prolonged survival of skin grafts following treatment with an antibody to a putative cell triggering...

4
Eur. J. Immunol. 1991.21: 3053-3056 Tcell function and QCA-1 3053 Short paper Richard AspinallA, Jaap KampingaAand Johan Van den BogaerdeO Quadrant*, Maris Lane, 'Ihunphgton, Cambridge, Dept. of Histology and Cell BiologyA University of Groningen, Groningen and Dept. of Rheumatologyo, Royal Postgraduate Medical School, London Prolonged survival of skin grafts following treatment with an antibody to a putative cell triggering molecule, QCA-l* The QCA-1 molecule (quiescent cell antigen-1) appears to be involved in the differentiation events undergone by T cell following occupancy of the antigen receptor. Here we show that modulation of the QCA-1antigenfrom the surfaceof the cell normally follows activation, and that treatment of animals with the antibodies against the QCA-1 molecule inhibits the normal response to an allograft without appearing to alter the number of peripheral Tcells or the expression by these cells of the a/P T cell antigen receptor. 1 Introduction The majority of T cells in the recirculating peripheral T cell pool are not proliferating and are in either the GI or, more commonly, the Go stage of the cell cycle [l]. Those cells in the Go stage require a minimum of two signals to enter the proliferative phase of the cycle [2]. The first signal is a multistep process in which the most critical step is the cross-linking of the TcR [3]. This is accomplished under physiological conditionsby antigen presented in the groove of a MHC molecule on the surface of an APC [4]. This critical step cannot be achieved efficiently without the participation of several other cell surface molecules, nota- bly those which increase the adhesiveness of intercellular conjugation (e.g. LFA-1) [5] and those which may increase both the avidity of binding and the efficiency of the transmissionof the activation signal (e.g. CD4,CD8) [6,7]. The cumulative effect of these interactions is to activate the cell and shift it from the Go to the GI stage. As a consequence of this first signal and consequent changes in the cell's metabolism, there are changes in the surface phenotype, including increased expression of sev- eral cell surface markers. One of these, the IL 2R, needs to be occupied by its ligand IL 2, before the cell can cross the Gl/S boundary of the cell cycle [S] .This provides the second signal for T cell activation and proliferation. Manipulation of the immune response to an antigen can be achieved by interfering with either of the two signals necessary for the cell to progress towards activation, using [I 98121 * This work was supported by the Medical Research Council of Great Britain, the Quadrant Research Foundation, the Bradlow Foundation and a grant from the Dutch Foundation for Medical and Health Research Medigon, which is subsidized by the Netherlands Organization for Advancement of Pure Research (ZWO, Grant 900-505-201). Correspondence: Richard Aspinall, Quadrant, Mans Lane, Tmm- pington, Cambridge, (332 2sY, GB Abbreviation: QCA-1: Quiescent cell antigen-1 mAb directed at the cell surface molecules involved [9-111. Recent studies have shown that a mAb to a novel lympho- cyte cell surface molecule (quiescent cell antigen-1, QCA-1) has little effect onTor NK cell-mediated lysis [12] but inhibits T cell proliferation to alloantigen or xeno antigen in vitro [13]. The QCA-1 molecule consists of two protein chains of M, 46 and 60 kDa and is expressed on the majority of peripheral Tand B cells. In this study the level of expression and possible function of the molecule are assessed during lymphocyte activation processes both in vitro and in vivo. 2 Materials and methods 2.1 Animals and preparation of antibodies for use in vivo The rat strains PVG (RTlC), DA (RTlaV1) and Lode (RTIC) were used at approximately 3 months of age. Antibodies for injection were precipitated from ascites fluids with ammonium sulfate. The precipitate was then resuspended, dialyzed against PBS, filter sterilized and stored at - 20°C or lower prior to use. Injection protocols are described in Sect. 3. 2.2 Cell culture and immunofluorescent staining of cells LN cells from PVG rats were cultured in Hams F12DMEM (Flow Labs, Rickmansworth, GB) supplemented with 5% (v/v) fetal bovine serum (Sera-Lab, Crawley Down, GB), 2.5 x lop5 M 2-ME (BDH Chemicals, Poole, GB), 2 m~ L-glutamine (Sigma, Poole, GB), penicillin at 100 U/ml, (Crystapen, Glaxo Laboratories, Greenford, GB), 0.1 mg/ml streptomycin (Flow), and 5 pg/ml Con A (Sigma), in an atmosphere of 5% COz in air at 37 "C. The cells were stained at different time intervals after the initiation of the culture. For fluorescent staining, approximately lo6 cells were incubated for 30 min on ice in the well of a 96-well plate (Nunc, Roskilde, Denmark) with 100 pl of PBS supplemented with 5% (v/v) FCS containing approximately 1 pg of first-stageantibody.The cells were then washed, and incubated for a further 30 min on ice with FITC-conjugated sheep anti-mouse IgG (Serotec, Oxford, GB). These cells were then washed twice and fixed by resuspending in 1% 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 OO14-2980/91/1212-3053$3 SO + .25/0

