Immunology, 1975, 29, 455.
The Activity of T and B Lymphocytes in Immunity and Tolerance to the Lymphocytic Choriomeningitis Virus in Mice M. VOLKERT, K. BRO-J0RGENSEN, 0. MARKER, B. RUBIN AND LOTTE TRIER Institute of Medical Microbiology, University ofCopenhagen, Immunobiologv Laboratory, Blood Bank and Blood Grouping Department, The State Serum Institute, Copenhagen, Denmark
(Received 9th October 1974; acceptedfor publication 20th February 1975) Summary. Treatment with anti-theta serum and the Wigzell column technique for cell separation was employed to study the separate functions of the B and T lymphocytes in the late states of immunity to the LCM virus in mice. The cell preparations examined were mixtures of spleen and lymph node cells from immune mice. The results revealed that the anti-viral effect of such cells after transfer to virus carriers was unimpaired in T cell-enriched and B cell-deprived cell preparations. The anti-viral effect was also retained in cell preparations deprived so much of B cells that no antibody was produced in the virus carrier mice receiving transplants of these cells. The results strongly indicate that the anti-viral effect of late immune cells is not only T cell-dependent but that it is also mediated solely by T cells and, moreover, that antibodies have no or very little influence on the virus elimination. The observation that antibody production could be caused neither by column-passed cells nor by anti-theta serum-treated cells, but was obtained by mixtures of these cells, demonstrates that co-operation between T and B cells is crucial for the LCM antibody response. Accordingly, the convincing demonstration of the absence in the persistent virus carriers of cells which, in respect of antibody production, are able to co-operate either with column-passed or with anti-theta serum-treated immune cells, implies that such animals are extremely deficient as regards immune function of both B and T LCM-primed lymphocytes. INTRODUCTION Recently data were presented (Volkert, Marker and Bro-J0rgensen, 1974) which strongly indicated that during a lymphocytic choriomeningitis (LCM) virus infection in adult mice two distinctly different populations of lymphocytes were involved in the immune reaction to the virus. One of them, the early immune cells, occurred in the early phase of the infection and was characterized by resistance to X-irradiation, cytotoxic activity against virus-infected target cells, anti-viral effect after transfer to acutely infected recipients and ability to protect such mice against the lethal outcome of the infection. In all probability the course of an acute infection is determined by the race between the Correspondence: Professor M. Volkert, Institute of Medical Microbiology, University of Copenhagen, 22 Juliane Manes Vej, DK-2100 Copenhagen 0, Denmark.
455
M. Volkert et al. 456 development of these early cells and the virus multiplication in vital organs. The early immune cells disappear when the virus has been eliminated, i.e. about 2-3 weeks after the infection. Then the late immune cells appear. It is unlikely that these cells descend from the early immune cells, and they possess none of the abilities which characterize the early cells. However, they have the capacity, after transfer to persistent virus carriers, to eliminate the chronic infection and cause antibody production. Probably the late immune cells have acquired immunological memory and are those which keep occult virus in check and protect against reinfection. The functions of each of the two populations of lymphocytes just mentioned were abolished by treatment with anti-theta serum, strongly indicating the dependence on T-cell activity. However, especially in the case of the late immune cells it was not known whether the T cells were acting alone, as helper cells for B cells or in conjunction with antibody. It is the purpose of this paper to present experimental results which elucidate aspects of these problems. In addition, the data presented strongly indicate that neither LCMprimed active T nor B cells develop in mice with persistent virus infection induced by inoculation of virus in newborn mice. These results give new support to the 25-year-old hypothesis of Burnet and Fenner (1949) concerning the development in mice of a tolerant state to the LCM virus-a hypothesis that has recently been challenged by some workers (Oldstone and Dixon, 1967, 1971).
MATERIALS AND METHODS Mice Except for virus titration purposes highly inbred C3H mice were used throughout the study. The mice receiving transplants were females aged 3-5 months, either acutely infected or persistent virus carriers. The acute infection was produced by intraperitoneal (i.p.) inoculation of 103 LD50 virus. The virus carriers were produced by i.p. inoculation of the newborn mice with 103 LD50 virus. Such mice carry virus in high titre in blood and organs throughout life, and do not develop any demonstrable immunity to the virus (Hotchin and Cinits, 1958; Volkert and Larsen, 1965). The experimental groups were chosen by random selection, and no group consisted of less than six mice. Donor mice for late immune cells were the mothers of infected babies about 2 months after the babies were born. Such mice have been infected by their babies and are highly immune (Volkert, 1962). In other cases virus carriers were also employed as cell donors.
