Human Ig in reconstituted SCID mice
Eur. J. Immunol. 1992. 22: 823-828
823
Mohammad R. AbediO', Birger Christenssonoa, Khalid B. Islamo0, Lennart Hammarstromon and C. I. Edvard Smithou
Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells*
Department of Clinical Immunologyo and Department of Pathologyo, Karolinska Institute at Huddinge Hospital, and Center for BioTechnology, NOVUMO, Huddinge
Immunodeficient C.B-17 scidlscid (SCID) mice were reconstituted with human peripheral blood mononuclear cells and analyzed for humoral immunity. The majority of adoptively transferred animals had serum levels of 1-4 mg/ml of human IgG 8-12 weeks after i.p. reconstitution with 20 x 10hPBMC,whereasthe IgM and especially IgA concentrations were considerably lower. The half-lives of human IgG, IgM, and IgA in SCID mice were 12 days, 36, and 23 hours, respectively. Furthermore, IgA was rapidly secreted into bile indicating that the low IgA concentration was mainly caused by a high turnover rate. IgG subclass distribution in mouse serum was similar to that found in the donor serum. Irradiation with 3 Gy prior to adoptive transfer resulted in increased levels of human IgG early after reconstitution, whereas both IgM and IgA concentrations were impaired. A polyclonal serum Ig pattern was found 4 weeks after transfer of human cells later frequently followed by a predominance of oligoclonal bands. Unexpectedly, oligoclonal bands were also found using donors with a negative Epstein-Barr virus serology. Human cells were found to reside in the peritoneal cavity for several months.Within 2 weeks of reconstitution, human cells were also identified in lymphoid structures in the vicinity of the liver hilus with a later spread to other lymphoid organs. Homing of human cells to skin and gut was not seen.
1 Introduction Certain studies of the human immune system can, for ethical reasons, not be performed in vivo, reducing such investigations to in vitro analysis or the use of animal models. However, hypersensitivity to foreign substances or sensitivity to drugs and toxic or infectious agents are often genetic in origin, and the establishment of an experimental model that permits the analysis of the human immune system is therefore instrumental. In 1988,two elegant novel models, based on the transfer of human lymphoid tissue cells to severe combined immunodeficient C.B- 17 scidlscid (SCID) mice, were reported [ l , 21. SCID mice lack a functional lymphoid system [3, 41 and the defect also results in an increased sensitivity to irradiation [S]. These new transfer techniques have so far proven useful for the analysis of infectious diseases [6, 71, tumors [8, 91, autoimmunity [lo, 111 and immunodeficiency [12, 131. Thus, although this technology allows long-term survival of human lymphoid cells, the regulation, both in terms of differentiation of lymphoid precursors, as well as the collaboration between mature lymphocytes and the interaction with accessory cells, may be restricted. In this study we report on the homing of human lymphocytes and the
synthesis and turnover of immunoglobulin in SCID mice repopulated with human PBMC through i.p. injection. We have also studied the influence of a previous EBV infection for the behavior of the human donor lymphocytes as well as the effect of pre-reconstitution irradiation.
2 Materials and methods 2.1 Reconstitution and immunization of animals C.B-17 SCID mice [3] were obtained from Stanford University, CA, by the courtesy of Drs. M. Lieberman and J. M. McCune, and bred in our animal facility. Male and female mice were reconstituted at 6-12 weeks of age and received antibiotics as described [l]. Irradiated mice received 3 Gy total body irradiation prior to reconstitution with human cells. Ficoll-Hypaque-separated PBMC from healthy donors were injected i.p. into SCID mice [2]. In most experiments, each mouse received 20 X lo6 PBMC suspended in 0.4 ml of normal saline. Reconstituted mice were immunized with 2 pg HBsAg (hepatitis B surface Ag, Engerix-B, Smithkline Biologicals, Rixensart, Belgium) in 0.1 ml suspension i.p. and were boosted with the same dose 2 weeks later. Serum Ig against HBsAg was detected in ELISA.
[I 98071
*
This work was supported by the Swedish Medical Research Council, the Palle Ferb Memqrial Foundation, the King Gustaf V Jubilee Foundation, the Ake Wiberg Foundation, Baxter Hyland Medical AB, and the Karolinska Institute. Scholar of the Ministry of Culture and Higher Education of the Islamic Republic of Iran.
