Eur. J. Immunol. 1990. 20: 1281-1288
Shu Guang LioAn, Tom H. M. Ottenhoff, Peter Van den Elsen.., Frits Koning., Li ZhangO, Tak Mako and Ren6 R. P. DeVries. Department of Immunohaematology and Blood Bank., University Hospital, Leiden and Ontario Cancer Instituteo, Toronto
Human suppressor Tcell clones lack CD28
Human suppressor T cell clones lack CD28* Previously we showed that certain T cell lines and clones from a lepromatous leprosy patient displayed a dose-dependent suppression of the proliferation of autologous T cells to Mycobucterium leprue (M. leprue) but not mitogen or an unrelated antigen. The latter cells were also cloned and did not display this suppressive activity, were CD4+ and proliferated vigorously to M. feprue presented by autologous HLA-DR molecules. We shall refer to these cells as T helper (Th) cells. Most of the suppressive Tcell clones (T,) were also CD4+ and also proliferated to M. leprue presented by HLA-DR, but much less strongly than T h Cells. In this study we report on our search for (a) the mechanism of this apparently antigen-specific suppression by T cells, and (b) a possible phenotypic difference between Th and T, clones. The two main conclusions are that T, clones possess a lytic machinery, but that M. leprue-specific suppression and cytotoxicity can be clearly dissociated, and that the only phenotypic difference between T h and T, is the presence of the CD28 marker on Th and its absence on T, clones.
1 Introduction Lepromatous leprosy patients usually display a striking Mycobucterium leprue (M. 1eprue)-specific T cell nonresponsiveness, which at least in some cases may be due to active suppression by T cells [1-4]. Sometimes lack of proliferation to M. leprue by cultured T cells from these patients is observed even when the PBMC do proliferate to M. leprue [3]. One such so-called borderline lepromatous leprosy patient whose peripheral T cells did proliferate against M. leprue as well as against other Ag like HSV and mitogen (PHA) was selected because this might enable us to study both T h and T, Tcell responses specific for M. leprue. Indeed we were able to grow M. leprue-reactive Tcell clones that suppressed the proliferation of other Tcell clones to M. 1eprue.Weshall refer to the former asT, clones and to the latter as Th clones. There is no doubt that Tcells may cause immunosuppression. However, it is still not known whether a separate subpopulation of T, cells exists. In this study the answer is [I 76831
*
A
1281
Financial support for this study was obtained from the Immunology of Leprosy (IMMLEP) component of the UNDPNorld B a n W H O Special Program for Research and Training in Tropical Diseases, the Netherlands Leprosy Relief Association (NSL), the Dutch Foundation for Medical and Health Research (MEDIGON) (Grant no. 900-509-099) and the Willem Meindert de Hoop Foundation. Recipient of a fellowship from the Chinese Ministry of Public Health. Supported by the Netherlands Organization for Research (H92-90). Present address: Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway.
affirmative on the basis of a functional and phenotypic analysis of T h and T, clones in this M. leprue system. Notably we exclude cytotoxicity as the mechanism of the M. leprue-specific suppression displayed by these T, clones and show that T, clones lack the CD28 marker on their surface in contrast to Th clones.
2 Materials and methods 2.1 T cell lines and clones
M. leprue-reactive T, and Th clones were generated from M. leprue-primed PBMC of a BL leprosy patient as previously described [3]. Two generations of Ts cell clones and two generations of T h cell clones were obtained from PBMC of a bacillus leprae leprosy patient (SC). An HSV Agreactive Th cell line was generated from the same patient (SC) as described previously but using HSVAg instead of M. leprue [3]. 2.2 Antigens Dharmendra lepromin (Dh) was kindly provided by Dr. Abe (National Institute of Leprosy Research, Tokyo, Japan) and Dr. Good (Centre for Infectious Disease, Centres for Disease Control, Atlanta, GA) and consisted of bacilli that were isolated from human lepromas. Soluble M. leprue Ag preparations purified from armadillo tissue (CD67.11, CD84 and CD104) were generously provided by Dr. Rees (IMMLEP M. Leprae Bank, London, GB). HSV was provided by Dr. UytdeHaag (National Institute of Public Health, Bilthoven, Netherlands). PHA was from Wellcome (Beckenham, GB).
Correspondence: RenC R. P. De Vries, Department of Immunoheamatology and Blood Bank, University Hospital, PO. Box 9600, NL-2300 RC Leiden, The Netherlands
2.3 Suppression assays
Abbreviations: BL: Borderline lepromatous Dh: Dharmendra lepromin FACS: Fluorescence-activated cell sorting M . lepme: Mycobacteriurn Ieprae Tc: Cytotoxic T cell
M. leprue-specific T, or T h clones to be tested for their suppressive capacity were titrated into the test assay (1 x lo4 or 5 x lo4 cells/well). When Ag-specific responses
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0606-1281$02.50/0
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S. Guang Li, T. H. M. Ottenhoff, PVan den Elsen et al.
Table 1. mAb used in this report
mAb
Molecule recognized
Source
mAb
Molecule recognized
9.3 TcR ~ 6 - 1 WT31 wT32 OKT3
CD28 TcR y/6 ERdp CD3 cD3
Leu3a Leu-2a Leu-Sb Leu-15 Leu-llb
CD4 CD8 CD2 CDll CD16
2H4 4B4 B9.12.1
CD45R CD29
Leu-19 Leu-7
056 CD57
HLA-cI~ssI
[81 [91 Sanbio 1101 Ortho Pharmaceuticals [111 [I21 B. Malissen
B8.11.2 II.B.3
backbone HLA-DR backbone HLA-DQwl and w4
1131
Anti-IL2R
CD25
E Koning
of PBMC were tested, 10' cells/well were cultured with Ag or mitogen. When Th clones ( M . leprue-reactive) or Th cell line (HSV-reactive) were used as responders, Ag ( M . leprue or HSV) or mitogen and 5 x lo4 autologous PBMC (20 Gy irradiated) as APC were added to lo4Th cell per well. From the start of the culture, cloned T, or Th cells were added.
Source
Becton Dickinson
2.8 Radiolabeling and immunoprecipitation of TcR Cell surface labeling with 1251,specific immunoprecipitation with a rabbit anti-CD36 antiserum and analysis by SDS-PAGE under reducing conditions were all carried out as described previously [ 171.
3 Results 2.4 Cytotoxicity assays Standard 'lcr-release assays [5] were employed to measure cytotoxicity of cloned effector cells. Target cells tested were autologous APC (adherent M@ pulsed or non-pulsed with M . leprue), an M . leprae-induced T cell line, an HSVinduced Tcell line, M. leprue-reactive clones derived from the same patient, allogeneic target cells, or NK- or activated killer (AK)-susceptible target cell lines (K-562 and Daudi). Assays were set up as described [6,7].
2.5 mAb The 1/100 or 1/200 dilution used in this study are compiled in Table 1.
2.6 FCM analysis Analysis of indirect surface immunofluorescence was carried out with a FACS'" (laser) and FACSTM(mercury lamp analyzer) cell sorter (both from Becton Dickinson, Sunnyvale, CA) as described elsewhere [14].
3.1 T, clones specifically suppress the proliferation of Th clones to M . leprae As shown in Fig. la, all eight T, clones tested suppressed t h e response to M . leprue of an autologous M . leprueinduced proliferative T h clone SCVIII3B6 but not its response to PHA, which confirmed our previous observations [3].To test whether this suppression was Ag specific, an HSV-reactive Th cell line was generated from the same patient (SC). As shown in Fig.lb, the proliferation of this HSV-reactiveTh cell line was not suppressed by these eight T, clones.Two other Th clones could not inhibit the response of the Th clone SCVIII3B6 towards M . leprue (Fig. lc). These twoTh clones also did not affect the HSV response of the HSV-reactive T cell line (data not shown). Finally, Fig. Id shows that two M . leprue-induced T, clones did not suppress the (low) response of another M . leprue-induced T,clone (SCIXlF6) to M . leprue.Thus, in conclusion we are dealing with Ag-specific suppression of the proliferative response of M . leprue-reactive Tcells exerted by some but not all autologous M . leprue-reactive T cell clones. Although these clones also suppress the response to other mycobacteria [3], we will refer to this Ag-specific suppression as M . leprue-specific suppression.
2.7 Southern blot analysis 3.2 Suppression is not due to cytotoxicity High-molecular weight DNA was prepared from lo7 Tcells/clone as described by Nicklas et al. [15]. Ten micrograms of DNAwas digested with Eco RI (Boehringer, Mannheim, FRG) according to the manufacturer's instructions. The restriction fragments were size-fractionated in a 0.8% agarose gel and blotted onto Gene Screen Plus paper. TcR 6 gene rearrangements were detected with the Bgl IIPst I Cp fragment of HPB-Pl [6].