Upload: richard-aspinall

Post on 11-Jun-2016

213 views

Category:

Documents


1 download

TRANSCRIPT

Eur. J. Immunol. 1991.21: 3053-3056 Tcell function and QCA-1 3053

Short paper

Richard AspinallA, Jaap KampingaA and Johan Van den BogaerdeO

Quadrant*, Maris Lane, 'Ihunphgton, Cambridge, Dept. of Histology and Cell BiologyA University of Groningen, Groningen and Dept. of Rheumatologyo, Royal Postgraduate Medical School, London

Prolonged survival of skin grafts following treatment with an antibody to a putative cell triggering molecule, QCA-l*

The QCA-1 molecule (quiescent cell antigen-1) appears to be involved in the differentiation events undergone by T cell following occupancy of the antigen receptor. Here we show that modulation of the QCA-1 antigen from the surface of the cell normally follows activation, and that treatment of animals with the antibodies against the QCA-1 molecule inhibits the normal response to an allograft without appearing to alter the number of peripheral Tcells or the expression by these cells of the a/P T cell antigen receptor.

1 Introduction

The majority of T cells in the recirculating peripheral T cell pool are not proliferating and are in either the GI or, more commonly, the Go stage of the cell cycle [l]. Those cells in the Go stage require a minimum of two signals to enter the proliferative phase of the cycle [2]. The first signal is a multistep process in which the most critical step is the cross-linking of the TcR [3]. This is accomplished under physiological conditions by antigen presented in the groove of a MHC molecule on the surface of an APC [4]. This critical step cannot be achieved efficiently without the participation of several other cell surface molecules, nota- bly those which increase the adhesiveness of intercellular conjugation (e.g. LFA-1) [5] and those which may increase both the avidity of binding and the efficiency of the transmission of the activation signal (e.g. CD4,CD8) [6,7]. The cumulative effect of these interactions is to activate the cell and shift it from the Go to the GI stage.

As a consequence of this first signal and consequent changes in the cell's metabolism, there are changes in the surface phenotype, including increased expression of sev- eral cell surface markers. One of these, the IL 2R, needs to be occupied by its ligand IL 2, before the cell can cross the Gl/S boundary of the cell cycle [S] .This provides the second signal for T cell activation and proliferation.

Manipulation of the immune response to an antigen can be achieved by interfering with either of the two signals necessary for the cell to progress towards activation, using

[I 98121 * This work was supported by the Medical Research Council of

Great Britain, the Quadrant Research Foundation, the Bradlow Foundation and a grant from the Dutch Foundation for Medical and Health Research Medigon, which is subsidized by the Netherlands Organization for Advancement of Pure Research (ZWO, Grant 900-505-201).

Correspondence: Richard Aspinall, Quadrant, Mans Lane, Tmm- pington, Cambridge, (332 2sY, GB

Abbreviation: QCA-1: Quiescent cell antigen-1

mAb directed at the cell surface molecules involved [9-111. Recent studies have shown that a mAb to a novel lympho- cyte cell surface molecule (quiescent cell antigen-1, QCA-1) has little effect onTor NK cell-mediated lysis [12] but inhibits T cell proliferation to alloantigen or xeno antigen in vitro [13]. The QCA-1 molecule consists of two protein chains of M, 46 and 60 kDa and is expressed on the majority of peripheral Tand B cells. In this study the level of expression and possible function of the molecule are assessed during lymphocyte activation processes both in vitro and in vivo.

2 Materials and methods

2.1 Animals and preparation of antibodies for use in vivo

The rat strains PVG (RTlC), DA (RTlaV1) and Lode (RTIC) were used at approximately 3 months of age. Antibodies for injection were precipitated from ascites fluids with ammonium sulfate. The precipitate was then resuspended, dialyzed against PBS, filter sterilized and stored at - 20°C or lower prior to use. Injection protocols are described in Sect. 3.

2.2 Cell culture and immunofluorescent staining of cells

LN cells from PVG rats were cultured in Hams F12DMEM (Flow Labs, Rickmansworth, GB) supplemented with 5% (v/v) fetal bovine serum (Sera-Lab, Crawley Down, GB), 2.5 x lop5 M 2-ME (BDH Chemicals, Poole, GB), 2 m~ L-glutamine (Sigma, Poole, GB), penicillin at 100 U/ml, (Crystapen, Glaxo Laboratories, Greenford, GB), 0.1 mg/ml streptomycin (Flow), and 5 pg/ml Con A (Sigma), in an atmosphere of 5% COz in air at 37 "C. The cells were stained at different time intervals after the initiation of the culture. For fluorescent staining, approximately lo6 cells were incubated for 30 min on ice in the well of a 96-well plate (Nunc, Roskilde, Denmark) with 100 pl of PBS supplemented with 5% (v/v) FCS containing approximately 1 pg of first-stage antibody.The cells were then washed, and incubated for a further 30 min on ice with FITC-conjugated sheep anti-mouse IgG (Serotec, Oxford, GB). These cells were then washed twice and fixed by resuspending in 1%

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 OO14-2980/91/1212-3053$3 S O + .25/0

3054

paraformaldehyde in PBS, and analyzed in a FACScan (Becton Dickinson, Mountain View, CA).

