T-cell clone anti-clone interactions. Effects on suppressor and helper activities

Journal of Autoimmunity (1989) 2 (Supplement),

3-14

T-cell Clone Anti-clone Interactions. Effects on Suppressor and Helper Activities

David Naor, Gail Essery, Melvyn Kahan and Marc Feldmann The Charing Cross Sunley Research Centre, Lurgan Avenue, Hammersmith, London W6 8LW, UK

An experimental model of two interacting clones of T cells is described, which may be used for defining and exploring the T-cell immunoregulatory network. Mx9/9 is a CD4 clone bearing an antigen receptor recognixed by the Mx9 anti-VP8 monoclonal antibody (MoAb). Anti-Vllll MoAbs activate and induce cell proliferation of this clone. Autologous clones were raised against Mx9/9 cells using the peripheral blood mononuclear (PBM) cells of the Mx9/9 clone donor (PBMu,). Some of these cloned anti-clone cells proliferated after stimulation with irradiated Mx9/9 cells, but not after sdmulation with other autologous cloned T cells or heterologous PBM, suggesting that these clones recognize the T cell receptor (TCR) of the Mx9/9 cells. The proliferation of the Mx9/9 stimulated cloned anticlone cells was blocked by anti-class II MoAbs, indicating that the autoreactive clones recognize their target antigen in conjunction with HLA Class II products. The ability of clone Mx9/9 to proliferate after stimulation with anti-V@ MoAb was inhibited when clone 121 cells were added to the cultures. However, clone 121 lost its suppressor function after 4 months in culture and instead gained the ability to enhance the proliferation of Mx9/9 cells in the presence of anti-VW MoAb. In contrast, clone 18 lacked suppressor activity at the early stage of the study but later acquired this function. We conclude that some autoreactive clones are not fully committed and may express more than a single function. Such cells cannot therefore be designated as ‘suppressor cells’, although they expressed suppressor potential at certain stages.

Correspondence to: Dr D. Naor, The Lautenberg Center for General and Tumour Immunology, The Hebrew University, Hadassah Medical School, Jerusalem, 91010, Israel. 3 0896--8411/89/0300S3+

12 s03.00/0

0 1989 Academic Press Limited

4

D. Naor et al. Introduction

The golden age of the ‘suppressor T cells’ of the 1970s and early ‘8Os, during which a large part of the cellular immunology literature concerned these cells, has passed. Far fewer papers describing suppressor T cells are now published, indicating that the number of workers exploring this field has substantially decreased, and those remaining face more difficulty in publishing their work. The suppressor T cell attracted much attention because it provided an elegant explanation of the well established phenomenon of immunosuppression. Furthermore, this cell provided a balance for the ‘helper cell’, an effect required in order to understand immunoregulation [ 11. This scheme of immunoregulation has lost favour, due to the many conflicting and unresolved findings in the field of suppressor-cell research. For example, there are reports that some suppressor T cells express the CD8+ phenotype (e.g. [2]), others the CD4+ phenotype (e.g. [3]), while still more are double positive (e.g. [4]). Some authors indicated that the suppressor cells recognize free antigen (e.g. [5]) whereas others presented evidence that they recognize antigen only in association with major histocompatibility complex (MHC) products [6,7]. The introduction of molecular biology techniques into the field of immunology, rather than resolving the problems of the suppressor cells as it did for other T cells, augmented the problems. Some workers reported that their suppressor T cells use T-cell receptor (TCR) a and p chains, similar to those expressed on helper or killer cells [8, 91. Others found that suppressor T cells have deleted the p chain genes, or do not rearrange the b chain germ line genes [lo, 111, and therefore do not have an active p chain gene [6]. A further report describes a suppressor T-cell line that transcribes the p chain gene but not the a or they chain genes, while a different line transcribes the p and y chain genes [ 121. In addition, the ‘J’ molecule which was initially considered a suppressor T-cell marker [5] was not be detected when the MHC was mapped at the DNA level [ 131. The confusion is compounded by lack of understanding of the mechanism of action of suppressor T cells, and by some amazingly complicated multicellular pathways described [ 1, 14, 151. Finally, some authors found that the same T-cell clone exhibited either suppressor or helper functions when subjected to different experimental parameters [ 16-201, indicating that the functional programme of these clones is not necessarily committed to either pathway. Another common question about suppressor cells concerns their cytotoxic function [ 16, 211. The question whether such cells should be categorized as suppressor cells will not be discussed here because the difference between suppression and cytolysis as a mechanism of inhibition may be merely semantic. However, the other questions raised above are relevant and need to be answered. G&an Mijller [22] has already reached the unequivocal conclusion that suppressor cells do not exist. However, other immunologists think that the issue is still inconclusive, because the research in the field is far from being completed (see editorial discussion in Scand.J. Immunol. 1988.27: 621-628). This paper describes a set of autoreactive clones which were raised against a CD4+ clone bearing a T-cell receptor recognized by anti-VP8 monoclonal antibody (MoAb) Mx9. Since the anti-clones were specific for the clone that stimulated them, but not other autologous clones, we predicted that they recognize the T-cell receptor of the stimulator clone and may regulate its function. Approaching this concept

