Eur. J. Immunol. 1991. 22: 1289-1296
Els J. M. Hogervorst., Claire J. P. Boog., Jos6e R A. Wagenam., Marca H. M. Wauben., Ruurd Van der Zee. and Willem Van Eden. Institute of Infectious Diseases and Immunology., Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht and Department of Bacteriologyv, National Institute of Public Health and Environmental Protection, Bilthoven
Regulation by an hsp65 epitope in adjuvant arthritis
1289
T cell reactivity to an epitope of the mycobacterial65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses* Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial65-kDa heat-shock protein (hsp65), the possible origin of A A was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)FI rats, although major histocompatibility complex class I1 identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.
1 Introduction Adjuvant arthritis (AA) is an extensively studied animal model for human rheumatoid arthritis and is characterized by an inflammatory reaction in the synovial tissue, leading to destruction of the joints, very similar to the human pathological conditions. A A is induced in rats by immunization with heat-killed Mycobacterium tuberculosis (MT) in oil [l]. Inbred rat strains have been found to differ in their susceptibility to A A , indicating the existence of genetic factors controlling resistance to AA. For example, Lewis rats are highly susceptible to AA, while Fisher F344 rats,
despite the fact that they share the same RT1.B and RT1.D MHCclass I1 loci with Lewis, are not susceptible to A A [2]. Interestingly enough, Fisher rats have been shown to be susceptible to A A when kept and bred under germ-free conditions [3]. Other susceptible rat strains are Wistar [4] and DA [2]. (Lewis XFisher)F~rats are resistant and Brown Norway (BN) rats are of intermediate susceptibility, as we have shown now.
The autoimmune nature of A A became apparent when it was found that the disease could be transferred by means of polyclonal lymphocytes obtained from diseased animals [ 5 ] . More recently, Holoshitz et al. [6], succeeded in selecting and propagating in vitro an arthritogenic T cell clone (A2b) with reactivity to MT. Further detailed specificity analysis of A2b was carried out, when it was [I 91161 found that A2b was capable of recognizing, besides MT, an * Part of this work was supported by Institut MCrieux, Lyon, antigen co-purified with cartilage proteoglycans [7]. This France. mimicry relationship between mycobacteria and a proteoglycan-associated self antigen, as defined at the level of the Correspondence: Willem Van Eden, Institute of Infectious Dis- arthritogenic effector cell (A2b) itself, was found to be eases and Immunology, Department of Immunology, Faculty of contained in a mycobacterial heat-shock protein (hsp). Veterinary Medicine, University of Utrecht, Yalelaan 1, NL3508 Subsequently both arthritogenic A2b and another protecTD Utrecht, The Netherlands tive Tcell clone A2c, were shown to recognize an epitope, Abbreviations: AA: Adjuvant arthritis MT: Mycobacterium comprizing seven amino acid (180-186) residues as present tuberculosis bsp66: 65-kDa Heat-shock protein of Mycobacter- in the 65-kDa hsp (hsp65) of Mycobacterium bovis BCG ium bovis BCG [8, 91. '
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
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E. J. M.Hogervorst, C.J. €? Boog, J. €? A.Wagenaar et al.
Because of their evolutionary conservation, hsp are likely candidates for having such mimicry relationships with self structures. Such theoretical considerations and the empirical findings in the AA model have stimulated the search for the involvement of hsp in arthritis and other autoimmune disorders. A further characteristic feature of hsp seems to be their immunological dominance. After exposure of both experimental animals and humans to bacterial organism, antibodies and Tcells specific for hsp are easily found [10-13].The latter phenomenon is unexpected and is difficult to reconcile with the conserved nature of the molecules. Nevertheless, also in Lewis rats after AA induction with MT, responses to hsp tend to dominate (Hogervorst et al., in preparation). In the present study, responses to the hsp65 and its critical 180-188 epitope were examined in rats with distinct susceptibilities to AA. We studied four strains [Lewis, Fisher, (Lewis x Fisher)F1 and BN] for such responses in the draining LN cell populations after immunization with MT. Lewis rats showed responses to the hsp 65 and also to the 180-188 epitope. BN rats, carrying an MHC haplotype that differs from that of Lewis rats, were found to be low responders to hsp65 and nonresponders to the 180-188 epitope. Fisher rats, however, which have the same MHC class11 antigens as Lewis rats but are resistant to AA, responded to hsp65 but did not respond to the 180-188 epitope. (Lewis x Fisher)F1 rats showed similar responses to those of Fisher rats. These results further indicate that responsiveness to the 180-188 epitope is related to arthritis susceptibility and that the nonresponsiveness of Fisher rats to the 180-188 epitope is not caused by defective antigen processing or antigen presentation nor by active T cell suppression. Moreover, Lewis rats were also found capable of down-regulating their responses to the 180-188 epitope. Such inhibition of 180-188-specific responses was found to be triggered by responding to hsp 65, probably due to the presence of other regulatory epitopes present in hsp 65. These findings may explain the earlier reported results of hsp 65-induced protection in athritis models [8, 14-16].
2 Materials and methods 2.1 Rats Inbred Lewis rats were obtained from the University of Limburg, Maastricht, The Netherlands. BN, Fisher F344 and (Lewis x Fisher)F1 rats were obtained from Olac/HarIan CPB, Zeist, The Netherlands. All rats were used at 6-8 weeks of age.
2.2 Antigens The following antigens were used for immunization of the animals or in proliferation assays: heat-killed MT H37Ra (Difco Laboratories, Detroit, MI), hsp 65 M. bovis BCG recombinant protein, cloned and purified as previously described [17] and synthetic peptides 180-188,180-196 and 183-196 prepared by solid-phase techniques according to Steward et al. [MI.
Eur. J. Immunol. 1991. 21: 1289-1296
2.3 Arthritis induction with MT Arthritis was induced by injection of MT. Rats were injected intracutaneously at the base of the tail with 100 p1 of 10 mg/ml MT in IFA. The rats were observed for clinical arthritis and were scored by grading each paw from 0 to 4 based on erythema, swelling and deformity of the joint. All four legs were scored, so the highest score achievable was 16 ~91.
2.4 Passive transfer of AA Rats were immunized with MT at day0, for arthritis induction. At day 14 the spleens were removed and the cells were cultured in a concentration of 2 x 106for 3 days in the presence of 1 pg/ml Con A (Sigma, St. Louis, MO). Dead cells were removed by centrifugation on a Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) gradient. Naive animals were injected i.v with 1 x lo8 to 5 x 108 cells. After 8 days arthritis started to develop.
2.5 T cell proliferation assay Rats were immunized in each hind footpad with 50 pl of 10 mg/ml MT in IFA or with 50 pg hsp65 or 180-196 peptide mixed with 1mg dimethyl dioctadecyl ammonium bromide (Eastman Kodak, Rochester, NY) in PBS [20]. Nine days after immunization draining LN were removed and cell suspensions were made. Proliferative responses were measured in flat-bottom microtiter plates (Costar, Cambridge, MA) in triplicate wells. Each well contained 2 x 105 cells suspended in 0.2 ml DMEM (Gibco, Grand Island, NY) supplemented with 10% FCS (Gibco), 5X M 2-ME, 2 mM L-glutamhe, 100 U/ml penicillin and 100 pglrnl streptomycin, in the presence or absence of antigen. The cells were cultured for 3 days and for the last 16 h pulsed with [3H]dThd.The cultures were harvested on fiberglass filters, dThd incorporation was measured in a liquid scintillation counter and stimulation induces were determined (SI: cpm test divided by cpm control without antigen). In v i m restimulation of the cells was done by culturing the cells in flasks (Costar) for 3 days in the presence of 10 pg/ml antigen (hsp 65 or 180-196 peptide) at a concentration of 5 x 106/ml. After 3 days dead cells were removed using a Ficoll-Isopaque gradient and fresh medium with 10% EL4 SN as IL2 source was added. Seven days later the cells were tested in a proliferation assay as described above, 2 x 104 cells/well were tested together with 1 x lo6 autologous accessory cells (thymocytes). Tcell clone A2b was raised and maintained as described [21,22].When T cell clone A2b was used, 2 x 104 cells were tested together with 1 x lo6 irradiated (2500 rad) thymocytes isolated from either Lewis, Fisher or BN rats, as previously described [8].
2.6 Suppression of T cell clone A241 by LN cells from MT-immunized Fisher rats A2b cells (2 x 104) were tested for proliferation in the presence of the 180-188 peptide, 1 x 106 syngeneic APC (irradiated thymocytes) and 1 x 104 irradiated or non-
Eur. J. Immunol. 1991. 21: 1289-1296
Regulation by an hsp65 epitope in adjuvant arthritis
irradiated LN cells from MT-immunized Fisher rats. Fisher rats were immunized with 1 mg MT in the hind footpads and 9 days later the popliteal LN cells were isolated. The cell population was enriched for CD4+ and CD4- cells by a magnetic cell sorter MACS (Becton Dickinson, Mountain View, CA). The MACS separation was performed according the instructions of Becton Dickinson. Briefly, 6 X lo7 cells were washed in MACS buffer (PBS; 0.2% BSA; 0.01% sodium azide), labeled with biotinylated W3/25 mAb (dilution 1/200) and incubated for 15 min on ice.The cells were washed, resuspended in 1 ml, 200 p1 FITC-avidin (Becton Dickinson) was added and the cells were incubated for 15 min on ice.The cells were washed and resuspended in 200 pl. MACS biotin microbeads (2 pl; Becton Dickinson) were added and the cells were incubated for 5 min and washed. The column was sterilized, before use, with 70% ethanol and washed with PBS and MACS buffer. The cells were resuspended in 0.5 ml MACS buffer and pipetted onto the column (A2; Becton Dickinson) and washed with MACS buffer. The effluent was collected as the nonmagnetic (CD4-) fraction and the weakly bound cells were collected by washing the column with MACS buffer. The column was taken out of the separator and the positive cells were quickly washed out. The purity of the resulting fractions was analyzed by FCM (FACScan, Becton Dickinson).
