiles from a representative experiment are shown in Fig. 1. TCR-yb+ lymphocytes were found to be in part CD4- CD8- but also in part to be CD8+. SFMC stimulation with the control antigen fl-galactosidase expressed from the cloning vector, pEX2, did not result in a predominantly TCR-yb+ T cell expansion: in the two subjects tested, 6-5% and 5-2% respectively of CD3+ cells were TCR-yb+ (data not shown). In 2 patients (patients 3 and 5), the number of TCR-yb+ cells following SFMC stimulation by human 65-kD hsp was determined on day 4 (before addition of exogenous IL2) and day 10 (before restimulation with autologous PBMC and antigen). On day 4, 19-5% (patient 5) and 22-2% (patient 3) of total CD3+ cells were TCR-yb+; on day 10, the percentage of TCR-yb+ T cells was 68-4% in patient 5 and 64-6% in patient 3. This kinetic study shows that the expansion of TCR-yb+ lymphocytes in hsp-stimulated SFMC was not dependent on the addition of exogenous IL-2. A comparison of either human or mycobacterial 65-kD hspstimulated lines showed a tendency to higher percentages of TCR-yb+ cells in the human 65-kD hsp-stimulated SF T cell lines (mean 67-7% TCR-yb+ T cells of total CD3+ cells versus 49-4%), but this difference was statistically not significant. In 65kD hsp-stimulated PB-derived T cell lines, the percentage of TCR-y6+ cells was slightly elevated in only two of five PBL lines (donors 2 and 3) in contrast to the expansion ofthese cells in SFderived lines. Furthermore, SFMC or PBMC stimulation with TT as a control antigen did not lead to a comparable increase of TCR-yb+ lymphocytes in the lines, but induced a predominantly CD4+ TCR-xap+ T cell response. Only in three TT-stimulated SF-derived lines could notable elevations in the percentage of TCR-y6+ cells (a maximum of 15-4% TCR-yb+ T cells versus

Table 1. Phenotypic analysis of different synovial fluid (SF) and peripheral blood (PB) T cell subpopulations after stimulation with various antigens T cell subpopulations day 15 (positive staining cells relative to the percentage of total CD3 + cells)

SFMC or PBMC unstimulated

Subjects Patient 1 RA, SFMC 2 PsA, SFMC 3 AS, SFMC 4 ReA, SFMC 5 ReA, SFMC 6 PsA, SFMC 7 ReA, SFMC 8 RA, PBMC 9 PsA, PBMC Donor 1 Healthy, PBMC 2 Healthy, PBMC 3 Healthy, PBMC

Human 65-kD hsp

myc 65-kD hsp

Live Yersinia

Dead Yersinia

TT

#

y

c4)

y5

a4

v6

a4

y

a4

y6

a4

yc

TiyA

90-4 98-2 95-4 92-5 85-3 94-0 95.9 92-6 95-6

8-2 3-2 5-6 7-8

41-6 97-6 22-7 39-2 12-7 18-1 3-1 92-7 86-0

59 2 4-2 80-4 70-9 85-1 76-7 97-6 6-7 3-6

40-8 98-8 35-9 91-7 46-1 45-7 26-5 98-6 ND

74-6 3-3 58-4 11-8 52-5 53-2 76-1 2-3 ND

98-2 96-4 99-0 ND 96-6 94-0 92-7 98-3 ND

2-3 9-6 2-0 ND 2-8 9-9 15-4 0-7 ND

87-3 93-6 91-5 78-6 78-4 ND ND 97-6 ND

11-6 3-8 8-8 11-3 22-9 ND ND 1-2 ND

57-1 92-5 77-6 17-0 33-2 ND ND ND ND

41-0 2-4 26-1 75-7 70-2 ND ND ND ND

42-1 1-8 20-1 71-7 63-5 ND ND ND ND

90-1 87-8 82-5

7-8 11-4 16-1

92-5 90-0 472

6-1 12-1 21 8

ND 994

ND 19 85

92-5 91-3

6-4 8-6 ND

ND ND ND

ND ND ND

ND ND ND

93-5

5-6 3-6 9-6 6-8 4-0

93.7

5-8 6-9

93-8

5.5

90 7

ND

SFMC or PBMC were typed either unstimulated or after stimulation for 15 days with human or mycobacterial 65-kD hsp, tetanus toxoid (TI),

dead (heat-killed) or live Yersinia enterocolitica 03. The data are expressed as the percentages of MoAb-defined positive staining cells relative to the percentage of total CD3 + T cells. RA, Rheumatoid arthritis; PsA, psoriatic arthritis; AS, ankylosing spondylitis; ReA, reactive arthritis.