Clone anti-clone interactions

5

experimentally, we tested the ability of each clone to suppress or enhance the activity of the autologous stimulator clone. The results of these assays raise interesting possible solutions to some aspects of the suppressor cell puzzle. Materials and methods Human T-cell clones h&9/9 is a human CD4+ T-cell clone [23] reacting with the Mx9 MoAb, specific for the VP8 family of the T-cell receptor (TCR)P chain [24, 251. When coated onto plastic, Mx9 MoAb stimulates the proliferation of Mx9/9 cells. Anti-V85 MoAb directed against a different VP family does not stimulate Mx9/9 cells under identical conditions [23]. T-cell clones were derived from the autologous peripheral blood mononuclear (PBM) cells of the donor (J.M.) of the Mx9/9 cells (PBM,,) after stimulation with the Mx9/9 clone. PBM,, (0.5 x lo6 cells) were incubated for 7 d with irradiated (5,000 rad) autologous Mx9/9 CD4+ clone cells (0.5 x lo6 cells) suspended in 1 ml RPM1 1640 containing 100/b human A’ serum. Recombinant human IL-2 (20 ng/ml) was added to the cultures for the last 2 d. At day 7,2 x lo5 PBM cells from the first culture were harvested, washed and re-incubated with lo6 feeder cells (irradiated PBM& and 2 x lo5 irradiated Mx9/9 CD4 clone cells for an additional 5 d, in RPM1 containing 10% A+ serum and 20 ng/ml IL-2. The cells harvested from the second culture were cloned in Terasaki plates at a ratio of 0.3 cells per well in the presence of 5,000 irradiated Mx9/9 cells, 5,000 irradiated PBM,, and 20 ng/ml IL-2. Of 1,440 wells, 70 (5 96) clones were recovered and ofthese 26 were successfully expanded, cryopreserved, and used in our subsequent studies. Monoclonal antibodies (MoAbs) Table

1 describes

the monoclonal

antibodies

used in the present

study.

Proliferation assay One to 1.5 x lo4 responder cells were incubated with 1 to 2.5 x lo4 irradiated (5,000 rads) stimulator T cells, or with irradiated (20,000 rads) EBV transformed cells, for 64 h in RPM1 1640 supplemented with penicillin/streptomycin and 107; A’ human serum. 3H-thymidine was added to the cultures 14 h before harvesting with a Titertek 550 cell harvester (Flow Laboratories). 3H-thymidine incorporation was measured with a LKB 1219 Rackbeta (Pharmacia Biotechnology, Uppsala, Sweden). Fluorescence activated cell sorter (FACS)

analysis

For indirect staining the cells were incubated for 20 to 30 min with the unconjugated MoAbs listed in Table 1, washed twice with phosphate buffered saline supplemented with 2y0 Fetal Calf Serum, blocked with 20% normal mouse serum (Sigma) and finally incubated with 1: 100 dilution of goat anti-mouse phycoerythrin (PE) conjugate (Southern Biotechnology Associates, Birmingham, USA). For direct staining 5 x lo5 cells were incubated for 20 to 30 min at 4°C with the optimal concentration of