R. Aspinall, J. Kampinga and J. Van den Bogaerde

3 OCn (a) I

300

Eur. J. Immunol. 1991.21: 3053-3056

(b)

2.3 Skingraftmg

30c3

1 Full-thickness skin grafts were carried out according to the method described previously [ 141. Statistical significance of differences in graft survival data was assessed by the Mann-Whitney U-test.

(c)

3 Results and discussion

3.1 Expression of QCA-1 in relation to activation

Previous studies [12, 131 suggested that the expression of the QCA-1 antigen on Tcells was related to the state of activation of the cell. To test this, lymphocytes were stimulated with mitogen in culture and then analyzed at various time intervals to determine the expression of the QCA-1 antigen. The results (Fig. 1) show a decline in the percentage of QCA-1+ cells in these cultures with time. Fig. 1 a shows that cells freshly prepared from LN contain 65% QCA-l+ cells, with a mean fluorescence of 123 units. After 24 h in culture with Con A (Fig. 1 b), 53% of the population are positive and the mean fluorescence has dropped to 105 units. By 48 h (Fig. 1 c) there are only 16% positive cells and the mean fluorescence has dropped to 98 units. The cells in Con A cultures at this time are predom- inantly Tcells and we know that in normal animals approximately 90% of the Tcells are QCA-l+, so we can discount the possibility that what we are seeing is a rapid growth in the cultures of QCA-1- cells. In these cultures the reduction both in the mean peak of fluorescence and the number of QCA-l+ cells is probably due to the modulation of the antigen from the surface during the activation process.

Similar results on the loss of the QCA-1 antigen from the cell surface have been noted following stimulation of lymphocytes with the anti-TcR a / p antibody R73 (T. Hiinig, personal communication). These data suggest that the loss of QCA-1 from the cell surface is not transitory and is dependent on the state of activation of the cell. The loss of this antigen on activation explains why in previous studies the addition of antibody to the QCA-1 molecule to cytotoxicT cell assays had no effect on either the specificity or the degree of lysis of the targets [12].

3.2 Effect of antibody in vivo

3.2.1 Graft survival

The results from mitogen stimulation studies on the expression of QCA-1 suggested that the most appropriate regimen to adopt was to begin treatment with anti-QCA-1 antibody before grafting, since alloreactive cells specific for the target in normal animals would be naive and therefore still express the QCA-1 antigen.

The strain combination chosen, DA donor to Lou reci- pient, is possibly the most difficult combination for skin grafts. The Lou strain (RT1" haplotype) carries the gene

Figure 1. Loss of QCA-1 antigen on cells following culture with Con, A for (b) 24 and (c) 48 h. Control cells which have not been cultured are shown in (a).

which determines vigorous in vivo responses to class I molecules of DA (RTla haplotype) rats, and the high- responder RTlu haplotype also has a threefold greater number of anti-RTla haplotype-reactive T cells than the low-responder RTIC haplotype [14]. In this strain, combi- nation skin grafts are rejected within 8-11 days in untreated animals (Fig. 2).

Eur. J. Immunol. 1991.21: 3053-3056 Tcell function and QCA-1 3055

% surviving grafts I

No McAb - -E- anti QCA-1

-3- anti CD4

20 4

m 0 1 , , I I -

0 5 10 15 20 25 30 35 40 Graft survival time (days)

Figure 2. The survival of skin grafts from allogeneic donors on rats which were treated with antibodies against the CD4 molecule, or with antibody against the QCA-1 molecule, or which received no treatment. The treatment regimen is described in Sect. 2.3.

Table 1. Skin graft survival on animals receiving different treat- ment regimens

mAb therapy Mean survival p values time (days)

k SEM (n = 6)

No therapy 8.8 f 0.6

Ant i-CD4 10.7 & 0.5 Not significant

Anti-QCA-1 16.5 & 1.9 0.009

The recipients were treated with 2 to 4mg of the anti- QCA-1 antibody 2-3 days prior to receiving the skin graft, and thereafter received this same dose every 3 to 4 days. The grafts on treated animals showed significantly pro- longed survival when compared with the untreated controls (Table 1). For comparison, groups of recipients were treated with two antibodies against non-overlapping deter- minants on the CD4 molecules and grafted with allogeneic skin according to a regimen described previously, which allows the prolonged survival of allogeneic hearts between these two strains and induces graft-specific tolerance [16]. In the experiments reported here there was no significant prolongation of the survival of skin allografts (Fig. 2 and Table l), a result which confirms an earlier report [17].