E. Hermann et al.

430 Human 65-kD hsp

myc 65-kD hop

Medium

TT

0-75

0 -9

0-T

TCR-a,

CD3 97-B

TCR--y8

17-0

75.7

TiyA 71-7

Live

-

Y.enterco/itico

AALLL ___~~~~~~~~ 1k

Medium

0-81

900

9646

960

_-8

25 4

878

73-1

; g

^

6

78

11-H 3 311

log fluorescence intensity

CD3 ft ;

9 91T8-

Fig. 2. Cell sorter analysis of T cell subpopulations of synovial fluid |(SF)-derived T cell lines after SFMC stimulation with live or dead

~ enterocolitica 03 or live Pseudomonas aeroginosa for 15 days . And EL JoYersinia is shown for test MoAb (antifluorescence

89-6

TCR-a3C 14-8

TCR-yI

4). Log .(patient ..,......... -; aaJo,,.,t.. *-

intensity CD3, anti-TCR-afl, anti-TCR-yb, TiyA) on the abscissa and the relative number of cells on the ordinate.

Clonal analysis of human 65-kD hsp-stimulated T cells Cloning of the SF-derived T cell line JPl (stimulated with the

human 65-kD hsp, with a percentage of 85% TCR-y6+ T cells)

produced 26 T lymphocyte clones (TLC) that could be expanded, phenotyped and tested in a proliferation assay. Of

2i5

31.7

89.8

29-2

33-4

16.3

CD4

CD8

X

these, 24 TLC were TCR-y5+ and two TLC TCR-cxf+ CD4+. From the same patient, the SF-derived line JP2 (stimulated with dead Yersinia) was cloned (17 TLC, all TCR-cxf+ CD4+). Cloning of the SF-derived T cell line HSI (stimulated with human 65-kD hsp, 97-6% TCR-ax+, 4-2% TCR-y6+) generated 23 TCR-cx/+ TLC, 22 of which were CD4+, one TLC was

CD8+.

The specific functional capability of the TLC was tested in a

log fluorescence intensity Fig. 1. Phenotypic analysis by FACS from one representative experiment (patient 7) showing the MoAb-defined expression of CD3, TCRa4, TCR-y5, CD4 and CD8 (expressed as log intensity of fluorescence) on the abscissa and the relative number of cells on the ordinate. SFMC were stimulated with human or mycobacterial 65-kD hsp or tetanus toxoid (TT) for 15 days. 9-6% in the unstimulated SFMC) be demonstrated. This argues against a simple non-specific bystander stimulation and passive expansion of pre-activated TCR-yb+ T cells in response to growth factors produced by the antigen-specific TCR-oxf+ cells within the mononuclear cell fraction. Expansion of TCR-y6+ lymphocytes after stimulation with live bacteria Besides stimulation with 65-kD hsp, SFMC infection with live Yersinia (Table 1 and Fig. 2) was also capable of expanding TCR-y6+ cells in four of five patients tested. This effect was independent of the underlying rheumatic disease; SFMC infection in two patients with ReA, one patient with AS, and one patient with RA showed similar results. The Yersinia-induced SF-derived TCR-yb+ cells were mainly TiyA+. A similar distribution of T cell subsets could be demonstrated in the T cell line derived from SFMC infected by Ps. aeruginosa (64-8% TCR-yb+, 59-7% TiyA+ versus 35 0% TCR-4,B+ cells). In contrast, heat-killed Yersinia stimulation of SFMC resulted in a predominantly TCR-cxf3 T cell response. We identified one patient with PsA (patient 2) whose SF TCR-y6+ cells were refractory to stimulation: neither SFMC stimulation with 65kD hsp nor infection with live Yersinia induced expansion of TCR-y6+ lymphocytes.