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Table 2. After development of arthritis in Fisher rats, proliferative responses to the 180-188 epitope may be seena)
Antigen None
MT hsp 65 180-188 183-196
Arthritic rats 7.7b) 125.6 74.2 173 6.6
17.SC) 129.5 69.6 425 ND
Non-arthritic rats 4.5f 3.9) 54.5 f 37.7 1 1 . 9 f 11.2 5 . 4 k 4.8 ND
a) Results in cpm X b) Inguinal L N cells were tested 50 days after MTadministration in the base of the tail (n = 1). c) Inguinal L N cells were tested 35 days after MTadministration in the base of the tail (n = 1). d) Popliteal L N cells were tested 9 days after MTadministrationin the hind footpad ( n = 10).
Con A-activated spleen or LN cells was successful in Lewis (9 our of 12) but not in Fisher or BN rats.
3.2 Responses to mycobactend antigens in rat strains with distinct AA susceptibility
To investigate whether the responsiveness to selected mycobacterial antigens could be related to A A susceptibility, different rat strains were tested for responsiveness to these antigens. Lewis, Fisher, (Lewis x Fisher)F1 and BN 3.1 Lewis, Fisher, (Lewis x Fisher)F1 and BN rats differ rats were immunized in the hind footpads with MTand the in AA susceptibility draining LN cells were tested for antigen-specific proliferThe rat strains used in the present experiments are shown in ative responses 9 days later. All four rat strains developed Table 1. Lewis rats were found to be very susceptible to vigorous responses to MT (SI: Lewis 17.3 k 6.0; Fisher AA; Fisher and (Lewis x Fisher)F1 rats, despite the fact 15.0 k 4.9; (Lewis x Fisher)F1 21.0 k 3.5; BN 16.5 f 7.8). that they carry the same MHC class I1 haplotype as Lewis However, marked differences in their responses to hsp 65 were generally resistant to A A and BN rats were found to were found (Fig. 1). Lewis, Fisher and (Lewis x Fisher)FI be intermediate susceptible. The numbers given in Table 1 LN cells showed strong positive responses to hsp65, relate to the success rates in our hands of inducing arthritis whereas BN rats did not. Lewis rats showed responses to experimentally. Whereas A A induction in Lewis rats was the 180-188 synthetic peptide, while Fisher, (Lewis X found to be very successful (85 cases out of 91 MT Fisher)F, and BN rats did not. Responses to the 183-196 immunizations), we succeeded in inducing A A in Fisher peptide were essentially negative in all strains. Cells from rats only in two exceptional cases. In these cases, shown in naive Lewis, Fisher and BN rats showed low proliferative this report, onset of disease (day 35 and 20, respectively) responses to hsp 65 (SI: 2) and no responses to the 180-188 was significantly delayed as compared to what is seen in peptide (not shown). Lewis rats (mean day 14). In (Lewis x Fisher)FI rats incidence rates were found similar to those seen in Fisher rats. In BN rats arthritis was found exclusively in male rats 3.3 Reactivity to the 180-188 epitope in arthritic Fisher rats and not in females. Severity was generally reduced (mean maximal arthritis score 6.8 f 0.5) as compared to that seen in Lewis (mean maximal arthritis score 10.7 f 0.3). So far responsiveness to the 180-188 epitope seemed presAttempts to induce disease by means of passive transfer of ent exclusively in Lewis rats and not in Fisher, (Lewis X
3 Results
Table 1. Susceptibility to A A in four different rat strains Strain
MHC
Lewis Fisher (Lewis x Fisher)FI BN
RTl’ RT1”’ RTlm RTln
Arthritis induced with MT by transfer 85/91 2/12 I/ 6 12/16b)
9/12 w 2 ND o/ 4
Mean arthritis score” 10.7 f 0.3 5.0 2.0 9.0 6.8 +_ 0.5
*
a) Mean maximum arthritis scores are shown of animals who developed arthritis after administration of MT. b) Four female and twelve male rats were used. All male rats developed arthritis, in contrast to female rats who are not susceptible.
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Eur. J. Immunol. 1991. 21: 1289-1296
E. J. M. Hogervorst, C. J. P. Boog, J. P. A. Wagenaar et al.
Fisher)FI and BN rats, irrespective of the fact whether arthritis was seen (BN) or not [Fisher and (Lewis x Fisher)Fl]. However, two Fisher rats developed arthritis after immunization with MT. These particular animals showed responses to hsp65 and also the 180-188 peptide (Table 2). Arthritis in the first animal started approximately at day 35 after MT immunization and reached a maximum score of 7 at day43. At day50, when the score decreased to 3, the animal was killed and the draining inguinal LN cells demonstrated a high proliferative response to hsp65 as well as to the 180-188 epitope peptide. In the other Fisher rat, arthritis was observed at day 20 with a maximum score of 3. At that moment this animal was tested for proliferative responses and had responses to the 180-188 nonapeptide similar to what was seen in the previous rat. 3.4 Nonresponsiveness of Fisher rats to the 180-188 peptide is not caused by lack of antigen presentation
We then examined whether the absence of proliferative responses to the 180-188 epitope in non-arthritic MTimmunized Fisher rats could be explained by differences between Lewis, Fisher and BN rats at the level of antigen presentation. Fisher and BN APC were tested for their ability to present the hsp 65 antigen to the Lewis A2bT cell clone (Table 3). As can be seen in this representative experiment, Fisher cells were capable of selecting and presenting the proper mycobacterial epitope for recognition by A2b but BN cells were not. The same results were found when Tcell clone A2c was tested (not shown). The low-grade proliferation found after stimulation A2b in the presence of the 180-188 epitope and BN APC is most likely caused by some degree of autologousT cell presentation by A2b itself. A2b is expressing MHC class I1 molecules and the 180-188 epitope does not need processing for recognition (Wauben et al., unpublished results).The fact that BN cells did not present, can be explained by the difference in the MHC of BN rats vs. that of Fisher and Lewis rats. 3.5 Non-responsiveness of Fisher to the 180-188 epitope is also not due to antigen-specific suppression Another explanation for the absence of responses to the 180-188 epitope in Fisher rats would be a specific suppression of 180-188 epitope-directed responses. When, however, the polyclonal responses to the 180-188 epitope of Table3. Fisher APC present peptide 180-188 to T cell clone A2b Antigen
None MT hsp 65 180-188
Proliferative responsesa) of T cell clone A2bh) in the presence of irradiated APC from Lewis Fisher BN
0.1 121 92
0.2 92 71
0.1 0.1 0.6
119
92
2.5
a) Results in cpm x b) T cell clone A2b was obtained from a Lewis rat.
Table 4. Proliferation of Lewis T cell clone A2b is not suppressed by cells from MT-immunized Fisher rats Added cells None
Proliferative responses of A2b.I
74.6 f 3.3
Non-irradiated Fisher cells
Non-separated
77.8 f 1.8 73.0 f 4.4 61.5 f 2.7 90.7 f 5.8
Irradiated Fisher cells CD4cD4+ CD4-/CD4+ mixture Non-separated
12.9 f 7.0 66.2 f 2.3 73.9 f 1.9 65.5 f 1.8
cD4CD4+ cD4-/CD4+ mixture
a) Clone A2b was tested for proliferation in the presence of 10 pg/ml of peptide 180-188, irradiated syngeneic APC (thymocytes) and (non-)irradiated popliteal LN cells from MTimmunized Fisher rats. Fisher rat cells were used which were separated in a CD4+ or CD4- enriched population by a MACS separator. Both populations were also mixed (CD4-/CD4+ mixture) and tested and also unseparated cells were used. Results are shown in cpm x
Lewis cells after MT immunization were tested in the presence of MT-primed Fisher cells, no evidence for suppression of the response was found (data not shown). Also at the level of clonal responses with specificity for the 180-188 epitope, we were unable to demonstrate that addition of nonresponder primed Fisher cells, resulted in suppression of the response of T cell clone A2b to the 180-188 peptide (Table 4). For this purpose we tested both unseparated fractions of LN cells from Fisher rats and also a CD4- or CD4+ enriched population. Separation was performed by magnetic cell sorting and the biotinylated anti-CD4 mAb W3/25. After separation, the cells in the “non-magnetic” CD4- fraction and the “magnetic” CD4+ fraction were enriched threefold. We used irradiated and non-irradiated Fisher cells because previously it was described that suppressor cells were radiation sensitive. We were able to use non-irradiated Fisher cells because, as we have shown, these cells do not proliferate themselves in the presence of the 180-188 epitope. None of the Fisher cell fractions suppressed the proliferative responses of Lewis T cell clone A2b. 3.6 In Lewis rats responses to hsp65 and the 1804% epitope seem to be under regulatory control
After immunizing animals with MT in the footpads, Lewis and Fisher rats showed responses to hsp 65, whereas BN rats did not (Fig. 1 and Fig. 2). Following in vitro restimulation with hsp 65, a distinct pattern of proliferative responses to this protein and the 180-188 or 180-196 peptide was seen. Lewis, Fisher and also BN rats, all demonstrated elevated responses to hsp 65. Remarkably enough, responses to hsp 65 in BN rats exceeded those found in Lewis and Fisher rats after hsp 65 restimulation. On the other hand striking differences were seen in the responses to the 180-196 peptide. Fisher and BN rats showed no response at all not
Regulation by an hsp65 epitope in adjuvant arthritis
Eur. J. Immunol. 1991. 21: 1289-1296
even after in virro restimulation with hsp65. Lewis rats showed a response to the 180-196 peptide which, however, decreased after restimulation in vitro with hsp65 (Fig. 2). So, unexpectedly, although responses to peptide 180-188 seem to dominate after MT immunization in Lewis rats, restimulation with the hsp 65 antigen, containing the 180-188 epitope, did not seem to enhance further responsiveness to the 180-196 epitope. Similarly, when animals were immunized in vivo with hsp65, no responses to the 180-196 epitope were seen, not even in Lewis rats (Fig. 3).