6

D. Naor et af.

Table 1. Description of the monoclonal antibodies used in the present study

Nomenclature

Specificity

Conjugated to

Mx9

42/1Cl Tii39 Tii22 HB-55 B7121.2 CRL-800 1 Leu3a G-17 Leu2a Leu2a WCHT-4 WT-31 TiaA TCRGl

vi5 class II

DQ

DR DP CD3 CD4 CD4 CD8 CD8 CD8 IL-2R c$ receptor VY9 C6

FITC6 PE’ FITC FITC PE FITC PE

Source Dr S. Carrel’ Dr A. W. Boylston’ Dr A. Ziegler3 Dr A. Ziegler ATCC* Dr J. Bodmer’ ATCC Becton Dickinson Dr J. A. Ledbetter’ Becton Dickinson Becton Dickinson Dr P. Beverley9 Becton Dickinson Dr W. Tax” Dr T. Hercend” Dr M. B. Brenner”

Reference 24,25 26 27 27 28 29 30

31 32 33

‘LudwigInstitutefor CancerResearch,Lausanne,Switzerland *St Mary’s Hospital, London, UK. ‘Phillips Universitit, Marburg, FRG. ‘ATCC, American Type Culture Collection. ‘Imperial Cancer Research Fund, London, UK. 6FITC, Fluorescein isothiocyanate. ‘PE, phycoerythrin. 80ncogen, Seattle, USA. 91mperial Cancer Research Fund, London, UK. “‘St Radboud Hospital, Nijmegen, The Netherlands. “Institut Gustave-Roussy, Villejuif, France. ‘*Dana Farber, Boston, USA.

the fluorochrome conjugated monoclonal antibodies listed in Table 1. The cells were washed twice and analysed using a FACStar (Becton Dickinson). Cells were enumerated relative to log integrated green and red fluorescence, forward angle light scatter, and right angle light (side) scatter. Ten thousand events of list mode correlated data were acquired and analysed using a Hewlett Packard computer and the Consort 30 data analysis program. For detecting the y&TCR a mixture of TiyA and TCRyl MoAbs [32,33] were used in the first layer staining. Results Characterization of the autoreactive clones Seventy autologous clones were isolated from PBM,, as described in Materials and methods. Since the clones were raised against the autologous Mx9/9 CD4+ clone we anticipated that at least some of them will be specific to Mx9/9 CD4+ clone, but not to other clones obtained from the same individual. The proliferative responses of 26 clones, stimulated with irradiated Mx9/9 cells and other autologous cloned cells or heterologous PBM, are summarized in Table 2.

7

Clone anti-clone interactions

Table 2. Summary of the proliferation responses of 26 clones stimulated with various autologous clone cells

Type I II

III

Responder clones 12,17,18,111,121, 21,27,211,212 19,116,117,124,128, 129,132,134,22,28, 215,234 13,15,217,225,231

Mx9/9

JM6

Stimulators’ Clone 12

PBM,

IL-2

+

-

-

-

+

+

+

+

+

-

-

-

+

‘JM6 is a PBM clone which was raised against autologousEBV-transformed B cells. Clones 12,17,18, 111 and 121 only were tested against clone 12 stimulator cells. Clone which was raised against autologous Mx9/9 cells. PBM, were obtained

12 is a specific autoreactive clone from an unrelated individual.