Correspondence: Mohammad R . Abedi, Department of Clinical Immunology, F-79 Huddinge Hospital, S-141 86 Huddinge, Sweden
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
2.2 ELISA Human IgG, IgM, and IgA were quantified by sandwich ELISA using rabbit anti-human IgG, IgM, and IgA (y, p, and a , Dakopatts, Glostrup, Denmark) as the capture agents and alkaline phosphatase (AP)-conjugated affinitypurified rabbit anti-human IgG, IgM, and IgA (Dakopatts) for detection. Affinity-purified mouse anti-human IgGl (HP6069) IgG2 (HP6014), IgG3 (HP6047), and IgG4 0014-2980/92/0303-0823$3.50 + .25/0
Eur. J. Immunol. 1992. 22: 823-828
M. R. Abedi, B. Christensson, K. B. Islam et al.
824
(HP6025) subclass (Hybridoma Reagent Laboratory, Baltimore, MD) were used for quantification of human IgG subclasses in ELISA. Specific antibodies against HBsAg were also determined by ELISA using HBsAg as the capture agent and anti-Ig as described above (Dakopatts) followed by AP-conjugated sheep anti-rabbit IgG (Sigma Chemical Company, St. Louis, MO).
2.4 Immunohistochemistry and immunofluorescence The presence and distribution of human lymphoid cells were assayed by conventional histological and immunohistochemical analysis of parenchymatous organs. Cytospins were prepared from peritoneal lavage cells and bone marrow using a cytocentrifuge (Shandon, Elliott, GB). Flow cytometric analysis was made using conventional techniques with FITC- and PE-conjugated mAb on a FACScan (Becton Dickinson, San Jose, CA).
2.3 Serum electrophoresis and immunofixation Using conventional methods, 5 pl of undiluted serum samples from repopulted SCID mice was used for electrophoresis. Rabbit Ig to human y and p chains (Dakopatts) were used for immunofixation.
2.5 Monoclonal antibodies Antibodies against human CD45 (LCA), CD14 (Leu-M3), CD3 (Leu-4), and CD19 (Leu-12) were obtained from
Donor1
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Figure I. (A, B, and C) Human IgG, IgM, and IgA levels in sera obtained every 2 weeks after transfer from individual SCID mice reconstituted with human PBMC from a single donor.The dotted lines represent Ig levels in non-irradiated animals and the straight lines Ig levels in SCID mice receiving 3-Gy total body irradiation prior to reconstitution. (D, E, and F) Biological half-lives of human IgG, IgM, and IgA in SCID mice.Values given are the average of two experiments. Open circles represent the first phase (distribution phase) and filled circles represent the biological half-life (second phase). (G) Serum human IgG subclass concentrations in sera obtained from individual SCID mice 8 weeks after reconstitution with human PBMC from four different donors. Proportion of serum IgG subclasses in the donors are shown in the upper part of the figure. (H) Human IgG subclass in mouse No. 2 from each donor (Fig. la ) 4,8, and 12 weeks after reconstitution.
Eur. J. Immunol. 1992. 22: 823-828
Human Ig in reconstituted SCID mice
Becton Dickinson. A FITC-conjugated rabbit anti-mouse Ig (Dakopatts) was used with mAb against human MHC class I (W6/32, Seralab, Crawley Down, GB) in an indirect technique. In addition, mAb against human CD20 (L26) and CD2 (MT910, Dakopatts) were used in immunohistochemical analysis of cytospins.
825
seropositive donors that showed very high IgM levels (Table 1). Reconstitution of animals with only 5 x lo6 PBMC resulted in significantly lower Ig levels than expected as compared to 20 X lo6 (not shown).When SCID sera were analyzed for the presence of mouse Ig, 5 g/l. Human Ig was quantified by ELISA at 1, 3, 8, 18, and 30 h, as well as 2, 4,7, 14, and 21 days and half-lives calculated as previously described [ 141.
In irradiated mice a tendency to a faster and higher IgG production early after reconstitution was seen compared to non-irradiated mice (Fig. 1A). However, at 4 weeks, there was no significant difference in the IgG levels. In fact, the IgG production in irradiated mice seemed to decrease more rapidly and the IgM and IgA production was constantly lower (Fig. 1B and C).