Lewis
To test the possibility of inducing 180-188- or 180-196specific T cells by mere immunization with the 180-196 peptide itself, animals from all three rat strains were immunized in the hind footpads with peptide 180-196 and tested for proliferative responses.The results given in Fig. 4 show that without in vitro restimulation responses to the 180-196 peptide were obtained in Lewis rats. Such responses were not found in BN rats not even after in vitro stimulation with hsp 65 or the 180-196 peptide. In contrast to Fisher cells, however, Lewis T cells showed a high increment in their responses to the 180-196 epitope after in vitro restimulation with this peptide. In accordance with the results shown in Figs. 2 and 3 in virro restimulation with hsp 65 decreased the proliferative responses to the 180-196 peptide. N o change in 180-196 induced responses was found in Fisher or BN rats after in vitro restimulation with hsp 65 or with the 180-196 peptide (Fig. 4).
Fisher
5 X
Z
4 -
QI-
; 2
Furthermore, while Fisher and BN rats showed enhanced responses to hsp65 after in vitro restimulation with the protein, Lewis rats showed no enhancement of responses before or after restimulation in virro (Fig. 3).Taken together, these findings suggest a regulatory effect of hsp65 on 180-196-specific T cell responses. Moreover, as suggested by findings presented in Fig. 3, such regulation could be especially pertinent to Lewis rats.
3.7 T ceUs responding to the 180-188 epitope can be demonstrated exclusively in Lewis rats
6 -
xz
1293
3 -
I-
rT)
2 -
1 -
hsp 6 5
180-188
183-196
4 Discussion
Figure 1. Proliferative responses in Lewis, Fisher, (Lewis X Fisher)F, and BN rats after immunization with whole mycobacteria. Popliteal LN cells were obtained from rats immunized 9 days earlier with 1 mg of MT in IFA in the hind footpad. Proliferative responses were measured with 2 x 106 celldml in the presence and absence of antigen. Various concentrations of hsp 65 peptide 180-188 and peptide 183-196 were tested. The results are shown for responses obtained with an optimal concentration of 20 pg/ml antigen. Results are given as stimulation indexes (SI f SE). * Significant difference when compared to Lewis (p< 0.05).
rat straln
p m n ig I
I
)
~
Agusedfor in* reslimlation
Ag used in poliferstion assay
None HSP65
HSP65 HSP65
HSP65
180-196 180196
None HSP65 HSP65
None
0
L
10
15
20
25
11.7
HSP65 HSP65 160-196 180196
8.5
HSP65 HSP65 180-196 180-196
BN
5
AA is most easily induced in susceptible Lewis rats, either by active immunization with mycobacterial antigen or by passive transfer of immune lymphocytes. By Tcell cloning of responder lymphocytes obtained from MT-immunized Lewis rats, T cell populations were obtained that either were arthritogenic (A2,A2b) or protective (A2c) upon transfer. When examined for antigenic specificity, all functional LewisTcells obtained thus far showed specificity for
19.6
J
Figure 2. Proliferative responses in MT-immunized Lewis, Fisher and BN rats. Rats were immunized with 1 mg MT in IFA in the footpads and 9 days later, the popliteal LN cells were tested for proliferation to hsp 65 and the 180-196 peptide (concentration of 25 Kglml). Cells were also restimulated in vitro with 10 pg/ml hsp65 and tested for their specificity as described in Sect. 2.5. The results are shown as SI If: SE.
1294
E. J. M. Hogervorst, C. J. F? Boog, J. F! A. Wagenaar et al. Ag used for
rat strain
p n !g m m
invilro restimulation
Ag used in proliferation assay
10
0
Eur. J. Immunol. 1991. 21: 1289-1296
20
50
40
30
60
None HSP6.5 HSP6.5
180-196 180-196
0.9
I
c
21.5
L
ffl
HSP65
[
None HSP65
HSP6.5 HSP65 180-196 180-196
48.4
Figure3. Proliferative responses in hsp65-immunized Lewis, Fisher and BN rats. Rats were immunized with 100 pg hsp65 in dimethyldioctadecyl ammonium bromide in the hind footpads and popliteal LN cells were tested for proliferative responses and restimulated in vitro according to the procedure described in Fig. 2.
rat strain
ptng in
yry~
Ag used for
invitro restirnulation
Agusedin proliferation assay
I
Lewk
180.196
[
180-196
10
20
30
40
50
60
70
I
I
1
I
I
I
I
I
S.I.
180-196 HSP65
180-196
1 9 6
180-196
180-196
BN
0
[ 1, HSPa
56.5
12.9
180-196 180-196 180-196
1.1
Figure 4. Proliferative responses in peptide 180-196-immunized Lewis, Fisher and BN rats. Rats were immunized with 50 pg of peptide 180-196 in dimethyl dioctadecyl ammonium bromide in the hind footpads and popliteal L N cells were tested for proliferative responses and in vitro restimulated according to the procedure described in Fig. 2.
the 180-188 sequence present in the hsp 65 of mycobacteria ([8], Hogervorst et al., unpublished results). The results of the present study show that rats that differ in their susceptibility to A A may have distinct Tcell responses to these mycobacterial antigens. Lewis rats, highly susceptible to AA, can be shown to recognize the nonapeptide present on hsp 65 of M . bovis BCG after MT immunization at the polyclonal level. However, Fisher rats, not susceptible to AA, failed to respond to the 180-188 epitope,while showing excellent responses to epitopes of hsp 65 different from 180-188. In Fisher rats we also showed that, even after in vivo immunization and subsequent in vitro restimulations with peptide 180-196, it was not possible to demonstrate the presence of 180-188-reactive T cells. Lewis and Fisher were reported to be MHC RT1 compatible by serology [23] and GvH reaction [24]. However, differences were found in MLC, induced by a distinction at the RT1.C (class I) locus [25-271. The major antigen studied here, hsp65, is an antigen predominantly presented by class I1 molecules [6, 28-30]. We demonstrated that Fisher rats
were able of presenting the 180-188 epitope to LewisTcell clone A2b and that arthritic Fisher rats did respond to this epitope.