Virus The LCM virus originated from Dr E. Traub (Ludwig-Maximillians Universitat, Munich, Germany). It was kept at - 700 as a 10 per cent spleen suspension from infected C3H mice. When necessary it was passed in mice, in recent years exclusively in C3H mice. Virus titrations were carried out by intracerebral (i.c.) inoculation of ordinary 12-15 g white Swiss mice. Titration end-points were calculated by the method described by Karber (1931), and the titres expressed as log10 LD50/0O03 ml i.c. dose. When inoculated by the i.p. route the virus was not lethal for C3H mice. Anti-theta serum (AOS) AOS was produced by immunizing AKR mice with C3H mouse thymocytes, and was tested for specificity and potency (Volkert et al., 1974). The data obtained strongly indi-
457 Lymphocytic Choriomeningitis Virus cated that the cytotoxicity of the serum was T cell-specific. In all experiments described in this paper, serum dilutions of 1: 2 were employed. The lymphoid cells in amounts of 2-5 x 108 were incubated in the serum at 40 for 1 hour. The cells were separated by gentle centrifugation and resuspended in tissue culture medium containing appropriate amounts of guinea-pig complement. After 45 minutes' incubation at 370 the cells were transplanted. Cell batches used as controls were prepared and treated in exactly the same way, except that the A0S was replaced by tissue culture medium.
Cell separation Two kinds of cell separation columns were employed in the present study: (1) glass bead columns coated with mouse immunoglobulin (MIg) anti-MIg complexes were used to retain B cells; (2) glass bead columns coated with human serum albumin (HSA) antiHSA complexes were used for sham treatment of the control preparations. They were prepared by the method described by Wigzell, Sundquist and Yoshida (1972) and Rubin and Wigzell (1973). In the first series of experiments the glass beads had a diameter of 500 pm (type I column). However, to obtain enrichment of the T cells in the following experiments glass beads with a diameter of 150 pm were employed (type II column). Cell passage was carried out at 40 at a flow rate of 2-5 ml/min, and a cell concentration of 2 x 107 cells/ml. Characterization of passed and retained cells has been discussed elsewhere (Wigzell et al., 1972; Rubin and Wigzell, 1973). In the present study non-passed and column-passed cells were tested for: (1) the number of 0-positive cells (T marker); (2) the number of EAC'-RFC (the marker for B cells having a receptor for activated complement factor C3). These tests were performed as described by Trier and Rubin (1974). Passage through type I MIg-anti-MIg columns increased the number of 0-positive cells by about 200 per cent and reduced the number of EAC'-RFC by more than 90 per cent. When type II columns were employed the results of the cell tests were slightly better, but in vivo experiments showed a striking difference. All preparations of late immune cells which had passed the MIg-anti-MIg type I column still had, after transfer to virus carriers, the ability to produce antibodies in relatively high titres. However, passage through MIg-anti-MIg type II columns could deprive the cell preparations of almost all their antibody-producing capacity. Passage through the control type I columns caused a moderate increase in the number of 0-positive cells, and only a few per cent reduction in the number of EAC'-RFC. Type II columns gave the same increase in the number of 0-positive cells and close to 25 per cent reduction of the EAC'-RFC. Neither column reduced the ability of the cell preparations to produce antibodies when transferred to virus carriers. Cell assay The cell preparations used were mixtures of spleen and lymph node cells. The method for preparation has been described elsewhere (Volkert, 1962). In all cases the cells were transplanted i.p. in volumes of 0-5 ml. The cell function was assayed by the ability to provoke an adoptive immune response after transfer to either virus carriers (Volkert, Larsen and Pfau, 1964; Volkert and Larsen, 1965) or to X-ray-irradiated, acutely infected normal adult mice. This response was measured by the anti-viral effect and/or the amount of complement-fixation (CF) antibodies produced. Where the virus carriers served as recipients the adoptive immunity could be expected to be at a maximum approximately 10 days after the cells had been D
458 M. Volkert et al. transferred (Volkert et al., 1974). Accordingly, the effect of the transplanted cells was tested on days 8-10 and, when necessary, an additional test was carried out on day 20. Where the X-ray-irradiated, acutely infected mice were concerned the irradiation (300 R) was carried out 24 hours before the infection, and the cells were transferred 2 days after, at a time when preliminary experiments had shown that the virus had multiplied enough to cause an antigenic stimulus of the transplanted cells. The antibodies produced by such a stimulus of immune cells are already present in high titre 6 days later, i.e. at a time when X-ray-irradiated, infected but otherwise untreated C3H mice cannot by themselves produce any detectable antibody to the virus. In the transplanted mice all the virus antibodies demonstrable 6 days after the cell transfer can therefore be considered to be due to the transplanted cells. Accordingly, the assay for the immunity transfer was carried out at the same time (for further details see Tables 3 and 4).