Since the observed suppression of theTcell proliferation to M . leprue might be explained by cytotoxicity directed against either the responding Ag-specificTcells or APC, we investigated whether (a) peripheral T cells activated by M . leprue or HSV, (b) M . leprue-reactive Th clones, (c) autologous EBV-BC pulsed or unpulsed with M . leprue, or (d) the NK target K-562 could be lysed by T, cells. We
Human suppressor T cell clones lack CD28
Eur. J. Immunol. 1990. 20: 1281-1288 120
both Th and T, clones are able to lyse M . leprue-pulsed Ma.
100 80 60
"1
30
-
20
7
s 2
10
n
0 2
-
0 c
0
1
5
4
a
25
looilc)
20
1s 10
3
2
-
1283
I/
M.LEPRAE
1
lo! 0,
-
1
S
0
T C E L L S ADDED X
1
S
lo-'
Figure 1. Ag-specific and "T,,"-specific suppression. (a) Agspecific suppression of an M . leprae- (.)-induced proliferative Th clone SCVIII3B6 but. not of PHA-induced (0) proliferation of the same Th clone by eight M . leprue-reactiveT, clones. The latter were added to 10" Th clone in concentrations of 0, 1 X lo4 or 5 x 10"cells/culture. (b) No suppression of HSV- (B) and PHAI the same eight (0)-induced proliferative Th cell line SC T H ~ vby M . leprue-reactiveT, clones. (c) No suppression of the M . leprae(A) and PHA- (A)-induced proliferative Th clone SCVIII3B6 by another two M . leprue-induced Th clones. (d) No suppression of M . leprae- (+)and PHA- (0)-induced proliferation of T, clone SCIXlF6 by two other M . leprae-inducedT, clones.The results are expressed as the mean cpm of triplicate cultures ([3H]dThd incorporation k SD).
reported previously that no specific lysis of these targets by the T, cell line and T, clones was observed [3, 181. The T, cells used in the present study tested after several restimulations and long-term culture (eleven months) did not lyse these targets except for the target K-562 (data not shown).
We then investigated in more detail whether T, clones possessed the capacity to kill T h cells. "Cr-labeled Th cells were incubated with autologous 7-day adherent unlabeled MQ in the presence or absence of M . leprue Ag.We argued that the target structures for suppression could possibly only be expressed by Ag-restimulated T, cells. However, no lysis was seen in these experiments either (Table 2). T, clones nevertheless possess the lytic machinery to kill appropriately presented target cells since they could lyse both Con A-treated Th clones as well as WT31-pretreated K-562 cells (data not shown). Both treatments are known to nonspecifically bridge effector and target cells, thus positioning ligand and receptor molecules for transducing lethal hit signals. In conclusion, we clearly could dissociate suppression from Ag-specific cytotoxicity exerted by T,.
3.3 FACS analysis: T, clones lack CD28 The FACS analysis data of cell surface markers for all clones are summarized in Table 3, whereas crucial data for a few representative clones are shown in Fig. 3. With regard to CD4/CD8, three phenotypes can be distinguished among the fifteen T, clones: CD4+CD8- (11/15). CD4-CD8+ (2/15) and CD4+CD8+(2/15). In contrast, all Th clones are CD4+CD8- (Table 3). The dual expression (CD4+CD8+)by two out of fifteenT, clones was confirmed by two-color immunofluorescence experiments (data not shown). Fig. 3 shows the fluorescence histograms of three representative T, and three T h clones. In order to see whether the surface phenotypes might change in time during in vitro culture, they were regularly checked after restimulation and found to retain their original CD4/CD8 phenotype after several expansions (data not shown). All M . leprue-reactiveTcel1 clones studied (Ts and Th) were CD2+. Neither Th nor T, clones expressed the CD45R Ag, whereas some Th and T, clones were found to express the CD29 Ag. NK cell markers, such as CD56, CD57, C D l l and CD16 (FcyR), were weakly or not expressed on all clones except for one Th clone (SCII2B2). Interestingly, the CD28 (9.3) Tcell surface Ag was present on all Th clones, but absent or only very weakly expressed on all Ts clones. This lack of CD28 expression was checked several times (data not shown) after restimulation with M. leprae Ag and was always observed in the presence of high expression of activation markers like HLA-DR and CD25 (Fig. 3). Thus, it was a constant characteristic of T, clones. On the other hand, all M . leprae-reactiveTcells that were suppressed were CD28+ .With reference to Fig. 1, it was interesting that the HSV line, which was not suppressed, also appeared to be CD28- (data not shown). The presence of CD28 might thus be necessary for suppression by CD28- cells to occur.
Since EBV-BC might not sufficiently process or present M . leprae Ag toT, cells and thus not be a suitable target,we therefore tested adherent M a as targets since it was found recently that Ag-specific CD4+ and CD8+ T, cells could lyse such targets [6]. As shown in Fig. 2, bothTh clones and Ts clones efficiently lysed adherent M a in an Ag-dependent 3.4 Both Th and T, clones express heterogeneous TcR a/P/CD3 complexes manner. This lysis was HLA class I1 (DR) restricted because DRZrestricted T, clones and Th clones only lysed DR2+ adherent MQ, whereas DR4-restricted T, clones and All clones were CD3+ and WT31+ (Table 3). Thus, both T, Th clones only lysed DR4+ M a (data not shown). Thus, and Th clones express a TcRa/fi/CD3 complex and no
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S. Guang Li,T. H. M. Ottenhoff, PVan den Elsen et al.
Table 2. M. leprue-reactive T, clones do not show M.
Effector cells Ts cell clones SCIlD8
SCIlG2
SCI3D3
scmcs
s m a
leprue-specific lysis of Ti, clones
IIgrget cells E/T 1OO:l 30: 1 10: 1 1oO:l 30: 1 10: 1 1oO:l 30: 1 10: 1 1Oo:l 30: 1 10: 1 1oO:l 30: 1 10:1
Th clonesakith M@ Th clones without M@ M. leprae Medium ConA WT31 Medium 1f lb) l f 2
o+o
2+1 0+1 Of1 0+1 6+8 1+1 2+1 2f2 2+1 If1 121 2+1
2+3 353 0+1 If1 If1 O f 1 If1 5f7 1f2 1+1
o+o
l f 2 1+1 l f 2 If1
44+16 43 f 13 3of 11 72f 8 68+16 56 15 59 f 27 45 f 16 3o+ 15 51 14 47 12 37 12 51 f 19 36 f 17 32+11
+
+
+ +
1+2 3+3 1+1 3+2 3+2 3f1 2+2 7+6 2+2 1f1 2i2 1+2 3f2 1+2 1f1
TcRy/6/CD3 complex. This was confirmed at the mRNA level by Northern blot analysis, which showed the presence of TcR a and P mRNA and absence of TcR 6 mRNA in both T h and T, clones (data not shown). DNA rearrangement and protein analysis of theTcR was performed t o check for heterogeneity and perhaps detect differences between T h and T, cells. The clones were digested with EcoRI and hybridized with a cDNA probe comprising the first C region of TcRP (TcR Cpl). As expected from the FACS analysis presented above,TcR Cp genes were rearranged in all clones (Fig. 4). Three DR4-restricted T, clones (lane 1-3) showed a predominantly rearranged Eco RI fragment of about 6 kb, whereas a DR2-restricted T, clone SCIX3G7 (lane 4) had undergone a rearrangement which leads to a distinct Eco RI fragment (8 kb) after hybridiza-
4+3
o+o
2+2 2*2 4+4 llt2 3-1-3 3f4 4f4 3f3 2f2 Sf4 3f3 4+4 1+1
a) Th clones had been restimulated and cultured in 20% IL2, and at day 10 were washed and labeled with slCr and added to the adherent M Q which had been incubated for 6 days, and 18 h before the assay pulsed with or without M. leprae Ag. Three Th clones were used as targets for testing T, clones. b) Percent of mean SICrrelease and SD of three Th clones.
tion with the Cg probe. In contrast, neither DR2-restricted Th clones (SCII2F5, SCVIII2D6, 3E7, SCIIlG6 and 2F6, lane5, 6, 7, 9, 10) nor the DRCrestricted Th clone S(JVIII3G4 (lane 8) showed the same rearrangements. In order to determine whether we could detect heterogeneity at the product level of TcR a@expressed on the surface of these clones, cell surface iodinated Ts and T h clones were lysed in digitonin-containing lysis buffer and TcR/CD3 complexes were recovered by immunoprecipitation with an anti-CD3 6 antiserum. These immunoprecipitates were analyzed on SDS-PAGE gels. As can be seen from Fig. 5, for all clones CD3-associated TcR structures could be detected and heterogeneity existed in the TcR complexes expressed by both T, and T h clones.