proliferation assay with human or mycobacterial 65-kD hsp or heat-killed Y. enterocolitica 03. The results are shown in Table 2. Stimulation indices greater than 5-0 were considered to be positive for the appropriate antigen.The majority (20 of 24) of TCR-y6+ TLC from line JPl did not show a proliferative response to the 65-kD antigen in the proliferation assay. Only four TLC responded weakly but specifically to the human 65kD hsp (SI between 5-4 and 7-6) though they were capable of proliferating in the presence of IL-2 (Table 3). In contrast to the non-responsiveness of TCR-yb+ lymphocytes, the majority of TCR-af+ TLC (14 of 23) of TLC derived from line HSl and one of the two TCR-aB+ TLC from line JPl proliferated vigorously in response to the 65-kD hsp antigen. Stimulation indices between 9-0 and 131 could be demonstrated. Most of these clones (14 of 16) were not specific for human 65kD hsp but cross-recognized the mycobacterial hsp homologue or heat-killed Yersinia or both. Ten out of 16 TCR-ac/+ TLC derived from line JP2 proliferated in response to Yersinia antigen, one of these TLC cross-recognized the human and mycobacterial 65-kD hsp. DISCUSSION To delineate the relationship of 65-kD hsp to TCR-y6+ T cells in the SF, we generated SF-derived T cell lines by stimulating mononuclear cells with human and mycobacterial 65-kD hsp and control antigens in vitro. Phenotyping of the 65-kD hspstimulated lines after 14 days revealed an expansion of TCR-y6+ T cells in six of the seven patients tested. This marked increase in percentage of TCR-y6+ lymphocytes was also shown after stimulation with live but not with heat-killed enterobacteria. The expansion of TCR-y6+ lymphocytes after stimulation with 65-kD hsp could be due to either an hsp-specific autono-

Synovial TCR-yb+ T cells and heat shock protein

431

Table 2. Proliferative response of T lymphocyte clones (TLC) generated from synovial fluid (SF)-derived T cell lines Proliferation in response to antigens (stimulation index (SI) > 5-0)

SF-derived line

No. of clones (TLC)

Human 65-kD hsp

myc 65-kD hsp

Yersinia enterocolitica

Pat. JP 1 (human 65 kD)

4TLC (yb+) 1 TLC (a4, CD4+) I TLC (ap, CD4+) 20 TLC (yb+)

+ + -

+ -

+ -

Pat. JP 2 (Y. enterocolitica)

9 TLC (a4), CD4+) 1 TLC (a4, CD4+) 6TLC (ap, CD4+)

+

+

+ +

Pat. HS 1 (human 65 kD)

5 TLC (a4, CD4+) 4 TLC (a4, CD4+)

+ +

+ +

3TLC (ap, CD4+) 2 TLC (a#, CD4+) I TLC (a#, CD4+) 7TLC (afl, CD4+)

+ + -

+ +

-

+

lTLC (a#, CD8+)

The T cell lines JP1 (human 65-kD hsp-stimulated), JP2 (stimulated with dead Yersinia), and HSI (human 65-kD hsp-stimulated) were cloned and the resulting TLC were tested on autologous T cell-depleted PBMC (E-) as APC for reactivity against the antigens indicated. Table 3. Proliferative activity of six T lymphocyte clones (TLC) generated from the synovial fluid (SF)-derived T cell line JPl

Proliferative response to TLC no. JP7 JP14 JP16 JPl9 JP22 JP23

TCR

a4+ Yb+ Yb+

afl+ Yb+ Yb+

Medium control

Human 65-kD hsp

myc 65-kD hsp

Yersinia enterocolitica

IL-2

186 122 198 152 57 75

4603 936 1482 5402 302 390

3636 178 267 273 90 91

378 340 183 138 158 172

3254 2636 4174 4213 860 1188

Results are expressed as mean ct/min of 3H-thymidine incorporation of cloned T cells. The remaining 20 clones derived from line JPI, all expressing the y5 TCR, proliferated after stimulation with IL-2, but not in response to any of the antigens tested (stimulation indices between 1 0 and 4-3).