X-irradiation and complement-fixation (CF) tests These were carried out as described by Volkert et al. (1974) and Marker and Volkert (1973). RESULTS T-CELL ACTIVITY IN PREPARATIONS OF LATE IMMUNE CELLS In the experiments described in the previous paper (Volkert et al., 1974) it was shown that the AOS treatment of preparations of late immune cells could abolish the anti-viral effect of these cells after transfer to virus carriers. However, adoptive immunization of virus carriers also causes antibody production, and the AOS effect on that cell function had not been determined. This was tested in the following experiment. Half a batch of late immune cells were treated with AOS and the other half was sham-treated. The cells were then transferred to virus carriers in amounts of 50 x 106 per mouse. The results are shown in Table 1. AOS abrogated not only the anti-viral effect but also almost completely the
ability to produce antibody. This finding leaves us with the question whether the virus elimination is brought about through T-cell action alone or indirectly by T-cell helper function for the B-cell production of the antibodies which, in the end, might be the decisive factor. In attempts to clarify this question experiments were carried out employing cell preparations enriched in T cells and mainly deprived of B cells. In the first series of experiments these cell preparations were produced by passing the cells through a MIg-anti-MIg type I column, and in the second series by passing them through a similar column of type II. The cell suspensions were then transferred to virus carriers. The cell dose transplanted was chosen to be close to the minimal dose of untreated cells required to cause a clear-cut anti-viral effect after transfer to similar recipients. From the results it appeared that the full anti-viral activity remained in cell populations which had passed a type I column. However, transplants of these cells were still able to produce antibodies in rather high titres. Representative results of the activity of the cell populations which had passed through a MIg-anti-MIg type II column are shown in Table 1. It appears that the passage through the MIg-anti-MIg type II column could remove from the cell preparations all their antibody producing capacity. Only one out of sixteen mice receiving the columnpassed cells was able to produce antibody, and in this case only to a titre of 8. However, despite the lack of antibody all recipient mice were able to eliminate the virus. Moreover,
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460 M. Volkert et al. the virus elimination took place at the same rate and to the same extent as in the groups of recipients which had received either sham-treated or untreated control cell preparations. In the latter groups all mice showed a vigorous antibody production by titres between 3000 and 8000. These results reveal that the cells connected with the anti-viral effect pass through the MIg-anti-MIg column, whereas the antibody-producing capacity is abolished. Furthermore, the data presented clearly demonstrate that antibodies, even when present in high titres, do not have any detectable influence on the anti-viral process of transplanted late immune cells in virus carriers. B-CELL ACTIVITY IN PREPARATIONS OF LATE IMMUNE CELLS The AOS experiment described above indicated that the LCM antibody production of the B cells was T cell-dependent. This matter was investigated further. For this purpose virus carriers were given transplants of various preparations of late immune cells, and 11 days later tested for antibodies. A typical experiment is recorded in Table 2. The first group TABLE 2 CO-OPERATION BETWEEN T CELLS AND B CELLS IN THE ADOPTIVE ANTIBODY RESPONSE OF LATE IMMUNE CELLS AFTER TRANSFER TO VIRUS CARRIERS
Recipients (number of virus carriers) 8 8 8 8 8 8 8 8
Transplants
3 x 106 2-5 x 106 0 5 x l06 0 -x106 3 x 106 3 x 106 3x 106 5 x 106
imm. T cells* imm. B cellst imm. B cells imm. B cells imm. T cells+2-5x 106 imm. B cells imm. T cells+0 5 x 106 imm. B cells imm. T cells+0 l x 106 imm. B cells control cellst
CF titres (day 1 1)
265
The Activity of T and B Lymphocytes in Immunity and Tolerance to the Lymphocytic Choriomeningitis Virus in Mice M. VOLKERT, K. BRO-J0RGENSEN, 0. MARKER, B. RUBIN AND LOTTE TRIER Institute of Medical Microbiology, University ofCopenhagen, Immunobiologv Laboratory, Blood Bank and Blood Grouping Department, The State Serum Institute, Copenhagen, Denmark