Figure2. Both M. leprue-reactive Th and T, clones lyse M. leprae-pulsed (hatched bars), or -nonpulsed (open bars) adherent monocytes. Within each group the effector (Th and T, clone)/target ratios were 50 : 1, 15 : 1 and 5 : 1, respectively.
Human supprewx Tcell cloncs lack CD28
Eur. J. Immunol. 1990. 20: 1281-1288
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Table 3. FACS analysis o f surface markcn on T, and Th clones
Name of clone
Typeof
cibne
T*
Tb
Mark ers: mAb:
Percentage of bbeting CD29 CD45R 0 2 8 cM5 HLA- CDS7 CD56 CD11 DR llF2 Leu-Sb Leu-3a Leu-Pa 4B4 2H4 9.3 IL2R B8.11.2 Leu-7 Leu-19 Leu-15
CD3 TcRdB R R y h CD2
OK73 WrJl
SCIlDlO SCUD2
% l o 0 97 99
2 1 0 0 1 98
SCIlD8 SCI103
1 0 0 9 7 9 8 9 0
0 1 0 0
SCIIE9 SCI3D3 SCIlGll
9 8 100
9
8
98
SCUIG6
98 99 98
89 9s 97
2 3
6 4
1 1 0 0 8 8 2 1 8 0 5 9 9 8 20
7 4
3 9 9 1 0 0 1 3 8 9 7 98 1
2 2
92 98 96
100 100 99
3 10
15
2
6 2 3
8
16
1 2 2 2 2 1 2 2 1 3 0 1 7 1 4 1 2 2 3 4 5 1 2 1
8 6 3 2 4 3 7 5
ND ND ND
98 64 92
ND
98 84
ND ND ND ND ND ND ND
N N ND ND N N ND ND
2 4 4 8
1 75 8 3
0
97
SCII2B2 SCII2F5 SCII2F6 SCVIIIZD3 SCVIIIZW SCVIII3B6 SCIrIII3E7 ScvlIt3F11 SCVIII3G4
7 8 N D
ND ND
97
82 9 3 N D
8 6 98
9 9 91
1 2
% 100 9
3 9 4 2
98
9
9
2 3
100 4
9
9
9 0 9 4 8 8 8 8 8 3 9 7 9 7 9 6 2 8 5 9 8 9 1 9 5 1 8 8 8 4 9 4 9 8
95 100
4
98
9
ND
93
97 91
1 10
m
100
98
94 100 95 100 98 100
87 100
ND ND
97
93 66 98 100
1 11 1
ND
ND 100
100
100
1
100
90
scm
9 0 N D nND 93
CD8
18 9
88 13 9 8 9 2 N D
SCIX1F6 SCIX2FM SCIx2CS SCIXZC8 SCIX205 SCIx2G6 SCIXZGlO SCIX3G7
CD4
100 ND 100
9
9 9 9 9 9 9
8 9 5 9 9 9
1 7 1 1 1 1 2 1
9
9
0
1 9 8 N D
100
4 Discussion
In order to further understand the role of Ts cells in the pathogenesis of lepromatous leprosy, we and others established T, lines and clones from peripheral blood [ 3 , 41 and lesions (21 of lepromatous leprosy patients. The functional 0
CD3
CD4
CD8
CD28
7
62 3 1
0
ND
ND ND
90
5
7 7 N D
6 ND
43
61 3 60 8
ND
98 95
80 87
ND
58
4 ND 9 ND
ND
15 76 82 17 42 36 8 3 3 6 51 78 67 99 95 ND 80 76
32
ND
99 66
66
9 8 N D
2
18 26
ND
ND
9 9 9 8 6 9 7 6 6 98 8 4
10
4
2
6 4
3 16
0 31
3 3
6 17 7
14 2 18
3 1 1 18 D 3 D 3
1 2
1
D D
3 6 63 33
1 60 6
5
0
12 4
16 ND 6 ND
1
1
N D N D
ND
2 ND
66 12 N D N D 7
0
1
11 2 1 1 1
8 ND 12 ND
N D N D
Leu-llb
39 24
4
4
CD16
1
10 3 ND 3
ND 5 ND
definition of the T, clones described by us is that they specifically suppress the proliferative response of autologous PBMC as well as Th clones to M . leprne Ag but not responses to other Ag such as HSVand the mitogen PHA. In the present study we report the most pertinent data on the mechanism of this suppression and the results of our Ta C
and Th cloncs. Cclls were incubated
Th
log fluorescence intensity
anti-lL 2R (anti-CD25) mAb at 1 : 200 dilution of ascites fluid. followed by incubation with FITC-conjugated goat anti-mouse Ig F(ab')z. Fluorescence analysis was performed using a FACS'".The characteristics of the cells samples used are: 1-3 T, clones SCIXlF6, SCIIDIO and 1G2: and 4-6 Th cloncs SCVIII2D3. 3B6 and 3F11.
1286
S. Guang Li,T. H. M. Ottenhoff, PVan den Elsen et al.
Eur. J. Immunol. 1990. 20: 1281-1288 A series of experiments has excluded competition for Ag [24] as the mechanism of the suppression phenomenon studied by us: adding a supra-optimal dose of M. leprae could not diminish the suppression, the T, cells were radiation sensitive, heat-inactivated T, clones lost their suppressive capacity, and recognition of Ag plus HLA by T, clones was not required for suppression to occur [19]. The latter observation was unexpected and suggested that for our T, clones activation and MHC restriction may be separated from the suppressive effect exerted by them.
Figure 4. Southern blot analysis of TcR p chain gene rearrangements. Eco RI digestion of DNA prepared from T, and Th clones were probed with the 32P-labeledTcR Cpprobe. Lanes 1-4 contain DNA fromT,clones SCIlD8, 1G2,3D3and SCIX3G7. Lanes 5-10 contain DNA from Th clones SCI12F5, SCVIII2D6, 3E7, 3G4, SCIIlG6and 2F6.The DNA sample of a control B cell line (NOL) is shown in lane 11. The sizes of the germ-line fragments are indicated on the right (kb).
Figure 5. SDS-PAGE analysis of immunoprecipitates carried out with an antLCD36 chain antiserum. Lanes 1-4 are Ts clones SCIlD8, 1G2, 3D3 and SCIX3G7. Lanes5-7 are Th clones SCII2F5, SCVIII2D6 and 3E7.The position of the molecular mass is indicated on the right.The position of TcR markers (kDa x and CD3 chains is indicated on the left.
analysis of the TcR and other cell surface molecules of T, clones as compared tOTh clones. For further details refer to the thesis of Shu Guang Li [19].
Next, we performed a series of experiments to study the effector phase of the suppression exerted by these Agspecific T, clones. HLA-DQ and class I are not involved as was shown by the failure to abolish the suppression with andi-DQ and class I mAb [19]. Because T, cells are restricted via HLA-DR in their activation phase [25], it was interesting to know if HLA-DR or any other HLA class I1 (DP or D) might also be involved in their effector phase. The observation that a DRCrestricted T, clone could only suppress the M. reprue responses of a Th cell line from the DRCmatched brother of the patient, but not of DR4M. feprue-reactive T h lines from other individuals, may indicate that the effector phase of the T, cells may be restricted by HLA-DR [19]. However, M . leprue or leprosy-related environmental factors accumulated in the family of the patient also may explain these data. We have reported previously [3, 181that theseT, clones are not cytotoxic for Th clones or M . leprue-pulsed B cell lines. Our present studies show that T, clones also can not lyse the Th clones that they suppress when Th cells are co-cultured with M. leprue and adherent MQ. T, cells could lyse Th clones nonspecifically (Con A dependent) and some of these T, clones tested after several restimulations and long-term culture obtained some degree of an NK-like activity.Thus,T, clones do possess cytolytic potential but do not use it in order to suppress. T, as well as Th clones could lyse adherent M. leprue-pulsed M@ in an Ag-specific HLA class 11-restrictedmanner. Killing of MQ as APC, however, is virtually excluded as the mechanism of suppression since the presentation of HSV Ag by the same MQ to HSVreactive Tcells is unaffected by the presence of M. leprue and M. leprue-reactive T, cells. If these T, cells would suppress M. leprue-reactiveThcell responses by killing their APC, T, cells would also be expected to suppress HSVdriven responses. Thus, although both Th and T, cells possess the capacity to lyse appropriately presented target cells,T, cells apparently do not use their cytolytic potential for suppressing M. leprue-reactive Th cell responses.