3.2.2 Lymphocyte populations

Analysis of the lymphoid populations in the blood during treatment revealed that antibody therapy with anti-CD4 antibody leads to a rapid depletion in the CD4+ population within 72 h of start of treatment.These cells returned once the treatment regime was stopped. This was expected and was in line with previous reports on treatment with anti-CD4 antibodies [16, 171.

FCM analysis of the cells taken from the peripheral blood of animals throughout the period of treatment with the anti-QCA-1 antibody revealed that the treatment produced no significant reduction in either the apparent number of Tcells in the blood nor the amount of TcR a l p molecules expressed on their surface.The data presented in Fig. 4 are profiles of peripheral blood cells from an animal bearing an intact allogeneic skin graft stained to reveal cells expressing the TcR dB using the R73 antibody. At no time was the number of R73+ cells <70% of the total lymphoid population. There appeared to be no marked reduction in

Figure 3. FCM profiles of peripheral blood cells stained for the expression of the TcR a l p (unshaded area) taken at various intervals, from an animal with an intact skin graft.The percentage of positive cells and the times after grafting were (a) 0 days, 86%; (b) 8 days, 83%; (c) 22 days, 82%; (d) 29 days, 82%; (e) 36 days, 74% .The shaded area shows the cells stained with the second-layer

3056

the total number of peripheral lymphoid cells, and our results reveal that both CD4+ and CD8+ cells were present in the peripheral blood (data not shown).

R. Aspinall, J. Kampinga and J.Van den Bogaerde

4 Concluding remarks

Treatment of animals with anti-QCA-1 antibody prolongs the survival of allogeneic skin grafts without the depletion in the peripheral T cell pool seen following treatment with anti-CD4 antibodies. The T cells in animals treated with anti-QCA-1 express the a@ form of the TcR. Either the alloreactive cells in treated animals cannot respond to the graft antigen because the QCA-1 molecule cannot play its normal role in the activation process because it is covered by antibody or absent from the cell surface, or thoseTcells which are specific for the antigens on the graft have been depleted from the peripheral lymphoid pool. Evidence at present favors the former hypothesis, since the QCA-1 molecule is lost following activation in vitro, and we have preliminary evidence which suggests that it is lost from cells in vivo following treatment with the antibody.

Received August 6, 1991.

Eur. J. Immunol. 1991. 21: 3053-3056

5 References

1

2

3

4

5 6

7 8 9

10

11

12 13

14

Klein, J., The Natural History of the Major Histocompatibility Complex John Wiley, New York 1987. Mueller, D. L., Jenkins, M. K., Schwartz, R. H., Annu. Rev. Immunol. 1989. 7: 445. Weiss, A., Imboden, J., Hardy, K., Manger, B. ,Terhorst, C. and Stobo, J., Annu. Rev. Immunol. 1986. 4: 593. Bjorkmann, l? J., Saper, M. A., Samroui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C., Nature 1987. 329: 512. Springer, T. A., Nature 1990. 340: 425. McDonald, H. R. and Nabholz, M., Annu. Rev. Cell Biol. 1986. 2: 231. Rudd, C., Immunol. Today 1990. 11: 400. Smith, K. A., Science 1988. 240: 1169. Cobbold, S . I?, Jayasuriya, A., Nash, A., Prospero,T. D. and Waldmann, H., Nature 1984. 312: 648. Hirsch, R., Eckhaus, M., Auchinloss Jr., H., Sachs, D. H. and Bluestone, J. A., J. Immunol. 1988. 140: 3766. Kupiec-Weglinski, J. W.,Tilney, N. L., Stunkel, K. G., Grutz- mann, R. ,Van der Meide, l? H., Di Stefano, R. and Diamant- stein, T., Transplantation 1989. 47: 11. Aspinall, R. and Kampinga, J., Thymus 1989. 13: 245. Kampinga, J., Kroese, F. G. M., Pol, G. H., Meedendorp, B., Van den Bogaerde, J., Nieuwenhuis, F!, Roser, B. and Aspinall, R., Int. Immunol. 1990. 2: 915. Butcher, G.W., Corvalan, J. R., Licence, D. R. and Howard, J. C., J. Exp. Med. 1982. 155: 303.

16 Bogaerde, J.,White, D., Kampinga, J., Roser, B. and Aspinall,

17 Herbert, J. and Roser, B., Transplantation 1988. 46: 128. R., Transplantation 1990. 50: 915.