(Received 9th October 1974; acceptedfor publication 20th February 1975) Summary. Treatment with anti-theta serum and the Wigzell column technique for cell separation was employed to study the separate functions of the B and T lymphocytes in the late states of immunity to the LCM virus in mice. The cell preparations examined were mixtures of spleen and lymph node cells from immune mice. The results revealed that the anti-viral effect of such cells after transfer to virus carriers was unimpaired in T cell-enriched and B cell-deprived cell preparations. The anti-viral effect was also retained in cell preparations deprived so much of B cells that no antibody was produced in the virus carrier mice receiving transplants of these cells. The results strongly indicate that the anti-viral effect of late immune cells is not only T cell-dependent but that it is also mediated solely by T cells and, moreover, that antibodies have no or very little influence on the virus elimination. The observation that antibody production could be caused neither by column-passed cells nor by anti-theta serum-treated cells, but was obtained by mixtures of these cells, demonstrates that co-operation between T and B cells is crucial for the LCM antibody response. Accordingly, the convincing demonstration of the absence in the persistent virus carriers of cells which, in respect of antibody production, are able to co-operate either with column-passed or with anti-theta serum-treated immune cells, implies that such animals are extremely deficient as regards immune function of both B and T LCM-primed lymphocytes. INTRODUCTION Recently data were presented (Volkert, Marker and Bro-J0rgensen, 1974) which strongly indicated that during a lymphocytic choriomeningitis (LCM) virus infection in adult mice two distinctly different populations of lymphocytes were involved in the immune reaction to the virus. One of them, the early immune cells, occurred in the early phase of the infection and was characterized by resistance to X-irradiation, cytotoxic activity against virus-infected target cells, anti-viral effect after transfer to acutely infected recipients and ability to protect such mice against the lethal outcome of the infection. In all probability the course of an acute infection is determined by the race between the Correspondence: Professor M. Volkert, Institute of Medical Microbiology, University of Copenhagen, 22 Juliane Manes Vej, DK-2100 Copenhagen 0, Denmark.
455
M. Volkert et al. 456 development of these early cells and the virus multiplication in vital organs. The early immune cells disappear when the virus has been eliminated, i.e. about 2-3 weeks after the infection. Then the late immune cells appear. It is unlikely that these cells descend from the early immune cells, and they possess none of the abilities which characterize the early cells. However, they have the capacity, after transfer to persistent virus carriers, to eliminate the chronic infection and cause antibody production. Probably the late immune cells have acquired immunological memory and are those which keep occult virus in check and protect against reinfection. The functions of each of the two populations of lymphocytes just mentioned were abolished by treatment with anti-theta serum, strongly indicating the dependence on T-cell activity. However, especially in the case of the late immune cells it was not known whether the T cells were acting alone, as helper cells for B cells or in conjunction with antibody. It is the purpose of this paper to present experimental results which elucidate aspects of these problems. In addition, the data presented strongly indicate that neither LCMprimed active T nor B cells develop in mice with persistent virus infection induced by inoculation of virus in newborn mice. These results give new support to the 25-year-old hypothesis of Burnet and Fenner (1949) concerning the development in mice of a tolerant state to the LCM virus-a hypothesis that has recently been challenged by some workers (Oldstone and Dixon, 1967, 1971).
MATERIALS AND METHODS Mice Except for virus titration purposes highly inbred C3H mice were used throughout the study. The mice receiving transplants were females aged 3-5 months, either acutely infected or persistent virus carriers. The acute infection was produced by intraperitoneal (i.p.) inoculation of 103 LD50 virus. The virus carriers were produced by i.p. inoculation of the newborn mice with 103 LD50 virus. Such mice carry virus in high titre in blood and organs throughout life, and do not develop any demonstrable immunity to the virus (Hotchin and Cinits, 1958; Volkert and Larsen, 1965). The experimental groups were chosen by random selection, and no group consisted of less than six mice. Donor mice for late immune cells were the mothers of infected babies about 2 months after the babies were born. Such mice have been infected by their babies and are highly immune (Volkert, 1962). In other cases virus carriers were also employed as cell donors.