BN rats which were susceptible, albeit to a lower degree than Lewis rats, were found to be nonresponders to the 180-188 epitope either after MT immunization or after immunization with peptide 180-196 itself and subsequent restimulation with this peptide in vitro. In BN rats the response to the 180-188 epitope was probably absent due to the fact that these rats have an MHC haplotype that differs from Lewis and therefore, were not able to present this epitope to their T cells. This would imply that BN rats possibly select an arthritogenic epitope in MT that differs from 180-188, present on the hsp65 or not. In this regard it may be of interest that we have seen responses in MTprimed BN T lymphocytes to hsp 70 that exceeded those to hsp 65 (unpublished results). Generation of arthritogenic BN T cell lines and determination of the fine specificity is currently being done. The preliminary investigations of arthritis development and responsiveness to hsp 65 in
Eur. J. Imrnunol. 1991.21: 1289-1296
(Lewis x Fisher)F1 rats, showed that the results resemble those of Fisher rats, indicating a similar immunoregulation. The susceptibility of rats to A A may be explained by an improper regulation of responses directed to epitopes of MT that are “risky”. The 180-188 epitope can be one of such epitopes because of its structural or sequential homology with self structures of the rat (for example the link protein of proteoglycansof the cartilage; [7, 311). However, n o sequence homology was recently found with rat hsp 60 ~321. As we have shown now, responses to the particular 180-188 peptide can be demonstrated in Lewis rats after MT immunization. Their MHC class 11-identical Fisher counterparts, however, when immunized in the same manner, are obviously capable of avoiding the recognition of this epitope. The finding that an exceptional arthritic Fisher rat had a remarkably high response to the 180-188 epitope was suggestive of the fact that Fisher rats control their risk to develop arthritis by their lack of responsiveness to the 180-188 epitope. The results obtained from (Lewis x Fisher)FI rats have indicated that such regulation is dominant and is compatible with the existence of Fisher background genes controlling arthritis resistance. Nevertheless, so far no arthritis has been seen, not even in Lewis rats, following immunization with hsp 65 itself or the 180-188 synthetic peptide. On the contrary, immunization with hsp65 [8] or the 180-188 peptide [33,34] has been found to induce protection instead. hsp 65 pretreatment resulted in resistance to the induction of not only AA, but also streptococcal cell-wall-induced arthritis (SCW) [ 151 and pristane-induced arthritis in mice [16]. In collagen type 11-induced arthritis disease reduction was reported [14,35]. Thus, although containing a potentially arthritogenic epitope, the immunological consequences of responses to hsp65 turn out to be of a complex nature. In this study, we showed that the response to the 180-196 peptide and hsp65 itself were subject to regulation, after stimulating the cells with hsp65. After in vivo immunization with MT or the 180-196 epitope the proliferative responses could be suppressed, in the presence of the hsp 65 molecule. These results suggested the presence of other epitopes on the hsp65 molecule, capable of controlling responses to the critical 180-188 epitope. An example of such another epitope has been characterized now by Cohen et al. [36] as recognized by he protective clone M1, which seems to be part of a so-called preemptive Tcell network involved in regulating responses to hsp 65. It will be of interest to understand how Fisher rats develop such effective means to become resistant to A A and also other forms of experimental arthritis, such as SCW arthritis, despite the fact of having the proper genetic background to present the 180-188 epitope of hsp65. First, there still might be a difference in processing or presentation of the antigen. We now have demonstrated that this is not the case, since both Lewis and Fisher APC can present epitope 180-188 of MT and hsp65 to T cell clone A2b equally well. A second explanation could be the induction of suppression in Fisher rats, exerted by epitope 180-188specific cells or T cells with specificity for other suppressive epitopes on hsp 65. Our experimental findings, however,
Regulation by an hsp65 epitope in adjuvant arthritis
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showed no evidence for the presence of cellular suppression in MT-immunized Fisher rats when tested in the presence of responding Lewis cells. A third possibility could be that Lewis and Fisher rats have differences in their Tcell repertoire. Kohashi et al. [3] have shown that Fisher rats when bred under germ-free conditions were susceptible to AA. When, however, Fisher rats became colonized, for instance with E. coli, they became resistant to AA.Thus, it appeared that antigens present in the colonizing bacterial flora were inducing tolerance, possibly by the selective elimination (functional or physical) of theT cells reactive to potentially arthritogenic antigens. It is possible that hsp are the common denominators in the findings of Kohashi et al. [3] and the present findings with respect to hsp 65.Tolerance induction was thoroughly studied in the autoimmune model of EAE, which showed that tolerance to self epitopes is determined in the thymus during embryonic development or in the first days of postnatal life [37,38]. In the EAE model it has been shown that this tolerance inducting can lead to changes at the level of Vg families of theTcR and that such changes can have consequence for disease susceptibility [39,40]. Irrespective of which explanation will finally turn out to be right, resistance to experimental arthritis can be acquired, either early in life by mere exposition to antigen present in the commensal flora (Fisher) or by active immunization with a purified bacterial hsp65, even in the presence of genes predisposing to disease (Lewis). Recent findings made in human patients, have indicated that in humans, especially in the affected joints, T cells responding to hsp65 were also present [lo, 12, 13, 411. Furthermore in synovial cells of juvenile rheumatoid arthritis patients responses to the 180-188 peptide, to the partially homologous sequence of the rat link protein of proteoglycan (Danielli et al., submitted) and to the human hsp60 (Pl) (De Graeff-Meeder et al., submitted) were demonstrated. This lends support to the possibility that in human arthritis, T cell reactivity with specificity for a comparable mimicry epitope of the hsp 65 molecule may also play a role. Be that as it may, the present findings may be expected to contribute to our understanding of the regulatory mechanisms that control responses to epitopes critical to autoimmunity, such as the 180-188 epitope. And this can eventually lead to the successful development of new means of specific immunological intervention in autoimmune diseases. We thank Prof. I . R. Cohen (Weizmann Institute. Rehovot. Israel) for making clone A26 available for these studies.
Received December 10, 1990; in revised form February 7, 1991.
5 References 1 Pearson, C. M., Proc. SOC.Exp. Biol. Med. 1956. 91: 95. 2 Battisto, J. R., Smith, R. N., Beckmann, K., Sternlicht, M. and Welles, W. L., Arthritis Rheum. 1982. 25: 1194. 3 Kohashi, O., Kuwata, J., Umehara, F., Takahashi, T. and Ozawa, A., Infect. Immun. 1979. 26: 791. 4 Lussier, A., De Medicis, R. and Tetreault, L., Int. J. Tissue Reac. 1984. VI: 195. 5 Waksman, B. H. and Wennersten, C., Int. Arch. Allergy Appl. Immunol. 1963. 23: 129.
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6 Holoshitz, J., Matitiau, A. and Cohen, I. R., J. Clin. Invest. 1984. 73: 211. 7 Van Eden,W. Holoshitz, J., Nevo, Z., Frenkel, A., Klajman, A. and Cohen, I. R., Proc. Natl. Acad. Sci. USA 1985. 82: 5117. 8 Van Eden, W.,Thole, J. E. R. ,Van der Zee, R., Noordzij, A., Van Embden, J. D. A., Hensen, E. J. and Cohen, I. R., Nature 1988. 331: 171. 9 Van der Zee, R.,Van Eden, W., Meloen, R. H., Noordzij, A. and Van Embden, J. D. A., Eur. J. Immunol. 1989. 19: 43. 10 Res, €! C. M., Breedveld, F. C.,Van Embden, J. D. A., Schaar, C. G.,Van Eden,W., Cohen, I. R. and DeVries, R. P. P., Lancet 1988. ii: 478. 11 Bahr, G. M., Rook, G. A. W., Al-Saffar, M.,Van Embden, J., Stanford, J. L. and Behbehani, K., Clin. Exp. Immunol. 1988. 74: 211. 12 Holoshitz, J., Drucker, I.,Yaretzky, A.,Van Eden,W., Klajman, A., Lapidot, Z., Frenkel, A. and Cohen, I. R., Lancet 1986. ii: 305. 13 Holoshitz, J., Koning, F., Coligan, J. E., De Bruyn, J. and Strober, S., Nature 1989. 339: 226. 14 Billingham, M. E. J., Carney, S., Butler, R. and Colston, M. J., J. Exp. Med. 1990. 171: 339. 15 Van de Broek, M. E , Hogervorst, E. J. M.,Van Bruggen, M. C. J., Van Eden, W.,Van der Zee, R. and Van den Berg, W. B., J. Exp. Med. 1989. 170: 449. 16 Thompson, S. J., Rook. G. A. W., Brealey, R. J.,Van der Zee, R. and Elson, C. J., Eur. J. Immunol. 1990. 20: 2479. 17 Thole, J. E. R., Keu1en.W. J., De Bruyn, J., Kolk, A. H. J., Groothuis, D. G., Benvald, L. G.,Tiesjema, R. H. and Van Embden, J. D. A., Infect. Immun. 1987. 55: 1466. 18 Steward, J. M. and Young, J. D., Solid phase peptide synthesis, Pearce Chemical Co., Illinois 1984. 19 Trentham, D. E.,Townes, A. S. and Kang, A. H., J. Exp. Med. Med. 1977. 146: 857. 20 Snippe, H. and Kraaijeveld, C.A., in Allison, A. C., Arnon, R., Gregoriadis, G., Playfair, J. H. I. and Post, P. (Eds.), Immunological adjuvants and vaccines, Plenum Press, New York 1989. 21 Ben-Nun, A.,Wekerle, H. and Cohen, I. R., Eur. J. Immunol. 1981. 11: 195.
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22 Holoshitz, J., Naparstek,Y., Ben-Nun, A. and Cohen, I. R.. Science 1983. 219: 56. 23 Palm, J. and Black, G., Transplantation 1971. 11: 184. 24 Elkins, W. L. and Palm, J., Ann. N X Acad. Sci. 1966. 129: 573. 25 Marshak, A., Doherty, l? C. and Wilson, D. B.. J. Exp. Med. 1977. 146: 1773. 26 Sieck,T., Marshak-Rothstein, A. and Wilson, D. B., Immunogenetics 1979. 9: 165. 27 Sieck,T. and Wilson, D. B., Immunogenetics 1981. 14: 293. 28 Emmrich, F.,Thole, J.,Van Embden, J. and Kaufmann, S. H. E., J. Exp. Med. 1986. 163: 1024. 29 Ottenhof,T., Kale Ab, B.,Van Embden, J. D. A.,Thole, J. and Kiessling, R., J. Exp. Med. 1988. 168: 1947. 30 Brett, S. J., Lamb, J. H., Rothbard, J. B., Mehlert, A. and Ivanyi, J., Eur. J. Immunol. 1989. 19: 1303. 31 Van Eden,W., Hogervorst, E. J. M., Hensen, E. J. ,Van der Zee, R., Van Embden, J. D. A. and Cohen, I. R., Curr. Top. Microbiol. Immunol. 1989. 145: 27. 32 Venner, T. J. and Gupta, R. S., Nucleic Acids Res. 1990. 18: 5309. 33 Yang, X.-D., Gasser, J., Riniker, B. and Feige, U., J. Autoimmun. 1990. 3: 11. 34 Yang, X.-D., Gasser, J. and Feige, U., Clin. Exp. Immunol. 1990. 81: 189. 35 Ito, J., Krco, C. J.,Yu, D., Luthra, H. S. and David, C., J. Cell. Biochem. 1991. 15A: 284. 36 Cohen, I. R., in Melchers, F., et al. (Eds.), Progress in Immunology Vll, Springer-Verlag, Berlin 1989, p. 867. 37 Kappler, J. W., Roehm, N. and Marrack, P., Cell 1987. 49: 273. 38 Taguchi, 0. and Nishizuka,Y., J. Exp. Med. 1987. 165: 146. 39 Chluba, J., Steeg, C., Becker, A.,Wekerle, H. andEpplen, J.T., Eur. J. Immunol. 1989. 19: 279. 40 Heber-Katz, E. and Acha-Orbea, H., Immunol. Today 1989. 10: 164. 41 Pope, R. M., Wallis, R. S., Sailer, D., Buchanan, T. M. and Pahlavani, M. A,, Cell. Immunol., in press.
