Eur. J. Immunol. 1991.21: 3053-3056
3053
Tcell function and QCA-1
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-1antigen 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 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, notably 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 several 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.
mAb directed at the cell surface molecules involved [9-111. Recent studies have shown that a mAb to a novel lymphocyte 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 Manipulation of the immune response to an antigen can be achieved by interfering with either of the two signals LN cells from PVG rats were cultured in Hams F12DMEM necessary for the cell to progress towards activation, using (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 [I 98121 mg/ml streptomycin (Flow), and 5 pg/ml Con A (Sigma), in * This work was supported by the Medical Research Council of an atmosphere of 5% COz in air at 37 "C.The cells were Great Britain, the Quadrant Research Foundation, the Bradlow stained at different time intervals after the initiation of the Foundation and a grant from the Dutch Foundation for Medical culture. For fluorescent staining, approximately lo6 cells and Health Research Medigon, which is subsidized by the were incubated for 30 min on ice in the well of a 96-well Netherlands Organization for Advancement of Pure Research plate (Nunc, Roskilde, Denmark) with 100 pl of PBS (ZWO, Grant 900-505-201). supplemented with 5% (v/v) FCS containing approximately Correspondence: Richard Aspinall, Quadrant, Mans Lane, Tmm- 1pg of first-stageantibody.The cells were then washed, and incubated for a further 30 min on ice with FITC-conjugated pington, Cambridge, (332 2sY, GB sheep anti-mouse IgG (Serotec, Oxford, GB). These cells were then washed twice and fixed by resuspending in 1% Abbreviation: QCA-1: Quiescent cell antigen-1 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
OO14-2980/91/1212-3053$3S O .25/0
3054
Eur. J. Immunol. 1991.21: 3053-3056
R. Aspinall, J. Kampinga and J. Van den Bogaerde
paraformaldehyde in PBS, and analyzed in a FACScan (Becton Dickinson, Mountain View, CA).
I
3 OCn
(a)
2.3 Skingraftmg
1
Full-thickness skin grafts were carried out according to the method described previously [141. Statistical significanceof differences in graft survival data was assessed by the Mann-Whitney U-test.
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. 1a 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. 1b), 53% of the population are positive and the mean fluorescence has dropped to 105 units. By 48 h (Fig. 1c) 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 predominantly 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.
300
30c3
(b)
(c)
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 specificfor the target in normal animals would be naive and therefore still express the QCA-1 antigen. The strain combination chosen, DA donor to Lou recipient, 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 highresponder RTlu haplotype also has a threefold greater number of anti-RTla haplotype-reactive T cells than the low-responder RTIChaplotype [14]. In this strain, combination skin grafts are rejected within 8-11 days in untreated animals (Fig. 2).
Tcell function and QCA-1
Eur. J. Immunol. 1991.21: 3053-3056
The recipients were treated with 2 to 4mg of the antiQCA-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 prolonged survivalwhen compared with the untreated controls (Table 1). For comparison, groups of recipients were treated with two antibodies against non-overlapping determinants 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].
% surviving grafts I
20
-
No McAb
-E-
anti QCA-1
-3-
anti CD4
4
01 0
5
10
15
,
,
I
I
20
25
30
35
m
-
40
Graft survival time (days)
3.2.2 Lymphocyte populations
Figure 2. The survival of skin graftsfrom 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 treatment regimens
mAb therapy
Mean survival
3055
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
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-1antibody 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
3053
Tcell function and QCA-1
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-1antigen 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 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, notably 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 several 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.
mAb directed at the cell surface molecules involved [9-111. Recent studies have shown that a mAb to a novel lymphocyte 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 Manipulation of the immune response to an antigen can be achieved by interfering with either of the two signals LN cells from PVG rats were cultured in Hams F12DMEM necessary for the cell to progress towards activation, using (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 [I 98121 mg/ml streptomycin (Flow), and 5 pg/ml Con A (Sigma), in * This work was supported by the Medical Research Council of an atmosphere of 5% COz in air at 37 "C.The cells were Great Britain, the Quadrant Research Foundation, the Bradlow stained at different time intervals after the initiation of the Foundation and a grant from the Dutch Foundation for Medical culture. For fluorescent staining, approximately lo6 cells and Health Research Medigon, which is subsidized by the were incubated for 30 min on ice in the well of a 96-well Netherlands Organization for Advancement of Pure Research plate (Nunc, Roskilde, Denmark) with 100 pl of PBS (ZWO, Grant 900-505-201). supplemented with 5% (v/v) FCS containing approximately Correspondence: Richard Aspinall, Quadrant, Mans Lane, Tmm- 1pg of first-stageantibody.The cells were then washed, and incubated for a further 30 min on ice with FITC-conjugated pington, Cambridge, (332 2sY, GB sheep anti-mouse IgG (Serotec, Oxford, GB). These cells were then washed twice and fixed by resuspending in 1% Abbreviation: QCA-1: Quiescent cell antigen-1 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
OO14-2980/91/1212-3053$3S O .25/0
3054
Eur. J. Immunol. 1991.21: 3053-3056
R. Aspinall, J. Kampinga and J. Van den Bogaerde
paraformaldehyde in PBS, and analyzed in a FACScan (Becton Dickinson, Mountain View, CA).
I
3 OCn
(a)
2.3 Skingraftmg
1
Full-thickness skin grafts were carried out according to the method described previously [141. Statistical significanceof differences in graft survival data was assessed by the Mann-Whitney U-test.
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. 1a 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. 1b), 53% of the population are positive and the mean fluorescence has dropped to 105 units. By 48 h (Fig. 1c) 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 predominantly 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.
300
30c3
(b)
(c)
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 specificfor the target in normal animals would be naive and therefore still express the QCA-1 antigen. The strain combination chosen, DA donor to Lou recipient, 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 highresponder RTlu haplotype also has a threefold greater number of anti-RTla haplotype-reactive T cells than the low-responder RTIChaplotype [14]. In this strain, combination skin grafts are rejected within 8-11 days in untreated animals (Fig. 2).
Tcell function and QCA-1
Eur. J. Immunol. 1991.21: 3053-3056
The recipients were treated with 2 to 4mg of the antiQCA-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 prolonged survivalwhen compared with the untreated controls (Table 1). For comparison, groups of recipients were treated with two antibodies against non-overlapping determinants 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].
% surviving grafts I
20
-
No McAb
-E-
anti QCA-1
-3-
anti CD4
4
01 0
5
10
15
,
,
I
I
20
25
30
35
m
-
40
Graft survival time (days)
3.2.2 Lymphocyte populations
Figure 2. The survival of skin graftsfrom 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 treatment regimens
mAb therapy
Mean survival
3055
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
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-1antibody 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