3 Results 3.1 Human Ig class levels in reconstituted SCID mice Human Ig was detected in the serum of reconstituted SCID mice already 1week after reconstitution. The highest levels of human IgG, as well as of IgM and IgA, were obtained at 8-12 weeks post-reconstitution (Fig. 1). The serum IgG levels reached 1-4 mg/ml (approximately 10 YO-40% of the normal human serum concentration), while IgM and IgA levels, at their peaks, were usually
Eur. J. Immunol. 1992. 22: 823-828
823
Mohammad R. AbediO', Birger Christenssonoa, Khalid B. Islamo0, Lennart Hammarstromon and C. I. Edvard Smithou
Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells*
Department of Clinical Immunologyo and Department of Pathologyo, Karolinska Institute at Huddinge Hospital, and Center for BioTechnology, NOVUMO, Huddinge
Immunodeficient C.B-17 scidlscid (SCID) mice were reconstituted with human peripheral blood mononuclear cells and analyzed for humoral immunity. The majority of adoptively transferred animals had serum levels of 1-4 mg/ml of human IgG 8-12 weeks after i.p. reconstitution with 20 x 10hPBMC,whereasthe IgM and especially IgA concentrations were considerably lower. The half-lives of human IgG, IgM, and IgA in SCID mice were 12 days, 36, and 23 hours, respectively. Furthermore, IgA was rapidly secreted into bile indicating that the low IgA concentration was mainly caused by a high turnover rate. IgG subclass distribution in mouse serum was similar to that found in the donor serum. Irradiation with 3 Gy prior to adoptive transfer resulted in increased levels of human IgG early after reconstitution, whereas both IgM and IgA concentrations were impaired. A polyclonal serum Ig pattern was found 4 weeks after transfer of human cells later frequently followed by a predominance of oligoclonal bands. Unexpectedly, oligoclonal bands were also found using donors with a negative Epstein-Barr virus serology. Human cells were found to reside in the peritoneal cavity for several months.Within 2 weeks of reconstitution, human cells were also identified in lymphoid structures in the vicinity of the liver hilus with a later spread to other lymphoid organs. Homing of human cells to skin and gut was not seen.
1 Introduction Certain studies of the human immune system can, for ethical reasons, not be performed in vivo, reducing such investigations to in vitro analysis or the use of animal models. However, hypersensitivity to foreign substances or sensitivity to drugs and toxic or infectious agents are often genetic in origin, and the establishment of an experimental model that permits the analysis of the human immune system is therefore instrumental. In 1988,two elegant novel models, based on the transfer of human lymphoid tissue cells to severe combined immunodeficient C.B- 17 scidlscid (SCID) mice, were reported [ l , 21. SCID mice lack a functional lymphoid system [3, 41 and the defect also results in an increased sensitivity to irradiation [S]. These new transfer techniques have so far proven useful for the analysis of infectious diseases [6, 71, tumors [8, 91, autoimmunity [lo, 111 and immunodeficiency [12, 131. Thus, although this technology allows long-term survival of human lymphoid cells, the regulation, both in terms of differentiation of lymphoid precursors, as well as the collaboration between mature lymphocytes and the interaction with accessory cells, may be restricted. In this study we report on the homing of human lymphocytes and the
synthesis and turnover of immunoglobulin in SCID mice repopulated with human PBMC through i.p. injection. We have also studied the influence of a previous EBV infection for the behavior of the human donor lymphocytes as well as the effect of pre-reconstitution irradiation.
2 Materials and methods 2.1 Reconstitution and immunization of animals C.B-17 SCID mice [3] were obtained from Stanford University, CA, by the courtesy of Drs. M. Lieberman and J. M. McCune, and bred in our animal facility. Male and female mice were reconstituted at 6-12 weeks of age and received antibiotics as described [l]. Irradiated mice received 3 Gy total body irradiation prior to reconstitution with human cells. Ficoll-Hypaque-separated PBMC from healthy donors were injected i.p. into SCID mice [2]. In most experiments, each mouse received 20 X lo6 PBMC suspended in 0.4 ml of normal saline. Reconstituted mice were immunized with 2 pg HBsAg (hepatitis B surface Ag, Engerix-B, Smithkline Biologicals, Rixensart, Belgium) in 0.1 ml suspension i.p. and were boosted with the same dose 2 weeks later. Serum Ig against HBsAg was detected in ELISA.
[I 98071
*
This work was supported by the Swedish Medical Research Council, the Palle Ferb Memqrial Foundation, the King Gustaf V Jubilee Foundation, the Ake Wiberg Foundation, Baxter Hyland Medical AB, and the Karolinska Institute. Scholar of the Ministry of Culture and Higher Education of the Islamic Republic of Iran.