Els J. M. Hogervorst., Claire J. P. Boog., Jos6e R A. Wagenam., Marca H. M. Wauben., Ruurd Van der Zee. and Willem Van Eden. Institute of Infectious Diseases and Immunology., Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht and Department of Bacteriologyv, National Institute of Public Health and Environmental Protection, Bilthoven
Regulation by an hsp65 epitope in adjuvant arthritis
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T cell reactivity to an epitope of the mycobacterial65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses* Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial65-kDa heat-shock protein (hsp65), the possible origin of A A was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)FI rats, although major histocompatibility complex class I1 identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.
1 Introduction Adjuvant arthritis (AA) is an extensively studied animal model for human rheumatoid arthritis and is characterized by an inflammatory reaction in the synovial tissue, leading to destruction of the joints, very similar to the human pathological conditions. A A is induced in rats by immunization with heat-killed Mycobacterium tuberculosis (MT) in oil [l]. Inbred rat strains have been found to differ in their susceptibility to A A , indicating the existence of genetic factors controlling resistance to AA. For example, Lewis rats are highly susceptible to AA, while Fisher F344 rats,
despite the fact that they share the same RT1.B and RT1.D MHCclass I1 loci with Lewis, are not susceptible to A A [2]. Interestingly enough, Fisher rats have been shown to be susceptible to A A when kept and bred under germ-free conditions [3]. Other susceptible rat strains are Wistar [4] and DA [2]. (Lewis XFisher)F~rats are resistant and Brown Norway (BN) rats are of intermediate susceptibility, as we have shown now.
The autoimmune nature of A A became apparent when it was found that the disease could be transferred by means of polyclonal lymphocytes obtained from diseased animals [ 5 ] . More recently, Holoshitz et al. [6], succeeded in selecting and propagating in vitro an arthritogenic T cell clone (A2b) with reactivity to MT. Further detailed specificity analysis of A2b was carried out, when it was [I 91161 found that A2b was capable of recognizing, besides MT, an * Part of this work was supported by Institut MCrieux, Lyon, antigen co-purified with cartilage proteoglycans [7]. This France. mimicry relationship between mycobacteria and a proteoglycan-associated self antigen, as defined at the level of the Correspondence: Willem Van Eden, Institute of Infectious Dis- arthritogenic effector cell (A2b) itself, was found to be eases and Immunology, Department of Immunology, Faculty of contained in a mycobacterial heat-shock protein (hsp). Veterinary Medicine, University of Utrecht, Yalelaan 1, NL3508 Subsequently both arthritogenic A2b and another protecTD Utrecht, The Netherlands tive Tcell clone A2c, were shown to recognize an epitope, Abbreviations: AA: Adjuvant arthritis MT: Mycobacterium comprizing seven amino acid (180-186) residues as present tuberculosis bsp66: 65-kDa Heat-shock protein of Mycobacter- in the 65-kDa hsp (hsp65) of Mycobacterium bovis BCG ium bovis BCG [8, 91. '
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
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Because of their evolutionary conservation, hsp are likely candidates for having such mimicry relationships with self structures. Such theoretical considerations and the empirical findings in the AA model have stimulated the search for the involvement of hsp in arthritis and other autoimmune disorders. A further characteristic feature of hsp seems to be their immunological dominance. After exposure of both experimental animals and humans to bacterial organism, antibodies and Tcells specific for hsp are easily found [10-13].The latter phenomenon is unexpected and is difficult to reconcile with the conserved nature of the molecules. Nevertheless, also in Lewis rats after AA induction with MT, responses to hsp tend to dominate (Hogervorst et al., in preparation). In the present study, responses to the hsp65 and its critical 180-188 epitope were examined in rats with distinct susceptibilities to AA. We studied four strains [Lewis, Fisher, (Lewis x Fisher)F1 and BN] for such responses in the draining LN cell populations after immunization with MT. Lewis rats showed responses to the hsp 65 and also to the 180-188 epitope. BN rats, carrying an MHC haplotype that differs from that of Lewis rats, were found to be low responders to hsp65 and nonresponders to the 180-188 epitope. Fisher rats, however, which have the same MHC class11 antigens as Lewis rats but are resistant to AA, responded to hsp65 but did not respond to the 180-188 epitope. (Lewis x Fisher)F1 rats showed similar responses to those of Fisher rats. These results further indicate that responsiveness to the 180-188 epitope is related to arthritis susceptibility and that the nonresponsiveness of Fisher rats to the 180-188 epitope is not caused by defective antigen processing or antigen presentation nor by active T cell suppression. Moreover, Lewis rats were also found capable of down-regulating their responses to the 180-188 epitope. Such inhibition of 180-188-specific responses was found to be triggered by responding to hsp 65, probably due to the presence of other regulatory epitopes present in hsp 65. These findings may explain the earlier reported results of hsp 65-induced protection in athritis models [8, 14-16].
2 Materials and methods 2.1 Rats Inbred Lewis rats were obtained from the University of Limburg, Maastricht, The Netherlands. BN, Fisher F344 and (Lewis x Fisher)F1 rats were obtained from Olac/HarIan CPB, Zeist, The Netherlands. All rats were used at 6-8 weeks of age.
2.2 Antigens The following antigens were used for immunization of the animals or in proliferation assays: heat-killed MT H37Ra (Difco Laboratories, Detroit, MI), hsp 65 M. bovis BCG recombinant protein, cloned and purified as previously described [17] and synthetic peptides 180-188,180-196 and 183-196 prepared by solid-phase techniques according to Steward et al. [MI.
Eur. J. Immunol. 1991. 21: 1289-1296
2.3 Arthritis induction with MT Arthritis was induced by injection of MT. Rats were injected intracutaneously at the base of the tail with 100 p1 of 10 mg/ml MT in IFA. The rats were observed for clinical arthritis and were scored by grading each paw from 0 to 4 based on erythema, swelling and deformity of the joint. All four legs were scored, so the highest score achievable was 16 ~91.
2.4 Passive transfer of AA Rats were immunized with MT at day0, for arthritis induction. At day 14 the spleens were removed and the cells were cultured in a concentration of 2 x 106for 3 days in the presence of 1 pg/ml Con A (Sigma, St. Louis, MO). Dead cells were removed by centrifugation on a Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) gradient. Naive animals were injected i.v with 1 x lo8 to 5 x 108 cells. After 8 days arthritis started to develop.
2.5 T cell proliferation assay Rats were immunized in each hind footpad with 50 pl of 10 mg/ml MT in IFA or with 50 pg hsp65 or 180-196 peptide mixed with 1mg dimethyl dioctadecyl ammonium bromide (Eastman Kodak, Rochester, NY) in PBS [20]. Nine days after immunization draining LN were removed and cell suspensions were made. Proliferative responses were measured in flat-bottom microtiter plates (Costar, Cambridge, MA) in triplicate wells. Each well contained 2 x 105 cells suspended in 0.2 ml DMEM (Gibco, Grand Island, NY) supplemented with 10% FCS (Gibco), 5X M 2-ME, 2 mM L-glutamhe, 100 U/ml penicillin and 100 pglrnl streptomycin, in the presence or absence of antigen. The cells were cultured for 3 days and for the last 16 h pulsed with [3H]dThd.The cultures were harvested on fiberglass filters, dThd incorporation was measured in a liquid scintillation counter and stimulation induces were determined (SI: cpm test divided by cpm control without antigen). In v i m restimulation of the cells was done by culturing the cells in flasks (Costar) for 3 days in the presence of 10 pg/ml antigen (hsp 65 or 180-196 peptide) at a concentration of 5 x 106/ml. After 3 days dead cells were removed using a Ficoll-Isopaque gradient and fresh medium with 10% EL4 SN as IL2 source was added. Seven days later the cells were tested in a proliferation assay as described above, 2 x 104 cells/well were tested together with 1 x lo6 autologous accessory cells (thymocytes). Tcell clone A2b was raised and maintained as described [21,22].When T cell clone A2b was used, 2 x 104 cells were tested together with 1 x lo6 irradiated (2500 rad) thymocytes isolated from either Lewis, Fisher or BN rats, as previously described [8].
2.6 Suppression of T cell clone A241 by LN cells from MT-immunized Fisher rats A2b cells (2 x 104) were tested for proliferation in the presence of the 180-188 peptide, 1 x 106 syngeneic APC (irradiated thymocytes) and 1 x 104 irradiated or non-
Eur. J. Immunol. 1991. 21: 1289-1296
Regulation by an hsp65 epitope in adjuvant arthritis
irradiated LN cells from MT-immunized Fisher rats. Fisher rats were immunized with 1 mg MT in the hind footpads and 9 days later the popliteal LN cells were isolated. The cell population was enriched for CD4+ and CD4- cells by a magnetic cell sorter MACS (Becton Dickinson, Mountain View, CA). The MACS separation was performed according the instructions of Becton Dickinson. Briefly, 6 X lo7 cells were washed in MACS buffer (PBS; 0.2% BSA; 0.01% sodium azide), labeled with biotinylated W3/25 mAb (dilution 1/200) and incubated for 15 min on ice.The cells were washed, resuspended in 1 ml, 200 p1 FITC-avidin (Becton Dickinson) was added and the cells were incubated for 15 min on ice.The cells were washed and resuspended in 200 pl. MACS biotin microbeads (2 pl; Becton Dickinson) were added and the cells were incubated for 5 min and washed. The column was sterilized, before use, with 70% ethanol and washed with PBS and MACS buffer. The cells were resuspended in 0.5 ml MACS buffer and pipetted onto the column (A2; Becton Dickinson) and washed with MACS buffer. The effluent was collected as the nonmagnetic (CD4-) fraction and the weakly bound cells were collected by washing the column with MACS buffer. The column was taken out of the separator and the positive cells were quickly washed out. The purity of the resulting fractions was analyzed by FCM (FACScan, Becton Dickinson).