Thus far, we have failed to demonstrate M. feprue-specific suppression using SN of activated T,. We assume that for M. leprue-specific suppression to occur, interaction be4.1 Suppression is neither due to cytolysis nor to tween T,, Th andlor APC is necessary and we do not know competition for growth factors or antigen whether a molecule released by T, cells may mediate the suppressive function. Due to the unexpected finding that Several groups reported that suppression may result from M. leprue-specific suppression is probably not dependent interference by T, cells with TL2 secretion. T, cells might on activation of T, cells by M. leprue we considered the lower IL2 synthesis [20-221 or bind IL2 and thus lower the existence of veto cells (reviewed in [26]). The Ts cells in our effective concentration of this lymphokine in the microen- system bear class I1 molecules which can pick up soluble vironment [23].We could not abolish the M. leprue-specific Ag, and might thus be recognized by the class 11-restricted, suppression by adding exogenous IL2 [19], indicating that Ag-specific helper cells. However, a veto phenomenon M . leprue-specific suppression is not caused by IL2 con- would not easily explain why only Th cells reactive with sumption. M. leprue and not T h cells against HSV are suppressed.
Eur. J. Immunol. 1990. 20: 1281-1288
In conclusion, these M. Zeprue-specific, cloned T, cells specifically suppress M . leprue-driven proliferation of autologousTh cells by a mechanism that does not involve any of the known Tcell functions, such as cytolysis or competition for growth factors or antigen.
4.2 The only phenotypic difference between Th and T, is the presence of CD28 on Th and its absence on T, The main conclusion from the TcR analysis is that both T, and Th clones have TcRa/p/CD3 complexes. In several other systems similar observations have now been reported for human T, clones [27-291. This contrasts with the observations made in mouse systems, where many T, clones and hybridomas lacked bonafide TcR [30]. The TcR DNA and protein analysis combined with the differences in restriction specificity of both Th and T, clones [25] indicate that we are dealing with at least several different T, and Th clones. Three sets of observations regarding the other cell surface markers of T, compared to T h warrant further discussion. First we showed that all clones are regular CD3+ and CD2+ Tcells. Although both Th and T, acquired NK-like activity after long-term culture in vitro [19], they lacked NK cell markers such as CD56, CD57, CDll and CD16. T, clones may be CD4-CD8+, CD4+CD8- or CD4+CD8+. The majority of T, clones in our system were CD4+CD8- which differs from several studies in which the CD4-CD8+ Tcells are in the majority [2, 4, 31, 32].Thus, these data support the notion that CD4CD8 phenotypes are certainly not always associated with functional characteristics. Second, the consistent lack of CD45R on both T, and T h clones supports the notion that T cells lose the CD45R determinant upon stimulation [33,34]. Some T, clones as well as Th clones showed expression of CD29 (4B4),which has been postulated to be associated with CD4+ T h cells [ 121. Obviously, all clones strongly expressed the classical early activation markers such as CD25 and HLA-DR on their cell surface (Table 3 and Fig. 3). Finally, and most importantly, we observed that the CD28 Ag was lacking on the surface of T, clones but expressed on the surface of M . leprue-reactive Th clones, whose proliferation to M . leprue could be suppressed by T, cells. Interestingly, the HSV line that was not suppressed by the T, clones was also CD28-. This suggests that the presence of CD28 on the target cells might be needed for suppression to be induced. It should be noted that HSV was the only non-mycobacterial antigen (our of 10 tested) to which the patient (SC) did respond. Since a control T cell that is CD28+ and reactive to a non-mycobacterial antigen is not presently available, we therefore cannot be sure that the suppression is Ag and CD28 or only CD28 specific. The CD28 Ag is a cell surface Ag which is present on approximately 70% of PBL and which is recognized by the mAb9.3 [8]. It has been demonstrated that the CD8+CD28+subset contains Th cells [ 3 5 ] , T, cells [36] and precursor T, cells, whereas the CD8+CD28-subset contains precursors for Ag-specific suppressor cells [36, 371. The molecule carrying the CD28 Ag is involved in T cell activation and the level of expression of CD28 may
Human suppressor T cell clones lack CD28
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determine whether a positive or a negative signal is generated through this cell surface structure [38]. Our data show an absence of CD28 on both CD8+ and CD4+ Ag-specificT, cells. This observation is compatible with and extends the observation that the predominant T, cells in lepromatous lesions are CD8+CD28- [2, 391 and that this mAb can distinguish CD8+ suppressor effector cells (CDll+CD28-) from T, cells (CD1l-CD28+) [40].The two CD8+T, clones were both CD11+and CD28- (seeTable 2), but most CD4+T, clones were CD11-. In addition, our data suggest that CD28 may not only be a phenotypic marker dividing M. leprue-reactive T cells into two subsets, one (CD28-) suppressing the proliferation of the other (CD28+),but may be functionally involved in the suppression of cells carrying this determinant. This hypothesis can be at least partially tested, e.g. by transfection of the CD28 gene [41] in CD28- cells. The authors would like to thank Prof. Dr. J. J. Van Rood for his support; patient S. C. for donating blood; E Wassenaar for assistance with the Southern blotting technique; J. Blom, D. Van der Harst and S. Van Luxemburg for cell surface phenotyping part of the clones; Drs. S. E Schlossman, B. Malissen, H. Spits, E Bach, H . Maeda, R. Winchester, A . Ziegler, J. Bodmer, A . Kolk, M . Codon and R . Knowles for generously supplying us with monoclonal antibodies; Dr. J. Borst for her advice during the study and critical evaluation of the manuscript; Drs. H. Bruning and B. Vandekerckhove for stimulating discussions; Drs. E H. J. Claas, B. Roep and J. Thorogood for their suggestions, and C. S. L . Mackenzie, I. S. Curiel* D. Van Gorp, E. Van der Willik and 7:Van Westerop for help in preparing the manuscript. Received May 31, 1989; in final revised form February 1. 1990.
5 References 1 Nathan, C. F., Kaplan, G., Levis,W. R., Nusrat, A.,Witmer,W. D., Sherwin, S. A., Job, C. K., Path, F. R. C., Horowitz, C. R., Steinman, R. M. andCohn, Z. A., N. Engl. J. Med. 1986,315: 6. 2 Modlin, R. L., Kato, H., Mehra,V., Nelson, E. E., Xue-dong, F., Rea,T. H., Pattengale, P. K. and Bloom, B. R., Nafure 1986. 322: 459. 3 Ottenhoff, T. H. M., Elferink, D. G., Klatser, P. R. and Dc Vries, R. R. P., Nature 1986. 322: 462. 4 Kikuchi, I., Ozawa,T., Hirayama, K. and Sasazuki,T., Lepr. Rev. 1986. 57: 139. 5 Goulmy, E., in Ferrone, S. and Solheim, B. G . (Eds.), HLA Typing: Methodology and Clinical Aspects, (Volume /I), CRC Press, New York 1982, p. 105. 6 Ottenhoff,T. H. M., Kaleab, B. ,Van Embden, J. D. A. ,Thole, J. E. R. and Kiessling, R., J. Exp. Med. 1988. 168: 1947. 7 Birhane Kale Ab., Kiesling, R.,Van Embden, J. D. A. ,Thole, J. E. R., Kumararatne. D. S., Pisa, P., Wondina, A. and 0ttenhoff.T. H. M., Eur. J. Immunol. 1990. 20: 369. 8 Hansen, J. A., Martin, P. J. and Nowinski, R. C., Immunogenetics 1980. 10: 241. 9 Borst, J.,Van Dongen, J. J. M., Bolhuis, R. L. H.. Peters, P. J., Hafler, D. A , , DeVries, E . and De Griend, R. J., J. Exp. Med. 1988. 167: 1625. 10 Tax,W. J. M., Reekers, I? P. M., Capel, P. J. A. and Koene, R. A. F,! Nature 1983. 304: 445. 11 Morimoto, C., Letvin, N. L., Distaso, J. A., A1drich.W. R. and Schlossman, S. F., J. Immunol. 1985. 134: 1508. 12 Morimoto, C., Letvin, N. L., Boyd, A.W., Hagan. M., Brown, H. M., Kornacki, M. N. and Schlosman, S. F., J. Immunol. 1985. 134: 3762.