Table 3. Stimulation of an autoreactive clone by autologous EBV-transformed

B cells’

Stimulators (cpm of ‘H-thymidine incorporation)’

JM6

Responder clone

Medium

Mx9/9 DR 3,3

I=“,, Dr 3,3

EB”,, DR 6,7

EB”Gki DR4,2

EB”iCI. DR3,4

DR 3,3

Medium Clone 111

151 962

911 4,974

2,096 11,203

1,896 2,524

1,914 2,280

694 2,082

507 1,393

ISimilar results were obtained with clones 12,18,17, and 121. ZThe SD does not exceed 10% of the arithmetic mean.

Three patterns of response were noted. Some of the clones (e.g. clones 18 and 111) proliferated after stimulation with Mx9/9 clone cells, but not after stimulation with the other autologous cloned T cells. Therefore they were specific to Mx9/9 clone. For instance, the specific autoreactive clone 111 incorporated 488 3H-thymidine cpm when incubated alone and 16,941 cpm when incubated with 20 ng IL-2. The same cloned cells incorported 3,781 cpm when incubated with irradiated Mx9/9 stimulator cells but only 861 and 1,088 cpm when incubated with irradiated JM6 or irradiated clone 12 stimulator cells, respectively (the 3H-thymidine incorporation of the irradiated stimulator cells incubated alone was 100 cpm or less). The distinct ability of specific autoreactive clones to proliferate after stimulation with Mx9/9 cells but not after stimulation with other autologous or heterologous stimulator cells was detected at both high (2.5: 1) and low (1:4) responder to stimulator ratios (results not shown). The other two types of clones responded to all stimulator cells (e.g. clone 28) or did not respond to any one of them (e.g. clone 231). Whereas clones 12 and JM6 derived from the same PBM donor (J.M.) failed to stimulate the proliferation of the autoreactive clones (e.g. clone 11 l), EBVtransformed B cells from the same individual (EBV,& induced a greater proliferative response than Mx9/9 cells (Table 3). This stimulatory effect was genetically

8

D. Naor et al. Ant1 DR

Anti

HE55

DO

Tti 22

20 0 L

I

0.1

IO

I

IO 1.0

1.0 Monoclonal

I

0.1 anhbody

concentration

Figure 1. The effect of anti-class II MoAbs on the proliferative responses of autoreactive clones stimulated with CD4+ Mx9/9 clone or EBV-transformed B cells. Clones 17 (0) and 18 (0) were incubated with autologous CD4+ Mx9/9 cells (A) or EBV-transformed B cells (B) in the presence of different concentrations of the indicated MoAbs. 3H-thymidine incorporation was determined as described in material and methods. Similar results were obtained with clones 12,111 and 12 1. The proliferative responses of clones 17 and 18 in the absence of MoAbs were 5,590 and 3,844 (A) or 6,780 and 5,444 (B) respectively. The standard deviation of each individual average does not exceed 10% of the mean.

restricted, since EBV-transformed B cells of individuals expressing different DR haplotypes failed to stimulate the autoreactive clones (Table 3). Similar results were obtained when clones 12, 17, 18 and 121 were used as responder cells (results not shown). Anti-DR but not anti-DQ MoAbs inhibited the proliferative responses of clones 17 and 18 stimulated by autologous EBV [Figure (lb)], indicating that HLA Class II antigens on the EBV-transformed cells are recognized by the autoreactive clones. FACS analysis revealed that all the clones raised from the Mx9/9 stimulated cultures of JM PBM express the CD3+ CD4+ CD8- phenotype except clone 27 which was CD3+ CD4- CDS+. Clones 12,17,18,111 and 121 (but presumably all the others too) expressed IL-2 receptor as indicated by FACS analysis. In addition, clones 17 and 121 were analysed by FACS for the expression of up or y6 antigen receptor. The up TCR but not the y6 TCR was detected on these cells (Figure 2). The response of some of the autoreactive clones to the Mx9/9 CD4+ clone in the presence of MoAbs against HLA Class II (DR, DQ) was investigated. Figure l(a)