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Table 2. After development of arthritis in Fisher rats, proliferative responses to the 180-188 epitope may be seena)
Antigen None
MT hsp 65 180-188 183-196
Arthritic rats 7.7b) 125.6 74.2 173 6.6
17.SC) 129.5 69.6 425 ND
Non-arthritic rats 4.5f 3.9) 54.5 f 37.7 1 1 . 9 f 11.2 5 . 4 k 4.8 ND
a) Results in cpm X b) Inguinal L N cells were tested 50 days after MTadministration in the base of the tail (n = 1). c) Inguinal L N cells were tested 35 days after MTadministration in the base of the tail (n = 1). d) Popliteal L N cells were tested 9 days after MTadministrationin the hind footpad ( n = 10).
Con A-activated spleen or LN cells was successful in Lewis (9 our of 12) but not in Fisher or BN rats.
3.2 Responses to mycobactend antigens in rat strains with distinct AA susceptibility
To investigate whether the responsiveness to selected mycobacterial antigens could be related to A A susceptibility, different rat strains were tested for responsiveness to these antigens. Lewis, Fisher, (Lewis x Fisher)F1 and BN 3.1 Lewis, Fisher, (Lewis x Fisher)F1 and BN rats differ rats were immunized in the hind footpads with MTand the in AA susceptibility draining LN cells were tested for antigen-specific proliferThe rat strains used in the present experiments are shown in ative responses 9 days later. All four rat strains developed Table 1. Lewis rats were found to be very susceptible to vigorous responses to MT (SI: Lewis 17.3 k 6.0; Fisher AA; Fisher and (Lewis x Fisher)F1 rats, despite the fact 15.0 k 4.9; (Lewis x Fisher)F1 21.0 k 3.5; BN 16.5 f 7.8). that they carry the same MHC class I1 haplotype as Lewis However, marked differences in their responses to hsp 65 were generally resistant to A A and BN rats were found to were found (Fig. 1). Lewis, Fisher and (Lewis x Fisher)FI be intermediate susceptible. The numbers given in Table 1 LN cells showed strong positive responses to hsp65, relate to the success rates in our hands of inducing arthritis whereas BN rats did not. Lewis rats showed responses to experimentally. Whereas A A induction in Lewis rats was the 180-188 synthetic peptide, while Fisher, (Lewis X found to be very successful (85 cases out of 91 MT Fisher)F, and BN rats did not. Responses to the 183-196 immunizations), we succeeded in inducing A A in Fisher peptide were essentially negative in all strains. Cells from rats only in two exceptional cases. In these cases, shown in naive Lewis, Fisher and BN rats showed low proliferative this report, onset of disease (day 35 and 20, respectively) responses to hsp 65 (SI: 2) and no responses to the 180-188 was significantly delayed as compared to what is seen in peptide (not shown). Lewis rats (mean day 14). In (Lewis x Fisher)FI rats incidence rates were found similar to those seen in Fisher rats. In BN rats arthritis was found exclusively in male rats 3.3 Reactivity to the 180-188 epitope in arthritic Fisher rats and not in females. Severity was generally reduced (mean maximal arthritis score 6.8 f 0.5) as compared to that seen in Lewis (mean maximal arthritis score 10.7 f 0.3). So far responsiveness to the 180-188 epitope seemed presAttempts to induce disease by means of passive transfer of ent exclusively in Lewis rats and not in Fisher, (Lewis X
3 Results
Table 1. Susceptibility to A A in four different rat strains Strain
MHC
Lewis Fisher (Lewis x Fisher)FI BN
RTl’ RT1”’ RTlm RTln
Arthritis induced with MT by transfer 85/91 2/12 I/ 6 12/16b)
9/12 w 2 ND o/ 4
Mean arthritis score” 10.7 f 0.3 5.0 2.0 9.0 6.8 +_ 0.5
*
a) Mean maximum arthritis scores are shown of animals who developed arthritis after administration of MT. b) Four female and twelve male rats were used. All male rats developed arthritis, in contrast to female rats who are not susceptible.
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E. J. M. Hogervorst, C. J. P. Boog, J. P. A. Wagenaar et al.
Fisher)FI and BN rats, irrespective of the fact whether arthritis was seen (BN) or not [Fisher and (Lewis x Fisher)Fl]. However, two Fisher rats developed arthritis after immunization with MT. These particular animals showed responses to hsp65 and also the 180-188 peptide (Table 2). Arthritis in the first animal started approximately at day 35 after MT immunization and reached a maximum score of 7 at day43. At day50, when the score decreased to 3, the animal was killed and the draining inguinal LN cells demonstrated a high proliferative response to hsp65 as well as to the 180-188 epitope peptide. In the other Fisher rat, arthritis was observed at day 20 with a maximum score of 3. At that moment this animal was tested for proliferative responses and had responses to the 180-188 nonapeptide similar to what was seen in the previous rat. 3.4 Nonresponsiveness of Fisher rats to the 180-188 peptide is not caused by lack of antigen presentation
We then examined whether the absence of proliferative responses to the 180-188 epitope in non-arthritic MTimmunized Fisher rats could be explained by differences between Lewis, Fisher and BN rats at the level of antigen presentation. Fisher and BN APC were tested for their ability to present the hsp 65 antigen to the Lewis A2bT cell clone (Table 3). As can be seen in this representative experiment, Fisher cells were capable of selecting and presenting the proper mycobacterial epitope for recognition by A2b but BN cells were not. The same results were found when Tcell clone A2c was tested (not shown). The low-grade proliferation found after stimulation A2b in the presence of the 180-188 epitope and BN APC is most likely caused by some degree of autologousT cell presentation by A2b itself. A2b is expressing MHC class I1 molecules and the 180-188 epitope does not need processing for recognition (Wauben et al., unpublished results).The fact that BN cells did not present, can be explained by the difference in the MHC of BN rats vs. that of Fisher and Lewis rats. 3.5 Non-responsiveness of Fisher to the 180-188 epitope is also not due to antigen-specific suppression Another explanation for the absence of responses to the 180-188 epitope in Fisher rats would be a specific suppression of 180-188 epitope-directed responses. When, however, the polyclonal responses to the 180-188 epitope of Table3. Fisher APC present peptide 180-188 to T cell clone A2b Antigen
None MT hsp 65 180-188
Proliferative responsesa) of T cell clone A2bh) in the presence of irradiated APC from Lewis Fisher BN
0.1 121 92
0.2 92 71
0.1 0.1 0.6
119
92
2.5
a) Results in cpm x b) T cell clone A2b was obtained from a Lewis rat.
Table 4. Proliferation of Lewis T cell clone A2b is not suppressed by cells from MT-immunized Fisher rats Added cells None
Proliferative responses of A2b.I
74.6 f 3.3
Non-irradiated Fisher cells
Non-separated
77.8 f 1.8 73.0 f 4.4 61.5 f 2.7 90.7 f 5.8
Irradiated Fisher cells CD4cD4+ CD4-/CD4+ mixture Non-separated
12.9 f 7.0 66.2 f 2.3 73.9 f 1.9 65.5 f 1.8
cD4CD4+ cD4-/CD4+ mixture
a) Clone A2b was tested for proliferation in the presence of 10 pg/ml of peptide 180-188, irradiated syngeneic APC (thymocytes) and (non-)irradiated popliteal LN cells from MTimmunized Fisher rats. Fisher rat cells were used which were separated in a CD4+ or CD4- enriched population by a MACS separator. Both populations were also mixed (CD4-/CD4+ mixture) and tested and also unseparated cells were used. Results are shown in cpm x
Lewis cells after MT immunization were tested in the presence of MT-primed Fisher cells, no evidence for suppression of the response was found (data not shown). Also at the level of clonal responses with specificity for the 180-188 epitope, we were unable to demonstrate that addition of nonresponder primed Fisher cells, resulted in suppression of the response of T cell clone A2b to the 180-188 peptide (Table 4). For this purpose we tested both unseparated fractions of LN cells from Fisher rats and also a CD4- or CD4+ enriched population. Separation was performed by magnetic cell sorting and the biotinylated anti-CD4 mAb W3/25. After separation, the cells in the “non-magnetic” CD4- fraction and the “magnetic” CD4+ fraction were enriched threefold. We used irradiated and non-irradiated Fisher cells because previously it was described that suppressor cells were radiation sensitive. We were able to use non-irradiated Fisher cells because, as we have shown, these cells do not proliferate themselves in the presence of the 180-188 epitope. None of the Fisher cell fractions suppressed the proliferative responses of Lewis T cell clone A2b. 3.6 In Lewis rats responses to hsp65 and the 1804% epitope seem to be under regulatory control
After immunizing animals with MT in the footpads, Lewis and Fisher rats showed responses to hsp 65, whereas BN rats did not (Fig. 1 and Fig. 2). Following in vitro restimulation with hsp 65, a distinct pattern of proliferative responses to this protein and the 180-188 or 180-196 peptide was seen. Lewis, Fisher and also BN rats, all demonstrated elevated responses to hsp 65. Remarkably enough, responses to hsp 65 in BN rats exceeded those found in Lewis and Fisher rats after hsp 65 restimulation. On the other hand striking differences were seen in the responses to the 180-196 peptide. Fisher and BN rats showed no response at all not
Regulation by an hsp65 epitope in adjuvant arthritis
Eur. J. Immunol. 1991. 21: 1289-1296
even after in virro restimulation with hsp65. Lewis rats showed a response to the 180-196 peptide which, however, decreased after restimulation in vitro with hsp65 (Fig. 2). So, unexpectedly, although responses to peptide 180-188 seem to dominate after MT immunization in Lewis rats, restimulation with the hsp 65 antigen, containing the 180-188 epitope, did not seem to enhance further responsiveness to the 180-196 epitope. Similarly, when animals were immunized in vivo with hsp65, no responses to the 180-196 epitope were seen, not even in Lewis rats (Fig. 3).