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13 Rebai, N., Malissen, B., Pierre, M., Acolla, R . S., Corte, G. and Mawas, C., Eur. J. Immunol. 1983. 13: 106. 14 Blom, J., Harrisson, C. M. H., Schuurman, R. K. B. and Schot, J. D. L., J. Immunol. Methods 1986. 95: 39. 15 Nicklas, J. A., Hunter,T. C., Sullivan, L. M., Berman, J. K., ONeill, J. €? and Albertini, R. O., Mutagenesis 1987. 2: 341. 16 Yoshikai,Y., Antoniou, D., Clark, S. P.,Yanagi,Y., Sangster, R., Van den Elsen, €?,Terhorst,C. and Mak,T.W., Nature 1984.312: 521. 17 Koning, F., Lew, A. M., Maloy,W. L. ,Valas, R. and Coligan, J. E., J. Immunol. 1988. 140: 3126. 18 De Vries, R. R. P., Ottenhoff, T. H. M., Shuguang, Li, and Young, R. A., Lepr. Rev. 1986. 57: (Suppl. 2) 113. 19 Li, S. G., Phenotypic and functional characterization of human suppressor Tcell clones, Thesis, University of Leiden, 1989. 20 Palacios, R. and Moller, G., J. Exp. Med. 1981. 153: 1360. 21 Gunther, J., Haas,W. and Von Boehmer, H., Eur. J. Immunol. 1982. 12: 247. 22 Susskind, B. M., Merluzzi,V. J., Faanes, R. B., Palladino, M. A. and Choi,Y. S., J. Irnmunol. 1983. 130: 527. 23 Gullberg, M. and Larsson, E. L., J. Immunol. 1982. 128: 746. 24 Ashwell, J. D., Fox, B. S. and Schwartz, R. H., J. Immunol. 1986. 136: 757. 25 Li, S. G., Elferink, D. G. and De Vries, R. R . P., Hum. Immunol. 1990, in press. 26 Fink, P. J., Shimonkevitz, R. P. and Bevan, M. J., Annu. Rev. Immunol. 1988. 6: 115. 27 Toyonaga, B.,Yanagi,Y, Suciu-Foca, N., Minden, M. and Mak, T.W.. Nature 1984. 311: 385.
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28 Modlin, R. L., Brenner, M. B., Krangel, M. S., Duby, A. D. and Bloom, B. R., Nature 1987.329: 541. 29 Takeuchi,T., Schlossman, S. F. and Morimoto, C., J. Irnrnunol. 1988. 141: 3010. 30 Moller, G., Scand. J. Immunol. 1988. 27: 247. 31 Hirayama, K., Matsushita, S., Kikuchi, I., Iuchi, M., Ohta, N. and Sasazuki, T., Nature 1987. 327: 426. 32 Matsushita, S., Morimoto, C., Schlossman, S. F. and Sasazuki, T., Hum. Immunol. 1988. 22: 1. 33 Koning, F., Bakker, A., Dubelaar, M., Lesslauer,W., Mullink, R., Schuits, R., Ligthart, G., Naipal, A. and Bruning, H., Hum. Immunol. 1986. 17: 325. 34 Sanders, M. E., Makgoba, M.W., Sharrow, S. O., Stephany, D., Springer,T. A.,Young, H. A. and Shaw, S., J. Immunol. 1988. 140: 1401. 35 Lum, L. G., Orcutt-Thordarson, N., Seigneuret, M. C. and Hansen, J. A., Cell. Immunol. 1982. 72: 122. 36 Fast, L. D., Hansen, J. A. and Newman,W., J. lrnrnunol. 1981. 127: 448. 37 Damle, N. K., Mohagheghpour, N. and Engleman, E. G., J. Immunol. 1984. 132: 644. 38 Lesslauer, W., Koning, F., Ottenhoff,T., Giphart, M., Goulmy, E. and Van Rood, J. J., Eur. J. Immunol. 1986. 16: 1289. 39 Modlin, R . L., Melancon-Kaplan, J.,Young, S. M. M., Pirmez, C., Kino, H., Convit, J., Rea,T. and Bloom, B. R., Proc. Natl. Acad. Sci. USA 1988. 85: 1213. 40 Yamada, H., Martin, P. J., Bean, M. A., Braun, M. P, Beatty, €? G., Sadamoto, K. andHansen, J. A., Eur. J. Immunol. 1985. 15: 1164. 41 Aruffo, A. and Seed, B., Proc. Natl. Acad. Sci. USA 1987.84: 8573.
Shu Guang LioAn, Tom H. M. Ottenhoff, Peter Van den Elsen.., Frits Koning., Li ZhangO, Tak Mako and Ren6 R. P. DeVries. Department of Immunohaematology and Blood Bank., University Hospital, Leiden and Ontario Cancer Instituteo, Toronto
Human suppressor Tcell clones lack CD28
Human suppressor T cell clones lack CD28* Previously we showed that certain T cell lines and clones from a lepromatous leprosy patient displayed a dose-dependent suppression of the proliferation of autologous T cells to Mycobucterium leprue (M. leprue) but not mitogen or an unrelated antigen. The latter cells were also cloned and did not display this suppressive activity, were CD4+ and proliferated vigorously to M. feprue presented by autologous HLA-DR molecules. We shall refer to these cells as T helper (Th) cells. Most of the suppressive Tcell clones (T,) were also CD4+ and also proliferated to M. leprue presented by HLA-DR, but much less strongly than T h Cells. In this study we report on our search for (a) the mechanism of this apparently antigen-specific suppression by T cells, and (b) a possible phenotypic difference between Th and T, clones. The two main conclusions are that T, clones possess a lytic machinery, but that M. leprue-specific suppression and cytotoxicity can be clearly dissociated, and that the only phenotypic difference between T h and T, is the presence of the CD28 marker on Th and its absence on T, clones.
1 Introduction Lepromatous leprosy patients usually display a striking Mycobucterium leprue (M. 1eprue)-specific T cell nonresponsiveness, which at least in some cases may be due to active suppression by T cells [1-4]. Sometimes lack of proliferation to M. leprue by cultured T cells from these patients is observed even when the PBMC do proliferate to M. leprue [3]. One such so-called borderline lepromatous leprosy patient whose peripheral T cells did proliferate against M. leprue as well as against other Ag like HSV and mitogen (PHA) was selected because this might enable us to study both T h and T, Tcell responses specific for M. leprue. Indeed we were able to grow M. leprue-reactive Tcell clones that suppressed the proliferation of other Tcell clones to M. 1eprue.Weshall refer to the former asT, clones and to the latter as Th clones. There is no doubt that Tcells may cause immunosuppression. However, it is still not known whether a separate subpopulation of T, cells exists. In this study the answer is [I 76831
*
A
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Financial support for this study was obtained from the Immunology of Leprosy (IMMLEP) component of the UNDPNorld B a n W H O Special Program for Research and Training in Tropical Diseases, the Netherlands Leprosy Relief Association (NSL), the Dutch Foundation for Medical and Health Research (MEDIGON) (Grant no. 900-509-099) and the Willem Meindert de Hoop Foundation. Recipient of a fellowship from the Chinese Ministry of Public Health. Supported by the Netherlands Organization for Research (H92-90). Present address: Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway.
affirmative on the basis of a functional and phenotypic analysis of T h and T, clones in this M. leprue system. Notably we exclude cytotoxicity as the mechanism of the M. leprue-specific suppression displayed by these T, clones and show that T, clones lack the CD28 marker on their surface in contrast to Th clones.
2 Materials and methods 2.1 T cell lines and clones
M. leprue-reactive T, and Th clones were generated from M. leprue-primed PBMC of a BL leprosy patient as previously described [3]. Two generations of Ts cell clones and two generations of T h cell clones were obtained from PBMC of a bacillus leprae leprosy patient (SC). An HSV Agreactive Th cell line was generated from the same patient (SC) as described previously but using HSVAg instead of M. leprue [3]. 2.2 Antigens Dharmendra lepromin (Dh) was kindly provided by Dr. Abe (National Institute of Leprosy Research, Tokyo, Japan) and Dr. Good (Centre for Infectious Disease, Centres for Disease Control, Atlanta, GA) and consisted of bacilli that were isolated from human lepromas. Soluble M. leprue Ag preparations purified from armadillo tissue (CD67.11, CD84 and CD104) were generously provided by Dr. Rees (IMMLEP M. Leprae Bank, London, GB). HSV was provided by Dr. UytdeHaag (National Institute of Public Health, Bilthoven, Netherlands). PHA was from Wellcome (Beckenham, GB).
Correspondence: RenC R. P. De Vries, Department of Immunoheamatology and Blood Bank, University Hospital, PO. Box 9600, NL-2300 RC Leiden, The Netherlands
2.3 Suppression assays
Abbreviations: BL: Borderline lepromatous Dh: Dharmendra lepromin FACS: Fluorescence-activated cell sorting M . lepme: Mycobacteriurn Ieprae Tc: Cytotoxic T cell
M. leprue-specific T, or T h clones to be tested for their suppressive capacity were titrated into the test assay (1 x lo4 or 5 x lo4 cells/well). When Ag-specific responses
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0606-1281$02.50/0
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Table 1. mAb used in this report
mAb
Molecule recognized
Source
mAb
Molecule recognized
9.3 TcR ~ 6 - 1 WT31 wT32 OKT3
CD28 TcR y/6 ERdp CD3 cD3
Leu3a Leu-2a Leu-Sb Leu-15 Leu-llb
CD4 CD8 CD2 CDll CD16
2H4 4B4 B9.12.1
CD45R CD29
Leu-19 Leu-7
056 CD57
HLA-cI~ssI
[81 [91 Sanbio 1101 Ortho Pharmaceuticals [111 [I21 B. Malissen
B8.11.2 II.B.3
backbone HLA-DR backbone HLA-DQwl and w4
1131
Anti-IL2R
CD25
E Koning
of PBMC were tested, 10' cells/well were cultured with Ag or mitogen. When Th clones ( M . leprue-reactive) or Th cell line (HSV-reactive) were used as responders, Ag ( M . leprue or HSV) or mitogen and 5 x lo4 autologous PBMC (20 Gy irradiated) as APC were added to lo4Th cell per well. From the start of the culture, cloned T, or Th cells were added.