Virus The LCM virus originated from Dr E. Traub (Ludwig-Maximillians Universitat, Munich, Germany). It was kept at - 700 as a 10 per cent spleen suspension from infected C3H mice. When necessary it was passed in mice, in recent years exclusively in C3H mice. Virus titrations were carried out by intracerebral (i.c.) inoculation of ordinary 12-15 g white Swiss mice. Titration end-points were calculated by the method described by Karber (1931), and the titres expressed as log10 LD50/0O03 ml i.c. dose. When inoculated by the i.p. route the virus was not lethal for C3H mice. Anti-theta serum (AOS) AOS was produced by immunizing AKR mice with C3H mouse thymocytes, and was tested for specificity and potency (Volkert et al., 1974). The data obtained strongly indi-
457 Lymphocytic Choriomeningitis Virus cated that the cytotoxicity of the serum was T cell-specific. In all experiments described in this paper, serum dilutions of 1: 2 were employed. The lymphoid cells in amounts of 2-5 x 108 were incubated in the serum at 40 for 1 hour. The cells were separated by gentle centrifugation and resuspended in tissue culture medium containing appropriate amounts of guinea-pig complement. After 45 minutes' incubation at 370 the cells were transplanted. Cell batches used as controls were prepared and treated in exactly the same way, except that the A0S was replaced by tissue culture medium.
Cell separation Two kinds of cell separation columns were employed in the present study: (1) glass bead columns coated with mouse immunoglobulin (MIg) anti-MIg complexes were used to retain B cells; (2) glass bead columns coated with human serum albumin (HSA) antiHSA complexes were used for sham treatment of the control preparations. They were prepared by the method described by Wigzell, Sundquist and Yoshida (1972) and Rubin and Wigzell (1973). In the first series of experiments the glass beads had a diameter of 500 pm (type I column). However, to obtain enrichment of the T cells in the following experiments glass beads with a diameter of 150 pm were employed (type II column). Cell passage was carried out at 40 at a flow rate of 2-5 ml/min, and a cell concentration of 2 x 107 cells/ml. Characterization of passed and retained cells has been discussed elsewhere (Wigzell et al., 1972; Rubin and Wigzell, 1973). In the present study non-passed and column-passed cells were tested for: (1) the number of 0-positive cells (T marker); (2) the number of EAC'-RFC (the marker for B cells having a receptor for activated complement factor C3). These tests were performed as described by Trier and Rubin (1974). Passage through type I MIg-anti-MIg columns increased the number of 0-positive cells by about 200 per cent and reduced the number of EAC'-RFC by more than 90 per cent. When type II columns were employed the results of the cell tests were slightly better, but in vivo experiments showed a striking difference. All preparations of late immune cells which had passed the MIg-anti-MIg type I column still had, after transfer to virus carriers, the ability to produce antibodies in relatively high titres. However, passage through MIg-anti-MIg type II columns could deprive the cell preparations of almost all their antibody-producing capacity. Passage through the control type I columns caused a moderate increase in the number of 0-positive cells, and only a few per cent reduction in the number of EAC'-RFC. Type II columns gave the same increase in the number of 0-positive cells and close to 25 per cent reduction of the EAC'-RFC. Neither column reduced the ability of the cell preparations to produce antibodies when transferred to virus carriers. Cell assay The cell preparations used were mixtures of spleen and lymph node cells. The method for preparation has been described elsewhere (Volkert, 1962). In all cases the cells were transplanted i.p. in volumes of 0-5 ml. The cell function was assayed by the ability to provoke an adoptive immune response after transfer to either virus carriers (Volkert, Larsen and Pfau, 1964; Volkert and Larsen, 1965) or to X-ray-irradiated, acutely infected normal adult mice. This response was measured by the anti-viral effect and/or the amount of complement-fixation (CF) antibodies produced. Where the virus carriers served as recipients the adoptive immunity could be expected to be at a maximum approximately 10 days after the cells had been D
458 M. Volkert et al. transferred (Volkert et al., 1974). Accordingly, the effect of the transplanted cells was tested on days 8-10 and, when necessary, an additional test was carried out on day 20. Where the X-ray-irradiated, acutely infected mice were concerned the irradiation (300 R) was carried out 24 hours before the infection, and the cells were transferred 2 days after, at a time when preliminary experiments had shown that the virus had multiplied enough to cause an antigenic stimulus of the transplanted cells. The antibodies produced by such a stimulus of immune cells are already present in high titre 6 days later, i.e. at a time when X-ray-irradiated, infected but otherwise untreated C3H mice cannot by themselves produce any detectable antibody to the virus. In the transplanted mice all the virus antibodies demonstrable 6 days after the cell transfer can therefore be considered to be due to the transplanted cells. Accordingly, the assay for the immunity transfer was carried out at the same time (for further details see Tables 3 and 4).