mous proliferation of these T cells or a passive expansion of TCR-yb+ cells in response to growth factors like IL-2 or other cytokines produced by hsp-reactive TCR-a4+ cells from the same SFMC population. The finding that an increase of TCRyb+ cells was not observed after SFMC stimulation with TT or with dead Yersinia organisms that induce a vigorous proliferative response in SF bulk cultures [14] suggests that the expansion of TCR-y5+ cells by 65-kD hsp or live Yersinia is antigenspecific and not due to a non-specific IL-2-driven growth of preactivated TCR-y5+ cells in the SF. However, different cytokine patterns released in response to the different antigens may also be involved in the preferential proliferation of SF TCR-y6+ lymphocytes. That human TCR-y5+ T cells are

principally capable of proliferating in response to mycobacterial hsp in the presence of autologous antigen-presenting cells has been shown by investigating a PPD-specific TCR-yb+ T cell line [25] and SF-derived TCR-yb+ T cell clones [11], although others have demonstrated that a large fraction of human PB TCR-yb+ T cells is activated by Mycobacterium tuberculosis but not by its 65-kD hsp [26]. Recent studies [27] have shown that mycobacterial 65-kD hsp-activated SFMC lines with a high percentage of TCR-y6+ T lymphocytes are able to specifically recognize 65kD hsp upon restimulation with this antigen. The clonal analysis of our T cell line JP1 does not answer the question of active or passive expansion of TCR-y5+ cells but indicates that both possibilities must be considered. The

432

E. Hermann et al.

minority of TCR-yb+ TLC (four of 24) showed a weak (SI between 5 2 and 7-6) but specific proliferative response to human 65-kD hsp without cross-recognizing mycobacterial hsp or heat-killed Yersinia organisms. Twenty TCR-yb+ TLC did not recognize 65-kD hsp in the proliferation assay at all although they were capable of proliferating in response to IL-2. In contrast, most TCR-a41+ TLC generated from hsp-stimulated lines proliferated vigorously in response to human 65-kD hsp and in part also recognized the mycobacterial homologue and/ or dead Yersinia. A possible explanation is that in the proliferation assays of TLC a factor is missing that is essential for the proliferation of TCR-yb+ cells but not of TCR-ap+ cells. This factor or ingredient is apparently present in the primary cultures of SFMC. The proliferative response of TCR-yb+ cells in the primary cultures could, for example, depend on factors released by TCR-aof+ cells. Our data underline the hypothesis that TCR-yb+ T cells can be activated-eventually in a helper-dependent way-by microorganisms other than Mycobacteria and that their role is probably not restricted to acquired resistance against tuberculosis [28]. After completion of our data, a recent publication [29] has demonstrated that monocytes infected with live but not with dead Mycobacteria or Salmonella induce expansion of human TCR-yb+ T cells from PBMC. The present study confirms and extends these results. Our finding that TCR-yb+ lymphocytes from the same mononuclear cell population are preferentially activated by live bacteria as well as by stimulation with self hsp but not with dead bacteria or with other recall antigens suggests that a common mechanism is involved in the selective expansion of these TCR-yb+ lymphocytes. It may be speculated that these cells play a physiological role in the early defence reactions directed against cells infected by intracellular bacteria or parasites or against autologous 'stressed' cells expressing self hsp. Hsp and the immune response directed against self hsp may help to protect inflamed tissues from detrimental consequences arising during infection and inflammation of different origin [30]. Alternatively, the host immune response may also be directed against stress-proteins expressed by the infecting intracellular micro-organism itself. It has been shown for Salmonella that abundant amounts of Salmonella stress-proteins (GroEl and DnaK) are expressed during infection within the host macrophages under conditions that do not involve thermoinduction [31] and that these proteins are also immunodominant within the antigen-presenting cell. Procaryotic and human hsp share more than 60% sequence homology on the amino acid level [32] and may thus be cross-recognized by hspspecific T cells [16]. A remarkable result is the enrichment of TCR-yb+ cells in cultures of SFMC upon stimulation with hsp or with live bacteria as compared with PBMC. SF APC in contrast to PB APC might preferentially support the presentation of65-kD hsp to TCR-yb+ cells and thus promote the expansion of this T cell