Clone anti-clone interactions

9

FLI

Figure 2. Clone 121 expresses aj3TCR. Clone 121 cells were stained both indirectly with anti-as TCR MoAbs followed by PE conjugated goat anti-mouse immunoglobulin (vertical axis) and directly with FITC conjugated anti-CD4 (horizontal axis). The double stained clone 121 was analysed by FACS. Clone 121 expresses ap receptor [panel (c)l. Panels (a), (b) and (d) are controls; panel (a), unstained clone 121; panel (b), clone 121 stained with the second layer indirect reagent alone (PE conjugated goat antimouse immunoglobulin); panel (d), clone 121 double stained with anti-CD4 and anti-y6 TCR Mabs.

describes the effect of the antibodies on the proliferation of two representative clones, 17 and 18. Anti-DR antibodies inhibited the proliferation of the Mx9/9 stimulated clones, whereas anti-DQ antibodies were ineffective, suggesting that the autoreactive clones recognize an antigen on Mx9/9 cells in conjunction with HLA-DR. The dualfunction

of the autoreactive

clones

Clones 18, 111 and 121 were tested several times over a 6-month period for their ability to influence the proliferation of the autologous Mx9/9 clone, which had been stimulated with anti-VP8 MoAb. The results described in Table 4 show that the unirradiated 121 cells suppressed the proliferation of the autologous Mx9/9 clone cells at the beginning of March and end of April 1988, but in contrast enhanced this response 4 months later. However, irradiated (5,000 rads) clone 121 cells suppressed the proliferation of the autologous Mx9/9 CD4+ clone over the entire 6-month period. This inhibition is not due to a crowding affect because similar concentration of 111 irradiated cells failed to suppress the Mx9/9 proliferation under identical conditions. The regulatory effects of clones 18 and 111 also varied when tested at different time points (Table 4). Discussion

The idiotype network theory proposed by Jeme [34] suggests that the homeostasis of the immune system is regulated at least in part by idiotype-anti-idiotype interactions. In modern terms, it means that the antigen receptors of one cell interact with

3,216+451

62,095+4,380

23,488 &- 1,914

34,439

672+22

1,731 f235

2,086 f 266

107

29.4.88

15.8.88

1.9.88

1,524

196%

2,583

2,797 + 58 113% 55,588+4,549 110%

aV@S + 18

Mx9/9 +

192%

176%

145% 52,718 + 3,779 f224% 51,391 t 149%

772 f 79

175%* 36,131 f978

aVPS+xr121

Mx9/9 +

791 f224

h4x9/9 + aVPS+ 121

t 169%

58,457

136%

147 39,954+5,112

1,714+68

aVpS+lll

Mx9/9+

e$fect of autoreactive

Mx9/9 +

f206%

71,076

aVPS+xrlll

clones’

539k300 835f132

2,974 + 860

alone

xr121 alone

Clone Clone 121

‘1 x 10’ h4x9/9 responder cells and 1 x lo4 stimulator cells were incubated in each well either alone or together in the presence or the absence of aVp8 MoAbs. 2The percent figures express either suppression (1) or enhancement (r) of the cell proliferation. a, anti; xr, x-irradiated.