Source
Becton Dickinson
2.8 Radiolabeling and immunoprecipitation of TcR Cell surface labeling with 1251,specific immunoprecipitation with a rabbit anti-CD36 antiserum and analysis by SDS-PAGE under reducing conditions were all carried out as described previously [ 171.
3 Results 2.4 Cytotoxicity assays Standard 'lcr-release assays [5] were employed to measure cytotoxicity of cloned effector cells. Target cells tested were autologous APC (adherent M@ pulsed or non-pulsed with M . leprue), an M . leprae-induced T cell line, an HSVinduced Tcell line, M. leprue-reactive clones derived from the same patient, allogeneic target cells, or NK- or activated killer (AK)-susceptible target cell lines (K-562 and Daudi). Assays were set up as described [6,7].
2.5 mAb The 1/100 or 1/200 dilution used in this study are compiled in Table 1.
2.6 FCM analysis Analysis of indirect surface immunofluorescence was carried out with a FACS'" (laser) and FACSTM(mercury lamp analyzer) cell sorter (both from Becton Dickinson, Sunnyvale, CA) as described elsewhere [14].
3.1 T, clones specifically suppress the proliferation of Th clones to M . leprae As shown in Fig. la, all eight T, clones tested suppressed t h e response to M . leprue of an autologous M . leprueinduced proliferative T h clone SCVIII3B6 but not its response to PHA, which confirmed our previous observations [3].To test whether this suppression was Ag specific, an HSV-reactive Th cell line was generated from the same patient (SC). As shown in Fig.lb, the proliferation of this HSV-reactiveTh cell line was not suppressed by these eight T, clones.Two other Th clones could not inhibit the response of the Th clone SCVIII3B6 towards M . leprue (Fig. lc). These twoTh clones also did not affect the HSV response of the HSV-reactive T cell line (data not shown). Finally, Fig. Id shows that two M . leprue-induced T, clones did not suppress the (low) response of another M . leprue-induced T,clone (SCIXlF6) to M . leprue.Thus, in conclusion we are dealing with Ag-specific suppression of the proliferative response of M . leprue-reactive Tcells exerted by some but not all autologous M . leprue-reactive T cell clones. Although these clones also suppress the response to other mycobacteria [3], we will refer to this Ag-specific suppression as M . leprue-specific suppression.
2.7 Southern blot analysis 3.2 Suppression is not due to cytotoxicity High-molecular weight DNA was prepared from lo7 Tcells/clone as described by Nicklas et al. [15]. Ten micrograms of DNAwas digested with Eco RI (Boehringer, Mannheim, FRG) according to the manufacturer's instructions. The restriction fragments were size-fractionated in a 0.8% agarose gel and blotted onto Gene Screen Plus paper. TcR 6 gene rearrangements were detected with the Bgl IIPst I Cp fragment of HPB-Pl [6].
Since the observed suppression of theTcell proliferation to M . leprue might be explained by cytotoxicity directed against either the responding Ag-specificTcells or APC, we investigated whether (a) peripheral T cells activated by M . leprue or HSV, (b) M . leprue-reactive Th clones, (c) autologous EBV-BC pulsed or unpulsed with M . leprue, or (d) the NK target K-562 could be lysed by T, cells. We
Human suppressor T cell clones lack CD28
Eur. J. Immunol. 1990. 20: 1281-1288 120
both Th and T, clones are able to lyse M . leprue-pulsed Ma.
100 80 60
"1
30
-
20
7
s 2
10
n
0 2
-
0 c
0
1
5
4
a
25
looilc)
20
1s 10
3
2
-
1283
I/
M.LEPRAE
1
lo! 0,
-
1
S
0
T C E L L S ADDED X
1
S
lo-'
Figure 1. Ag-specific and "T,,"-specific suppression. (a) Agspecific suppression of an M . leprae- (.)-induced proliferative Th clone SCVIII3B6 but. not of PHA-induced (0) proliferation of the same Th clone by eight M . leprue-reactiveT, clones. The latter were added to 10" Th clone in concentrations of 0, 1 X lo4 or 5 x 10"cells/culture. (b) No suppression of HSV- (B) and PHAI the same eight (0)-induced proliferative Th cell line SC T H ~ vby M . leprue-reactiveT, clones. (c) No suppression of the M . leprae(A) and PHA- (A)-induced proliferative Th clone SCVIII3B6 by another two M . leprue-induced Th clones. (d) No suppression of M . leprae- (+)and PHA- (0)-induced proliferation of T, clone SCIXlF6 by two other M . leprae-inducedT, clones.The results are expressed as the mean cpm of triplicate cultures ([3H]dThd incorporation k SD).
reported previously that no specific lysis of these targets by the T, cell line and T, clones was observed [3, 181. The T, cells used in the present study tested after several restimulations and long-term culture (eleven months) did not lyse these targets except for the target K-562 (data not shown).
We then investigated in more detail whether T, clones possessed the capacity to kill T h cells. "Cr-labeled Th cells were incubated with autologous 7-day adherent unlabeled MQ in the presence or absence of M . leprue Ag.We argued that the target structures for suppression could possibly only be expressed by Ag-restimulated T, cells. However, no lysis was seen in these experiments either (Table 2). T, clones nevertheless possess the lytic machinery to kill appropriately presented target cells since they could lyse both Con A-treated Th clones as well as WT31-pretreated K-562 cells (data not shown). Both treatments are known to nonspecifically bridge effector and target cells, thus positioning ligand and receptor molecules for transducing lethal hit signals. In conclusion, we clearly could dissociate suppression from Ag-specific cytotoxicity exerted by T,.
3.3 FACS analysis: T, clones lack CD28 The FACS analysis data of cell surface markers for all clones are summarized in Table 3, whereas crucial data for a few representative clones are shown in Fig. 3. With regard to CD4/CD8, three phenotypes can be distinguished among the fifteen T, clones: CD4+CD8- (11/15). CD4-CD8+ (2/15) and CD4+CD8+(2/15). In contrast, all Th clones are CD4+CD8- (Table 3). The dual expression (CD4+CD8+)by two out of fifteenT, clones was confirmed by two-color immunofluorescence experiments (data not shown). Fig. 3 shows the fluorescence histograms of three representative T, and three T h clones. In order to see whether the surface phenotypes might change in time during in vitro culture, they were regularly checked after restimulation and found to retain their original CD4/CD8 phenotype after several expansions (data not shown). All M . leprue-reactiveTcel1 clones studied (Ts and Th) were CD2+. Neither Th nor T, clones expressed the CD45R Ag, whereas some Th and T, clones were found to express the CD29 Ag. NK cell markers, such as CD56, CD57, C D l l and CD16 (FcyR), were weakly or not expressed on all clones except for one Th clone (SCII2B2). Interestingly, the CD28 (9.3) Tcell surface Ag was present on all Th clones, but absent or only very weakly expressed on all Ts clones. This lack of CD28 expression was checked several times (data not shown) after restimulation with M. leprae Ag and was always observed in the presence of high expression of activation markers like HLA-DR and CD25 (Fig. 3). Thus, it was a constant characteristic of T, clones. On the other hand, all M . leprae-reactiveTcells that were suppressed were CD28+ .With reference to Fig. 1, it was interesting that the HSV line, which was not suppressed, also appeared to be CD28- (data not shown). The presence of CD28 might thus be necessary for suppression by CD28- cells to occur.
Since EBV-BC might not sufficiently process or present M . leprae Ag toT, cells and thus not be a suitable target,we therefore tested adherent M a as targets since it was found recently that Ag-specific CD4+ and CD8+ T, cells could lyse such targets [6]. As shown in Fig. 2, bothTh clones and Ts clones efficiently lysed adherent M a in an Ag-dependent 3.4 Both Th and T, clones express heterogeneous TcR a/P/CD3 complexes manner. This lysis was HLA class I1 (DR) restricted because DRZrestricted T, clones and Th clones only lysed DR2+ adherent MQ, whereas DR4-restricted T, clones and All clones were CD3+ and WT31+ (Table 3). Thus, both T, Th clones only lysed DR4+ M a (data not shown). Thus, and Th clones express a TcRa/fi/CD3 complex and no
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Table 2. M. leprue-reactive T, clones do not show M.