subpopulation. Although hsp-reactive TCR-yb+ lymphocytes are present in the peripheral blood of healthy individuals, it is likely that such cells are preactivated in vivo at the site of rheumatic inflammation due to prior contact with bacterial or self hsp. The same immune cells that recognize bacteria-infected cells may be capable of eliminating stressed cells within the inflammatory milieu of synovitis. Only one of the SF tested (patient 2) was repeatedly shown to selectively expand TCR-af CD4+ T cells upon stimulation with the various antigens or live

bacteria. At present we have no explanation for this single observation. In peripheral blood, the predominant population of TCRy6 + cells expresses the V62 gene product which most frequently associates a V9/JP/C I encoded y chain recognized by anti-TiyA, and a V2/(D)/J1/C encoded 6 chain [33]. This population of Vy9-V62 expressing TCR-yb+ cells was also found to be the most prominent population in the SF-derived bacteria-stimulated TCR-yb+ cells in our studies. Reactivity with self or bacterial hsp may be a possible basis for several physiological TCR-y6+ lymphocyte functions. Hspspecific TCR-yb+ as well as TCR-43+ T cells may be present in the peripheral blood of normal individuals and may play a role in the defence of bacterial infections and in the elimination of damaged cells. The expansion of TCR-yb+ cells after stimulation of SFMC with self hsp indicates in vivo preactivation and special properties of these cells within the inflammatory milieu of active arthritis. Cross-reactivity of heterologous and self hsp may be critical in the inflamed joint and lead to autoaggressive reactions and maintenance of chronic inflammation. On the other hand, hsp-responding T cells together with other regulatory immune elements may prevent uncontrolled autoaggressive responses [33]. Further in vitro and animal in vivo studies of hspreactive TCR-yb+ and TCR-a#f+ T lymphocytes may provide us with an answer to the question of how these cells interact with their targets and what their functional significance is in the normal and 'stressed' immune system. ACKNOWLEDGMENTS This work was supported by the Kurt-Eberhard Bode Stiftung in the Stifterverband fur die Deutsche Wissenschaft (E.H.), by the Deutsche Forschungsgemeinschaft SFB 311 (E.H., A.W.L., K.-H. M.z.B., B.F.), by the Institute Merieux (R.v.d.Z.), by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Disease, and by the Concerted Action on Immunotherapy ofChronic Arthritis of the European Community.

REFERENCES I Brenner MB, McLean J, Dialynas DPB et al. Identification of a putative T cell receptor. Nature 1986; 322:145-9. 2 Stingl G, Koning F, Yamada H et al. Thy-i + dendritic epithelial cells express T3 antigen and the T-cell receptor y chain. Proc Natl Acad Sci USA 1987; 84:4586-90. 3 Bonneville M, Janeway CA, Ito K et al. Intestinal intraepithelial lymphocytes are a distinct set ofy6 T cells. Nature 1988; 336:479-81. 4 Goodman T, Lefrancois L. Expression of the y/5 T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 1988; 333: 855-8. 5 Groh V, Porcelli S, Fabbi M et al. Human lymphocytes bearing T cell receptor y/b are phenotypically diverse and evenly distributed throughout the lymphoid system. J Exp Med 1989; 169:1277-94. 6 Bucy RP, Chen CH, Cooper MD. Tissue localization and CD8 accessory molecule expression of Tyb cells in humans. J Immunol 1989; 142:3045-50. 7 Broker B, Lydyard PM, Emmrich F. The role of y6 T cells in the normal and disordered immune system. Klin Wochenschr 1990;

68:489-95. 8 Brennan FM, Londei M, Jackson AM et al. T cells expressing y3 chain receptors in rheumatoid arthritis. J Autoimmun 1988; 1:31926. 9 Haas W, Kaufmann S. Martinez-A C. The development and functi6n of y6 T cells. Immunol Today 1990; 11:340-3.