X-irradiation and complement-fixation (CF) tests These were carried out as described by Volkert et al. (1974) and Marker and Volkert (1973). RESULTS T-CELL ACTIVITY IN PREPARATIONS OF LATE IMMUNE CELLS In the experiments described in the previous paper (Volkert et al., 1974) it was shown that the AOS treatment of preparations of late immune cells could abolish the anti-viral effect of these cells after transfer to virus carriers. However, adoptive immunization of virus carriers also causes antibody production, and the AOS effect on that cell function had not been determined. This was tested in the following experiment. Half a batch of late immune cells were treated with AOS and the other half was sham-treated. The cells were then transferred to virus carriers in amounts of 50 x 106 per mouse. The results are shown in Table 1. AOS abrogated not only the anti-viral effect but also almost completely the
ability to produce antibody. This finding leaves us with the question whether the virus elimination is brought about through T-cell action alone or indirectly by T-cell helper function for the B-cell production of the antibodies which, in the end, might be the decisive factor. In attempts to clarify this question experiments were carried out employing cell preparations enriched in T cells and mainly deprived of B cells. In the first series of experiments these cell preparations were produced by passing the cells through a MIg-anti-MIg type I column, and in the second series by passing them through a similar column of type II. The cell suspensions were then transferred to virus carriers. The cell dose transplanted was chosen to be close to the minimal dose of untreated cells required to cause a clear-cut anti-viral effect after transfer to similar recipients. From the results it appeared that the full anti-viral activity remained in cell populations which had passed a type I column. However, transplants of these cells were still able to produce antibodies in rather high titres. Representative results of the activity of the cell populations which had passed through a MIg-anti-MIg type II column are shown in Table 1. It appears that the passage through the MIg-anti-MIg type II column could remove from the cell preparations all their antibody producing capacity. Only one out of sixteen mice receiving the columnpassed cells was able to produce antibody, and in this case only to a titre of 8. However, despite the lack of antibody all recipient mice were able to eliminate the virus. Moreover,
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460 M. Volkert et al. the virus elimination took place at the same rate and to the same extent as in the groups of recipients which had received either sham-treated or untreated control cell preparations. In the latter groups all mice showed a vigorous antibody production by titres between 3000 and 8000. These results reveal that the cells connected with the anti-viral effect pass through the MIg-anti-MIg column, whereas the antibody-producing capacity is abolished. Furthermore, the data presented clearly demonstrate that antibodies, even when present in high titres, do not have any detectable influence on the anti-viral process of transplanted late immune cells in virus carriers. B-CELL ACTIVITY IN PREPARATIONS OF LATE IMMUNE CELLS The AOS experiment described above indicated that the LCM antibody production of the B cells was T cell-dependent. This matter was investigated further. For this purpose virus carriers were given transplants of various preparations of late immune cells, and 11 days later tested for antibodies. A typical experiment is recorded in Table 2. The first group TABLE 2 CO-OPERATION BETWEEN T CELLS AND B CELLS IN THE ADOPTIVE ANTIBODY RESPONSE OF LATE IMMUNE CELLS AFTER TRANSFER TO VIRUS CARRIERS
Recipients (number of virus carriers) 8 8 8 8 8 8 8 8
Transplants
3 x 106 2-5 x 106 0 5 x l06 0 -x106 3 x 106 3 x 106 3x 106 5 x 106
imm. T cells* imm. B cellst imm. B cells imm. B cells imm. T cells+2-5x 106 imm. B cells imm. T cells+0 5 x 106 imm. B cells imm. T cells+0 l x 106 imm. B cells control cellst
CF titres (day 1 1)
265