Lewis
To test the possibility of inducing 180-188- or 180-196specific T cells by mere immunization with the 180-196 peptide itself, animals from all three rat strains were immunized in the hind footpads with peptide 180-196 and tested for proliferative responses.The results given in Fig. 4 show that without in vitro restimulation responses to the 180-196 peptide were obtained in Lewis rats. Such responses were not found in BN rats not even after in vitro stimulation with hsp 65 or the 180-196 peptide. In contrast to Fisher cells, however, Lewis T cells showed a high increment in their responses to the 180-196 epitope after in vitro restimulation with this peptide. In accordance with the results shown in Figs. 2 and 3 in virro restimulation with hsp 65 decreased the proliferative responses to the 180-196 peptide. N o change in 180-196 induced responses was found in Fisher or BN rats after in vitro restimulation with hsp 65 or with the 180-196 peptide (Fig. 4).
Fisher
5 X
Z
4 -
QI-
; 2
Furthermore, while Fisher and BN rats showed enhanced responses to hsp65 after in vitro restimulation with the protein, Lewis rats showed no enhancement of responses before or after restimulation in virro (Fig. 3).Taken together, these findings suggest a regulatory effect of hsp65 on 180-196-specific T cell responses. Moreover, as suggested by findings presented in Fig. 3, such regulation could be especially pertinent to Lewis rats.
3.7 T ceUs responding to the 180-188 epitope can be demonstrated exclusively in Lewis rats
6 -
xz
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3 -
I-
rT)
2 -
1 -
hsp 6 5
180-188
183-196
4 Discussion
Figure 1. Proliferative responses in Lewis, Fisher, (Lewis X Fisher)F, and BN rats after immunization with whole mycobacteria. Popliteal LN cells were obtained from rats immunized 9 days earlier with 1 mg of MT in IFA in the hind footpad. Proliferative responses were measured with 2 x 106 celldml in the presence and absence of antigen. Various concentrations of hsp 65 peptide 180-188 and peptide 183-196 were tested. The results are shown for responses obtained with an optimal concentration of 20 pg/ml antigen. Results are given as stimulation indexes (SI f SE). * Significant difference when compared to Lewis (p< 0.05).
rat straln
p m n ig I
I
)
~
Agusedfor in* reslimlation
Ag used in poliferstion assay
None HSP65
HSP65 HSP65
HSP65
180-196 180196
None HSP65 HSP65
None
0
L
10
15
20
25
11.7
HSP65 HSP65 160-196 180196
8.5
HSP65 HSP65 180-196 180-196
BN
5
AA is most easily induced in susceptible Lewis rats, either by active immunization with mycobacterial antigen or by passive transfer of immune lymphocytes. By Tcell cloning of responder lymphocytes obtained from MT-immunized Lewis rats, T cell populations were obtained that either were arthritogenic (A2,A2b) or protective (A2c) upon transfer. When examined for antigenic specificity, all functional LewisTcells obtained thus far showed specificity for
19.6
J
Figure 2. Proliferative responses in MT-immunized Lewis, Fisher and BN rats. Rats were immunized with 1 mg MT in IFA in the footpads and 9 days later, the popliteal LN cells were tested for proliferation to hsp 65 and the 180-196 peptide (concentration of 25 Kglml). Cells were also restimulated in vitro with 10 pg/ml hsp65 and tested for their specificity as described in Sect. 2.5. The results are shown as SI If: SE.
1294
E. J. M. Hogervorst, C. J. F? Boog, J. F! A. Wagenaar et al. Ag used for
rat strain
p n !g m m
invilro restimulation
Ag used in proliferation assay
10
0
Eur. J. Immunol. 1991. 21: 1289-1296
20
50
40
30
60
None HSP6.5 HSP6.5
180-196 180-196
0.9
I
c
21.5
L
ffl
HSP65
[
None HSP65
HSP6.5 HSP65 180-196 180-196
48.4
Figure3. Proliferative responses in hsp65-immunized Lewis, Fisher and BN rats. Rats were immunized with 100 pg hsp65 in dimethyldioctadecyl ammonium bromide in the hind footpads and popliteal LN cells were tested for proliferative responses and restimulated in vitro according to the procedure described in Fig. 2.
rat strain
ptng in
yry~
Ag used for
invitro restirnulation
Agusedin proliferation assay
I
Lewk
180.196
[
180-196
10
20
30
40
50
60
70
I
I
1
I
I
I
I
I
S.I.
180-196 HSP65
180-196
1 9 6
180-196
180-196
BN
0
[ 1, HSPa
56.5
12.9
180-196 180-196 180-196
1.1
Figure 4. Proliferative responses in peptide 180-196-immunized Lewis, Fisher and BN rats. Rats were immunized with 50 pg of peptide 180-196 in dimethyl dioctadecyl ammonium bromide in the hind footpads and popliteal L N cells were tested for proliferative responses and in vitro restimulated according to the procedure described in Fig. 2.
the 180-188 sequence present in the hsp 65 of mycobacteria ([8], Hogervorst et al., unpublished results). The results of the present study show that rats that differ in their susceptibility to A A may have distinct Tcell responses to these mycobacterial antigens. Lewis rats, highly susceptible to AA, can be shown to recognize the nonapeptide present on hsp 65 of M . bovis BCG after MT immunization at the polyclonal level. However, Fisher rats, not susceptible to AA, failed to respond to the 180-188 epitope,while showing excellent responses to epitopes of hsp 65 different from 180-188. In Fisher rats we also showed that, even after in vivo immunization and subsequent in vitro restimulations with peptide 180-196, it was not possible to demonstrate the presence of 180-188-reactive T cells. Lewis and Fisher were reported to be MHC RT1 compatible by serology [23] and GvH reaction [24]. However, differences were found in MLC, induced by a distinction at the RT1.C (class I) locus [25-271. The major antigen studied here, hsp65, is an antigen predominantly presented by class I1 molecules [6, 28-30]. We demonstrated that Fisher rats
were able of presenting the 180-188 epitope to LewisTcell clone A2b and that arthritic Fisher rats did respond to this epitope.
BN rats which were susceptible, albeit to a lower degree than Lewis rats, were found to be nonresponders to the 180-188 epitope either after MT immunization or after immunization with peptide 180-196 itself and subsequent restimulation with this peptide in vitro. In BN rats the response to the 180-188 epitope was probably absent due to the fact that these rats have an MHC haplotype that differs from Lewis and therefore, were not able to present this epitope to their T cells. This would imply that BN rats possibly select an arthritogenic epitope in MT that differs from 180-188, present on the hsp65 or not. In this regard it may be of interest that we have seen responses in MTprimed BN T lymphocytes to hsp 70 that exceeded those to hsp 65 (unpublished results). Generation of arthritogenic BN T cell lines and determination of the fine specificity is currently being done. The preliminary investigations of arthritis development and responsiveness to hsp 65 in
Eur. J. Imrnunol. 1991.21: 1289-1296
(Lewis x Fisher)F1 rats, showed that the results resemble those of Fisher rats, indicating a similar immunoregulation. The susceptibility of rats to A A may be explained by an improper regulation of responses directed to epitopes of MT that are “risky”. The 180-188 epitope can be one of such epitopes because of its structural or sequential homology with self structures of the rat (for example the link protein of proteoglycansof the cartilage; [7, 311). However, n o sequence homology was recently found with rat hsp 60 ~321. As we have shown now, responses to the particular 180-188 peptide can be demonstrated in Lewis rats after MT immunization. Their MHC class 11-identical Fisher counterparts, however, when immunized in the same manner, are obviously capable of avoiding the recognition of this epitope. The finding that an exceptional arthritic Fisher rat had a remarkably high response to the 180-188 epitope was suggestive of the fact that Fisher rats control their risk to develop arthritis by their lack of responsiveness to the 180-188 epitope. The results obtained from (Lewis x Fisher)FI rats have indicated that such regulation is dominant and is compatible with the existence of Fisher background genes controlling arthritis resistance. Nevertheless, so far no arthritis has been seen, not even in Lewis rats, following immunization with hsp 65 itself or the 180-188 synthetic peptide. On the contrary, immunization with hsp65 [8] or the 180-188 peptide [33,34] has been found to induce protection instead. hsp 65 pretreatment resulted in resistance to the induction of not only AA, but also streptococcal cell-wall-induced arthritis (SCW) [ 151 and pristane-induced arthritis in mice [16]. In collagen type 11-induced arthritis disease reduction was reported [14,35]. Thus, although containing a potentially arthritogenic epitope, the immunological consequences of responses to hsp65 turn out to be of a complex nature. In this study, we showed that the response to the 180-196 peptide and hsp65 itself were subject to regulation, after stimulating the cells with hsp65. After in vivo immunization with MT or the 180-196 epitope the proliferative responses could be suppressed, in the presence of the hsp 65 molecule. These results suggested the presence of other epitopes on the hsp65 molecule, capable of controlling responses to the critical 180-188 epitope. An example of such another epitope has been characterized now by Cohen et al. [36] as recognized by he protective clone M1, which seems to be part of a so-called preemptive Tcell network involved in regulating responses to hsp 65. It will be of interest to understand how Fisher rats develop such effective means to become resistant to A A and also other forms of experimental arthritis, such as SCW arthritis, despite the fact of having the proper genetic background to present the 180-188 epitope of hsp65. First, there still might be a difference in processing or presentation of the antigen. We now have demonstrated that this is not the case, since both Lewis and Fisher APC can present epitope 180-188 of MT and hsp65 to T cell clone A2b equally well. A second explanation could be the induction of suppression in Fisher rats, exerted by epitope 180-188specific cells or T cells with specificity for other suppressive epitopes on hsp 65. Our experimental findings, however,
Regulation by an hsp65 epitope in adjuvant arthritis
1295
showed no evidence for the presence of cellular suppression in MT-immunized Fisher rats when tested in the presence of responding Lewis cells. A third possibility could be that Lewis and Fisher rats have differences in their Tcell repertoire. Kohashi et al. [3] have shown that Fisher rats when bred under germ-free conditions were susceptible to AA. When, however, Fisher rats became colonized, for instance with E. coli, they became resistant to AA.Thus, it appeared that antigens present in the colonizing bacterial flora were inducing tolerance, possibly by the selective elimination (functional or physical) of theT cells reactive to potentially arthritogenic antigens. It is possible that hsp are the common denominators in the findings of Kohashi et al. [3] and the present findings with respect to hsp 65.Tolerance induction was thoroughly studied in the autoimmune model of EAE, which showed that tolerance to self epitopes is determined in the thymus during embryonic development or in the first days of postnatal life [37,38]. In the EAE model it has been shown that this tolerance inducting can lead to changes at the level of Vg families of theTcR and that such changes can have consequence for disease susceptibility [39,40]. Irrespective of which explanation will finally turn out to be right, resistance to experimental arthritis can be acquired, either early in life by mere exposition to antigen present in the commensal flora (Fisher) or by active immunization with a purified bacterial hsp65, even in the presence of genes predisposing to disease (Lewis). Recent findings made in human patients, have indicated that in humans, especially in the affected joints, T cells responding to hsp65 were also present [lo, 12, 13, 411. Furthermore in synovial cells of juvenile rheumatoid arthritis patients responses to the 180-188 peptide, to the partially homologous sequence of the rat link protein of proteoglycan (Danielli et al., submitted) and to the human hsp60 (Pl) (De Graeff-Meeder et al., submitted) were demonstrated. This lends support to the possibility that in human arthritis, T cell reactivity with specificity for a comparable mimicry epitope of the hsp 65 molecule may also play a role. Be that as it may, the present findings may be expected to contribute to our understanding of the regulatory mechanisms that control responses to epitopes critical to autoimmunity, such as the 180-188 epitope. And this can eventually lead to the successful development of new means of specific immunological intervention in autoimmune diseases. We thank Prof. I . R. Cohen (Weizmann Institute. Rehovot. Israel) for making clone A26 available for these studies.
Received December 10, 1990; in revised form February 7, 1991.
5 References 1 Pearson, C. M., Proc. SOC.Exp. Biol. Med. 1956. 91: 95. 2 Battisto, J. R., Smith, R. N., Beckmann, K., Sternlicht, M. and Welles, W. L., Arthritis Rheum. 1982. 25: 1194. 3 Kohashi, O., Kuwata, J., Umehara, F., Takahashi, T. and Ozawa, A., Infect. Immun. 1979. 26: 791. 4 Lussier, A., De Medicis, R. and Tetreault, L., Int. J. Tissue Reac. 1984. VI: 195. 5 Waksman, B. H. and Wennersten, C., Int. Arch. Allergy Appl. Immunol. 1963. 23: 129.
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E. J. M. Hogervorst, C. J. P. Boog, J. P. A. Wagenaar et al.
6 Holoshitz, J., Matitiau, A. and Cohen, I. R., J. Clin. Invest. 1984. 73: 211. 7 Van Eden,W. Holoshitz, J., Nevo, Z., Frenkel, A., Klajman, A. and Cohen, I. R., Proc. Natl. Acad. Sci. USA 1985. 82: 5117. 8 Van Eden, W.,Thole, J. E. R. ,Van der Zee, R., Noordzij, A., Van Embden, J. D. A., Hensen, E. J. and Cohen, I. R., Nature 1988. 331: 171. 9 Van der Zee, R.,Van Eden, W., Meloen, R. H., Noordzij, A. and Van Embden, J. D. A., Eur. J. Immunol. 1989. 19: 43. 10 Res, €! C. M., Breedveld, F. C.,Van Embden, J. D. A., Schaar, C. G.,Van Eden,W., Cohen, I. R. and DeVries, R. P. P., Lancet 1988. ii: 478. 11 Bahr, G. M., Rook, G. A. W., Al-Saffar, M.,Van Embden, J., Stanford, J. L. and Behbehani, K., Clin. Exp. Immunol. 1988. 74: 211. 12 Holoshitz, J., Drucker, I.,Yaretzky, A.,Van Eden,W., Klajman, A., Lapidot, Z., Frenkel, A. and Cohen, I. R., Lancet 1986. ii: 305. 13 Holoshitz, J., Koning, F., Coligan, J. E., De Bruyn, J. and Strober, S., Nature 1989. 339: 226. 14 Billingham, M. E. J., Carney, S., Butler, R. and Colston, M. J., J. Exp. Med. 1990. 171: 339. 15 Van de Broek, M. E , Hogervorst, E. J. M.,Van Bruggen, M. C. J., Van Eden, W.,Van der Zee, R. and Van den Berg, W. B., J. Exp. Med. 1989. 170: 449. 16 Thompson, S. J., Rook. G. A. W., Brealey, R. J.,Van der Zee, R. and Elson, C. J., Eur. J. Immunol. 1990. 20: 2479. 17 Thole, J. E. R., Keu1en.W. J., De Bruyn, J., Kolk, A. H. J., Groothuis, D. G., Benvald, L. G.,Tiesjema, R. H. and Van Embden, J. D. A., Infect. Immun. 1987. 55: 1466. 18 Steward, J. M. and Young, J. D., Solid phase peptide synthesis, Pearce Chemical Co., Illinois 1984. 19 Trentham, D. E.,Townes, A. S. and Kang, A. H., J. Exp. Med. Med. 1977. 146: 857. 20 Snippe, H. and Kraaijeveld, C.A., in Allison, A. C., Arnon, R., Gregoriadis, G., Playfair, J. H. I. and Post, P. (Eds.), Immunological adjuvants and vaccines, Plenum Press, New York 1989. 21 Ben-Nun, A.,Wekerle, H. and Cohen, I. R., Eur. J. Immunol. 1981. 11: 195.
Eur. J. Immunol. 1991. 21: 1289-1296
22 Holoshitz, J., Naparstek,Y., Ben-Nun, A. and Cohen, I. R.. Science 1983. 219: 56. 23 Palm, J. and Black, G., Transplantation 1971. 11: 184. 24 Elkins, W. L. and Palm, J., Ann. N X Acad. Sci. 1966. 129: 573. 25 Marshak, A., Doherty, l? C. and Wilson, D. B.. J. Exp. Med. 1977. 146: 1773. 26 Sieck,T., Marshak-Rothstein, A. and Wilson, D. B., Immunogenetics 1979. 9: 165. 27 Sieck,T. and Wilson, D. B., Immunogenetics 1981. 14: 293. 28 Emmrich, F.,Thole, J.,Van Embden, J. and Kaufmann, S. H. E., J. Exp. Med. 1986. 163: 1024. 29 Ottenhof,T., Kale Ab, B.,Van Embden, J. D. A.,Thole, J. and Kiessling, R., J. Exp. Med. 1988. 168: 1947. 30 Brett, S. J., Lamb, J. H., Rothbard, J. B., Mehlert, A. and Ivanyi, J., Eur. J. Immunol. 1989. 19: 1303. 31 Van Eden,W., Hogervorst, E. J. M., Hensen, E. J. ,Van der Zee, R., Van Embden, J. D. A. and Cohen, I. R., Curr. Top. Microbiol. Immunol. 1989. 145: 27. 32 Venner, T. J. and Gupta, R. S., Nucleic Acids Res. 1990. 18: 5309. 33 Yang, X.-D., Gasser, J., Riniker, B. and Feige, U., J. Autoimmun. 1990. 3: 11. 34 Yang, X.-D., Gasser, J. and Feige, U., Clin. Exp. Immunol. 1990. 81: 189. 35 Ito, J., Krco, C. J.,Yu, D., Luthra, H. S. and David, C., J. Cell. Biochem. 1991. 15A: 284. 36 Cohen, I. R., in Melchers, F., et al. (Eds.), Progress in Immunology Vll, Springer-Verlag, Berlin 1989, p. 867. 37 Kappler, J. W., Roehm, N. and Marrack, P., Cell 1987. 49: 273. 38 Taguchi, 0. and Nishizuka,Y., J. Exp. Med. 1987. 165: 146. 39 Chluba, J., Steeg, C., Becker, A.,Wekerle, H. andEpplen, J.T., Eur. J. Immunol. 1989. 19: 279. 40 Heber-Katz, E. and Acha-Orbea, H., Immunol. Today 1989. 10: 164. 41 Pope, R. M., Wallis, R. S., Sailer, D., Buchanan, T. M. and Pahlavani, M. A,, Cell. Immunol., in press.