Synovial TCR-yb3+ T cells and heat shock protein 10 Smith M, Br6ker B, Moretta L et al. Tyb cells and their subsets in blood and synovial tissue from rheumatoid arthritis patients. Scand J Immunol 1990; 32:585-93. 11 Holoshitz J, Koning F, Coligan JE et al. Isolation of CD4-CD8mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 1989; 339:226-9. 12 Res PCM, Schaar CG, Breedveld FC et al. Synovial fluid T cell reactivity against 65kDa heat shock protein of bacteria in early chronic arthritis. Lancet 1988; ii:478-80. 13 Gaston JSH, Life PF, Bailey L et al. In vitro responses to a 65kilodalton protein by synovial T cells from inflammatory arthritis patients. J Immunol 1989; 143:2494-500. 14 Hermann E, Mayet W-J, Lohse AW et al. Proliferative response of synovial fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65 kD mycobacterial heat shock protein. Med Microbiol Immunol 1990; 179:215-24. 15 Gaston JSH, Life PF, Jenner PJ et al. Recognition of a mycobacteria-specific epitope in the 65-kDa heat-shock protein by synovial fluid-derived T cell clones. J Exp Med 1990; 171:831-43. 16 Hermann E, Lohse AW, van der Zee R et al. Synovial fluid derived Yersinia reactive T-cells responding to human 65 kD heat shock protein and heat-stressed antigen presenting cells. Eur J Immunol 1991; 21:2139-43. 17 Rajasekar R, Gek-Kee S, Augustin A. Self heat shock and y T-cell reactivity. Proc Natl Acad Sci USA 1990; 87:1767-71. 18 Born W, Hall L, Dallas A et al. Recognition of a peptide antigen by heat shock-reactive yb T lymphocytes. Science 1990; 249:67-69. 19 Karlsson-Parra A, S6derstrom K, Ferm M et al. Presence of human 65 kD heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients. Scand J Immunol 1990; 31:283-8. 20 Van Eden W, Thole JER, van der Zee R et al. Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 1988; 331:171-3. 21 Thole JE, Keulen WJ, De Bruyn J et al. Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in Escherichia coliK-12. Infect Immun 1987; 55: 1466-75.

433

22 De Graeff-Meeder ER, Van der Zee R, Rijkers GT et al. Recognition of human 60 kD heat shock protein by mononuclear cells from patients with juvenile chronic arthritis. Lancet 1991; 337:1368-72. 23 Triebel F, Faure F, Grazzani M et al. A unique V-J-C rearranged gene encodes y protein expressed on the majority of CD3 + TCR a#lcirculating lymphocytes. J Exp Med 1988; 168:1-14. 24 Holzmann B, Johnson JJ. A beta-galactosidase linked immunoassay for the analysis of antigens on individual cells. J Immunol Meth 1983; 60:359-67. 25 Haregewoin A, Soman G, Hom RC et al. Human yb+ T cells respond to mycobacterial heat-shock protein. Nature 1989; 340: 309-12. 26 Kabelitz D, Bender A, Schondelmaier S et al. A large fraction of human peripheral blood y/6 + T cells is activated by Mycobacterium tuberculosis but not by its 65-kD heat shock protein. J Exp Med 1990; 171:667-79. 27 Soderstrom J, Halapi E, Nilsson E et al. Synovial cells responding to a 65-kDa mycobacterial heat shock protein have a high proportion of a TCRy6 subtype uncommon in peripheral blood. Scand J Immunol 1990; 32:503-15. 28 Munk ME, Gatrill AJ, Kaufmann SHE. Target cell lysis and IL-2 secretion by y/6 T lymphocytes after activation with bacteria. J Immunol 1990; 145:2434-9. 29 Havlir DV, Ellner JJ, Chervenak KA et al. Selective expansion of human y5 T cells by monocytes infected with live Mycobacterium tuberculosis. J Clin Invest 1991; 87:729-33. 30 Kaufmann SHE. Heat shock proteins: a missing link in the hostparasite relationship? Med Microbiol Immunol 1990; 179:61-69. 31 Buchmeier NA, Heffron F. Induction of Salmonella stress proteins upon infection of macrophages. Science 1990; 248:730-2. 32 Jindal S, Dudani AK, Singh B et al. Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. Mol Cell Biol 1989; 9:2279-83. 33 Triebel F, Hercend T. Subpopulations of human peripheral T y6 lymphocytes. Immunol Today 1989; 10:186-8.