alone

6.3.88

Date

h&9/9 + aVB8

Mx9/9

Table 4. The regulatory bifunctional

Clone anti-clone interactions

11

the receptors of a second cell, and the receptor of a third cell, in turn, can interact with the receptor of the second one and so on. It is assumed that these mutual interactions keep the immune system consistantly regulated and stable. Lamb and Feldmann [35] demonstrated that such receptor-anti-receptor interactions can exist at the level of T-cell clones. They presented evidence that a T-cell clone which was raised against an autologous helper-cell clone specific for the influenza virus suppressed the ability of these helper cells to provide help to autologous B cells. The B cells could not produce anti-influenza antibodies when mixed with both the helper and the antihelper clones. Since the anti-helper clone was stimulated by the helper clone but not by other autologous cells, and the helper clone was stimulated by X-irradiated ‘anti-helper’, TCR anti-TCR interaction was proposed [35]. It is likely that such TCR-anti TCR interactions are also exhibited by some of the autoreactive clones raised against the autologous Mx9/9 CD4 clone. These autoreactive clones proliferated after stimulation with Mx9/9 cells but not after stimulation with other clones derived from the same individual (Table 2). Note that clones 12,17, 18, 111 and 121 which are family members of Type I autoreactive clones, did not activate one another (Table 2), and they failed to proliferate after stimulation with anti-VP8 MoAb (results not shown). This is not surprising, because they were induced with the Mx9/9 clone. The autologous clone JM6, generated with autologous EBV-transformed B cells, proliferated after stimulation with both Mx9/9 clone and anti-Mx9/9 clones (results not shown), indicating that the JM6 is a ‘non-specific’ autoreactive clone. In contrast, the anti-Mx9/9 clones did not stimulate the proliferation of Mx9/9 clone (results not shown), indicating that although they express stimulatory capacity, they were unable to stimulate the clone which generated them. The fact that Mx9/9 clone stimulated the proliferation of the anti-Mx9/9 clones but failed to be stimulated by them implies that the stimulatory signal is unidirectional. The only known difference between the Mx9/9 clone which stimulated the autoreactive clones and other autologous clones which did not stimulate them is the Mx9/9 TCR which is recognized by the anti-V/38 antibody. We suggest, therefore, that the autoreactive clones specifically recognize the Mx9/9 TCR. Furthermore, they appear to recognize the Mx9/9 TCR in conjunction with Class II MHC products, since MoAbs against HLA-DR inhibited the proliferation of the autoreactive clones after stimulation with Mx9/9 cells. Interestingly, EBV-transformed autologous B cells, but not heterologous EBV-transformed cells stimulated the autoreactive clones and this stimulation was blocked by anti-Class II MoAbs. These findings suggest that activation of TCR expressing high af%nity to MHC plus antigen can be mimicked by activation with high concentrations of the appropriate MHC alone, as proposed by Doherty and Bennick [36]. What is the consequence of the TCR-anti-TCR interactions? The idiotype network concept suggests that these interactions can regulate the immune responses, i.e. they amplify weak reponses and suppress strong ones, in attempts to stabilize the system. By incubating anti-VP8 stimulated Mx9/9 cells with the autoreactive clones we could test the effect of TCR-anti-TCR interactions under defined experimental conditions. We have found that the same unirradiated clone (e.g. clones 111 and 121) suppressed the Mx9/9 proliferation when tested at an early time but enhanced the response when tested again after 4 months. However, the irradiated 121 clone

12

D. Naor et al.

suppressed the Mx9/9 proliferation both at early and late times. This observation clearly indicates that at least some clones which can inhibit are not fully committed to express a single function, i.e. they can either suppress or enhance the immune response, possibly by delivering antagonistic lymphokines or other types of signal. The fact that clone 121 cells lost their late helper function after x-irradiation, but instead express suppressor function suggests that under normal conditions the radiosensitive helper function of clone 121 dominates the radioresistant suppressor function. The suppressor function of the clone could only be expressed when the helper function was blocked by x-irradiation. The regulatory effect of many of our first panel of clones has not yet been investigated. They may express bifunctional or multifunctional programs or may be committed to a single program of either suppression or help. Whatever the results would be, our experimental model emphasizes the complexity of the immunoregulatory system and further indicates how much effort still needs to be invested in an attempt to enlighten this controversial issue.

Acknowledgements

The authors thank the donors of the monoclonal antibodies listed in Table 1. This work was supported by grants from the NufField Foundation, BP, ICI plc, the Sunley Trust and the Michael and Anna Wix Charitable Trust. David Naor holds the Milton Winograd professorial chair of Cancer studies, The Hebrew University of Jerusalem.

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14 29.

30.

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32. 33.

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