Effector cells Ts cell clones SCIlD8
SCIlG2
SCI3D3
scmcs
s m a
leprue-specific lysis of Ti, clones
IIgrget cells E/T 1OO:l 30: 1 10: 1 1oO:l 30: 1 10: 1 1oO:l 30: 1 10: 1 1Oo:l 30: 1 10: 1 1oO:l 30: 1 10:1
Th clonesakith M@ Th clones without M@ M. leprae Medium ConA WT31 Medium 1f lb) l f 2
o+o
2+1 0+1 Of1 0+1 6+8 1+1 2+1 2f2 2+1 If1 121 2+1
2+3 353 0+1 If1 If1 O f 1 If1 5f7 1f2 1+1
o+o
l f 2 1+1 l f 2 If1
44+16 43 f 13 3of 11 72f 8 68+16 56 15 59 f 27 45 f 16 3o+ 15 51 14 47 12 37 12 51 f 19 36 f 17 32+11
+
+
+ +
1+2 3+3 1+1 3+2 3+2 3f1 2+2 7+6 2+2 1f1 2i2 1+2 3f2 1+2 1f1
TcRy/6/CD3 complex. This was confirmed at the mRNA level by Northern blot analysis, which showed the presence of TcR a and P mRNA and absence of TcR 6 mRNA in both T h and T, clones (data not shown). DNA rearrangement and protein analysis of theTcR was performed t o check for heterogeneity and perhaps detect differences between T h and T, cells. The clones were digested with EcoRI and hybridized with a cDNA probe comprising the first C region of TcRP (TcR Cpl). As expected from the FACS analysis presented above,TcR Cp genes were rearranged in all clones (Fig. 4). Three DR4-restricted T, clones (lane 1-3) showed a predominantly rearranged Eco RI fragment of about 6 kb, whereas a DR2-restricted T, clone SCIX3G7 (lane 4) had undergone a rearrangement which leads to a distinct Eco RI fragment (8 kb) after hybridiza-
4+3
o+o
2+2 2*2 4+4 llt2 3-1-3 3f4 4f4 3f3 2f2 Sf4 3f3 4+4 1+1
a) Th clones had been restimulated and cultured in 20% IL2, and at day 10 were washed and labeled with slCr and added to the adherent M Q which had been incubated for 6 days, and 18 h before the assay pulsed with or without M. leprae Ag. Three Th clones were used as targets for testing T, clones. b) Percent of mean SICrrelease and SD of three Th clones.
tion with the Cg probe. In contrast, neither DR2-restricted Th clones (SCII2F5, SCVIII2D6, 3E7, SCIIlG6 and 2F6, lane5, 6, 7, 9, 10) nor the DRCrestricted Th clone S(JVIII3G4 (lane 8) showed the same rearrangements. In order to determine whether we could detect heterogeneity at the product level of TcR a@expressed on the surface of these clones, cell surface iodinated Ts and T h clones were lysed in digitonin-containing lysis buffer and TcR/CD3 complexes were recovered by immunoprecipitation with an anti-CD3 6 antiserum. These immunoprecipitates were analyzed on SDS-PAGE gels. As can be seen from Fig. 5, for all clones CD3-associated TcR structures could be detected and heterogeneity existed in the TcR complexes expressed by both T, and T h clones.
Figure2. Both M. leprue-reactive Th and T, clones lyse M. leprae-pulsed (hatched bars), or -nonpulsed (open bars) adherent monocytes. Within each group the effector (Th and T, clone)/target ratios were 50 : 1, 15 : 1 and 5 : 1, respectively.
Human supprewx Tcell cloncs lack CD28
Eur. J. Immunol. 1990. 20: 1281-1288
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Table 3. FACS analysis o f surface markcn on T, and Th clones
Name of clone
Typeof
cibne
T*
Tb
Mark ers: mAb:
Percentage of bbeting CD29 CD45R 0 2 8 cM5 HLA- CDS7 CD56 CD11 DR llF2 Leu-Sb Leu-3a Leu-Pa 4B4 2H4 9.3 IL2R B8.11.2 Leu-7 Leu-19 Leu-15
CD3 TcRdB R R y h CD2
OK73 WrJl
SCIlDlO SCUD2
% l o 0 97 99
2 1 0 0 1 98
SCIlD8 SCI103
1 0 0 9 7 9 8 9 0
0 1 0 0
SCIIE9 SCI3D3 SCIlGll
9 8 100
9
8
98
SCUIG6
98 99 98
89 9s 97
2 3
6 4
1 1 0 0 8 8 2 1 8 0 5 9 9 8 20
7 4
3 9 9 1 0 0 1 3 8 9 7 98 1
2 2
92 98 96
100 100 99
3 10
15
2
6 2 3
8
16
1 2 2 2 2 1 2 2 1 3 0 1 7 1 4 1 2 2 3 4 5 1 2 1
8 6 3 2 4 3 7 5
ND ND ND
98 64 92
ND
98 84
ND ND ND ND ND ND ND
N N ND ND N N ND ND
2 4 4 8
1 75 8 3
0
97
SCII2B2 SCII2F5 SCII2F6 SCVIIIZD3 SCVIIIZW SCVIII3B6 SCIrIII3E7 ScvlIt3F11 SCVIII3G4
7 8 N D
ND ND
97
82 9 3 N D
8 6 98
9 9 91
1 2
% 100 9
3 9 4 2
98
9
9
2 3
100 4
9
9
9 0 9 4 8 8 8 8 8 3 9 7 9 7 9 6 2 8 5 9 8 9 1 9 5 1 8 8 8 4 9 4 9 8
95 100
4
98
9
ND
93
97 91
1 10
m
100
98
94 100 95 100 98 100
87 100
ND ND
97
93 66 98 100
1 11 1
ND
ND 100
100
100
1
100
90
scm
9 0 N D nND 93
CD8
18 9
88 13 9 8 9 2 N D
SCIX1F6 SCIX2FM SCIx2CS SCIXZC8 SCIX205 SCIx2G6 SCIXZGlO SCIX3G7
CD4
100 ND 100
9
9 9 9 9 9 9
8 9 5 9 9 9
1 7 1 1 1 1 2 1
9
9
0
1 9 8 N D
100
4 Discussion
In order to further understand the role of Ts cells in the pathogenesis of lepromatous leprosy, we and others established T, lines and clones from peripheral blood [ 3 , 41 and lesions (21 of lepromatous leprosy patients. The functional 0
CD3
CD4
CD8
CD28
7
62 3 1
0
ND
ND ND
90
5
7 7 N D
6 ND
43
61 3 60 8
ND
98 95
80 87
ND
58
4 ND 9 ND
ND
15 76 82 17 42 36 8 3 3 6 51 78 67 99 95 ND 80 76
32
ND
99 66
66
9 8 N D
2
18 26
ND
ND
9 9 9 8 6 9 7 6 6 98 8 4
10
4
2
6 4
3 16
0 31
3 3
6 17 7
14 2 18
3 1 1 18 D 3 D 3
1 2
1
D D
3 6 63 33
1 60 6
5
0
12 4
16 ND 6 ND
1
1
N D N D
ND
2 ND
66 12 N D N D 7
0
1
11 2 1 1 1
8 ND 12 ND
N D N D
Leu-llb
39 24
4
4
CD16
1
10 3 ND 3
ND 5 ND
definition of the T, clones described by us is that they specifically suppress the proliferative response of autologous PBMC as well as Th clones to M . leprne Ag but not responses to other Ag such as HSVand the mitogen PHA. In the present study we report the most pertinent data on the mechanism of this suppression and the results of our Ta C
and Th cloncs. Cclls were incubated
Th
log fluorescence intensity
anti-lL 2R (anti-CD25) mAb at 1 : 200 dilution of ascites fluid. followed by incubation with FITC-conjugated goat anti-mouse Ig F(ab')z. Fluorescence analysis was performed using a FACS'".The characteristics of the cells samples used are: 1-3 T, clones SCIXlF6, SCIIDIO and 1G2: and 4-6 Th cloncs SCVIII2D3. 3B6 and 3F11.
1286
S. Guang Li,T. H. M. Ottenhoff, PVan den Elsen et al.
Eur. J. Immunol. 1990. 20: 1281-1288 A series of experiments has excluded competition for Ag [24] as the mechanism of the suppression phenomenon studied by us: adding a supra-optimal dose of M. leprae could not diminish the suppression, the T, cells were radiation sensitive, heat-inactivated T, clones lost their suppressive capacity, and recognition of Ag plus HLA by T, clones was not required for suppression to occur [19]. The latter observation was unexpected and suggested that for our T, clones activation and MHC restriction may be separated from the suppressive effect exerted by them.
Figure 4. Southern blot analysis of TcR p chain gene rearrangements. Eco RI digestion of DNA prepared from T, and Th clones were probed with the 32P-labeledTcR Cpprobe. Lanes 1-4 contain DNA fromT,clones SCIlD8, 1G2,3D3and SCIX3G7. Lanes 5-10 contain DNA from Th clones SCI12F5, SCVIII2D6, 3E7, 3G4, SCIIlG6and 2F6.The DNA sample of a control B cell line (NOL) is shown in lane 11. The sizes of the germ-line fragments are indicated on the right (kb).
Figure 5. SDS-PAGE analysis of immunoprecipitates carried out with an antLCD36 chain antiserum. Lanes 1-4 are Ts clones SCIlD8, 1G2, 3D3 and SCIX3G7. Lanes5-7 are Th clones SCII2F5, SCVIII2D6 and 3E7.The position of the molecular mass is indicated on the right.The position of TcR markers (kDa x and CD3 chains is indicated on the left.
analysis of the TcR and other cell surface molecules of T, clones as compared tOTh clones. For further details refer to the thesis of Shu Guang Li [19].
Next, we performed a series of experiments to study the effector phase of the suppression exerted by these Agspecific T, clones. HLA-DQ and class I are not involved as was shown by the failure to abolish the suppression with andi-DQ and class I mAb [19]. Because T, cells are restricted via HLA-DR in their activation phase [25], it was interesting to know if HLA-DR or any other HLA class I1 (DP or D) might also be involved in their effector phase. The observation that a DRCrestricted T, clone could only suppress the M. reprue responses of a Th cell line from the DRCmatched brother of the patient, but not of DR4M. feprue-reactive T h lines from other individuals, may indicate that the effector phase of the T, cells may be restricted by HLA-DR [19]. However, M . leprue or leprosy-related environmental factors accumulated in the family of the patient also may explain these data. We have reported previously [3, 181that theseT, clones are not cytotoxic for Th clones or M . leprue-pulsed B cell lines. Our present studies show that T, clones also can not lyse the Th clones that they suppress when Th cells are co-cultured with M. leprue and adherent MQ. T, cells could lyse Th clones nonspecifically (Con A dependent) and some of these T, clones tested after several restimulations and long-term culture obtained some degree of an NK-like activity.Thus,T, clones do possess cytolytic potential but do not use it in order to suppress. T, as well as Th clones could lyse adherent M. leprue-pulsed M@ in an Ag-specific HLA class 11-restrictedmanner. Killing of MQ as APC, however, is virtually excluded as the mechanism of suppression since the presentation of HSV Ag by the same MQ to HSVreactive Tcells is unaffected by the presence of M. leprue and M. leprue-reactive T, cells. If these T, cells would suppress M. leprue-reactiveThcell responses by killing their APC, T, cells would also be expected to suppress HSVdriven responses. Thus, although both Th and T, cells possess the capacity to lyse appropriately presented target cells,T, cells apparently do not use their cytolytic potential for suppressing M. leprue-reactive Th cell responses.
Thus far, we have failed to demonstrate M. feprue-specific suppression using SN of activated T,. We assume that for M. leprue-specific suppression to occur, interaction be4.1 Suppression is neither due to cytolysis nor to tween T,, Th andlor APC is necessary and we do not know competition for growth factors or antigen whether a molecule released by T, cells may mediate the suppressive function. Due to the unexpected finding that Several groups reported that suppression may result from M. leprue-specific suppression is probably not dependent interference by T, cells with TL2 secretion. T, cells might on activation of T, cells by M. leprue we considered the lower IL2 synthesis [20-221 or bind IL2 and thus lower the existence of veto cells (reviewed in [26]). The Ts cells in our effective concentration of this lymphokine in the microen- system bear class I1 molecules which can pick up soluble vironment [23].We could not abolish the M. leprue-specific Ag, and might thus be recognized by the class 11-restricted, suppression by adding exogenous IL2 [19], indicating that Ag-specific helper cells. However, a veto phenomenon M . leprue-specific suppression is not caused by IL2 con- would not easily explain why only Th cells reactive with sumption. M. leprue and not T h cells against HSV are suppressed.
Eur. J. Immunol. 1990. 20: 1281-1288
In conclusion, these M. Zeprue-specific, cloned T, cells specifically suppress M . leprue-driven proliferation of autologousTh cells by a mechanism that does not involve any of the known Tcell functions, such as cytolysis or competition for growth factors or antigen.
4.2 The only phenotypic difference between Th and T, is the presence of CD28 on Th and its absence on T, The main conclusion from the TcR analysis is that both T, and Th clones have TcRa/p/CD3 complexes. In several other systems similar observations have now been reported for human T, clones [27-291. This contrasts with the observations made in mouse systems, where many T, clones and hybridomas lacked bonafide TcR [30]. The TcR DNA and protein analysis combined with the differences in restriction specificity of both Th and T, clones [25] indicate that we are dealing with at least several different T, and Th clones. Three sets of observations regarding the other cell surface markers of T, compared to T h warrant further discussion. First we showed that all clones are regular CD3+ and CD2+ Tcells. Although both Th and T, acquired NK-like activity after long-term culture in vitro [19], they lacked NK cell markers such as CD56, CD57, CDll and CD16. T, clones may be CD4-CD8+, CD4+CD8- or CD4+CD8+. The majority of T, clones in our system were CD4+CD8- which differs from several studies in which the CD4-CD8+ Tcells are in the majority [2, 4, 31, 32].Thus, these data support the notion that CD4CD8 phenotypes are certainly not always associated with functional characteristics. Second, the consistent lack of CD45R on both T, and T h clones supports the notion that T cells lose the CD45R determinant upon stimulation [33,34]. Some T, clones as well as Th clones showed expression of CD29 (4B4),which has been postulated to be associated with CD4+ T h cells [ 121. Obviously, all clones strongly expressed the classical early activation markers such as CD25 and HLA-DR on their cell surface (Table 3 and Fig. 3). Finally, and most importantly, we observed that the CD28 Ag was lacking on the surface of T, clones but expressed on the surface of M . leprue-reactive Th clones, whose proliferation to M . leprue could be suppressed by T, cells. Interestingly, the HSV line that was not suppressed by the T, clones was also CD28-. This suggests that the presence of CD28 on the target cells might be needed for suppression to be induced. It should be noted that HSV was the only non-mycobacterial antigen (our of 10 tested) to which the patient (SC) did respond. Since a control T cell that is CD28+ and reactive to a non-mycobacterial antigen is not presently available, we therefore cannot be sure that the suppression is Ag and CD28 or only CD28 specific. The CD28 Ag is a cell surface Ag which is present on approximately 70% of PBL and which is recognized by the mAb9.3 [8]. It has been demonstrated that the CD8+CD28+subset contains Th cells [ 3 5 ] , T, cells [36] and precursor T, cells, whereas the CD8+CD28-subset contains precursors for Ag-specific suppressor cells [36, 371. The molecule carrying the CD28 Ag is involved in T cell activation and the level of expression of CD28 may
Human suppressor T cell clones lack CD28
1287
determine whether a positive or a negative signal is generated through this cell surface structure [38]. Our data show an absence of CD28 on both CD8+ and CD4+ Ag-specificT, cells. This observation is compatible with and extends the observation that the predominant T, cells in lepromatous lesions are CD8+CD28- [2, 391 and that this mAb can distinguish CD8+ suppressor effector cells (CDll+CD28-) from T, cells (CD1l-CD28+) [40].The two CD8+T, clones were both CD11+and CD28- (seeTable 2), but most CD4+T, clones were CD11-. In addition, our data suggest that CD28 may not only be a phenotypic marker dividing M. leprue-reactive T cells into two subsets, one (CD28-) suppressing the proliferation of the other (CD28+),but may be functionally involved in the suppression of cells carrying this determinant. This hypothesis can be at least partially tested, e.g. by transfection of the CD28 gene [41] in CD28- cells. The authors would like to thank Prof. Dr. J. J. Van Rood for his support; patient S. C. for donating blood; E Wassenaar for assistance with the Southern blotting technique; J. Blom, D. Van der Harst and S. Van Luxemburg for cell surface phenotyping part of the clones; Drs. S. E Schlossman, B. Malissen, H. Spits, E Bach, H . Maeda, R. Winchester, A . Ziegler, J. Bodmer, A . Kolk, M . Codon and R . Knowles for generously supplying us with monoclonal antibodies; Dr. J. Borst for her advice during the study and critical evaluation of the manuscript; Drs. H. Bruning and B. Vandekerckhove for stimulating discussions; Drs. E H. J. Claas, B. Roep and J. Thorogood for their suggestions, and C. S. L . Mackenzie, I. S. Curiel* D. Van Gorp, E. Van der Willik and 7:Van Westerop for help in preparing the manuscript. Received May 31, 1989; in final revised form February 1. 1990.
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