Correspondence: Mohammad R . Abedi, Department of Clinical Immunology, F-79 Huddinge Hospital, S-141 86 Huddinge, Sweden
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
2.2 ELISA Human IgG, IgM, and IgA were quantified by sandwich ELISA using rabbit anti-human IgG, IgM, and IgA (y, p, and a , Dakopatts, Glostrup, Denmark) as the capture agents and alkaline phosphatase (AP)-conjugated affinitypurified rabbit anti-human IgG, IgM, and IgA (Dakopatts) for detection. Affinity-purified mouse anti-human IgGl (HP6069) IgG2 (HP6014), IgG3 (HP6047), and IgG4 0014-2980/92/0303-0823$3.50 + .25/0
Eur. J. Immunol. 1992. 22: 823-828
M. R. Abedi, B. Christensson, K. B. Islam et al.
824
(HP6025) subclass (Hybridoma Reagent Laboratory, Baltimore, MD) were used for quantification of human IgG subclasses in ELISA. Specific antibodies against HBsAg were also determined by ELISA using HBsAg as the capture agent and anti-Ig as described above (Dakopatts) followed by AP-conjugated sheep anti-rabbit IgG (Sigma Chemical Company, St. Louis, MO).
2.4 Immunohistochemistry and immunofluorescence The presence and distribution of human lymphoid cells were assayed by conventional histological and immunohistochemical analysis of parenchymatous organs. Cytospins were prepared from peritoneal lavage cells and bone marrow using a cytocentrifuge (Shandon, Elliott, GB). Flow cytometric analysis was made using conventional techniques with FITC- and PE-conjugated mAb on a FACScan (Becton Dickinson, San Jose, CA).
2.3 Serum electrophoresis and immunofixation Using conventional methods, 5 pl of undiluted serum samples from repopulted SCID mice was used for electrophoresis. Rabbit Ig to human y and p chains (Dakopatts) were used for immunofixation.
2.5 Monoclonal antibodies Antibodies against human CD45 (LCA), CD14 (Leu-M3), CD3 (Leu-4), and CD19 (Leu-12) were obtained from
Donor1
Donor2
i
Donor3
Donor4
1 2 3
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11.9 days 2000 1000
125'0
- 3 8 0
c
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Half-life: 23.1 hours
10
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g 2 I 250
1 0
0
5
10
15
20
Weeks after reconstitution
0
1
2
3
4
Days after injection
4012
Weeks after reconstitution
Figure I. (A, B, and C) Human IgG, IgM, and IgA levels in sera obtained every 2 weeks after transfer from individual SCID mice reconstituted with human PBMC from a single donor.The dotted lines represent Ig levels in non-irradiated animals and the straight lines Ig levels in SCID mice receiving 3-Gy total body irradiation prior to reconstitution. (D, E, and F) Biological half-lives of human IgG, IgM, and IgA in SCID mice.Values given are the average of two experiments. Open circles represent the first phase (distribution phase) and filled circles represent the biological half-life (second phase). (G) Serum human IgG subclass concentrations in sera obtained from individual SCID mice 8 weeks after reconstitution with human PBMC from four different donors. Proportion of serum IgG subclasses in the donors are shown in the upper part of the figure. (H) Human IgG subclass in mouse No. 2 from each donor (Fig. la ) 4,8, and 12 weeks after reconstitution.
Eur. J. Immunol. 1992. 22: 823-828
Human Ig in reconstituted SCID mice
Becton Dickinson. A FITC-conjugated rabbit anti-mouse Ig (Dakopatts) was used with mAb against human MHC class I (W6/32, Seralab, Crawley Down, GB) in an indirect technique. In addition, mAb against human CD20 (L26) and CD2 (MT910, Dakopatts) were used in immunohistochemical analysis of cytospins.
825
seropositive donors that showed very high IgM levels (Table 1). Reconstitution of animals with only 5 x lo6 PBMC resulted in significantly lower Ig levels than expected as compared to 20 X lo6 (not shown).When SCID sera were analyzed for the presence of mouse Ig, 5 g/l. Human Ig was quantified by ELISA at 1, 3, 8, 18, and 30 h, as well as 2, 4,7, 14, and 21 days and half-lives calculated as previously described [ 141.
In irradiated mice a tendency to a faster and higher IgG production early after reconstitution was seen compared to non-irradiated mice (Fig. 1A). However, at 4 weeks, there was no significant difference in the IgG levels. In fact, the IgG production in irradiated mice seemed to decrease more rapidly and the IgM and IgA production was constantly lower (Fig. 1B and C).
3 Results 3.1 Human Ig class levels in reconstituted SCID mice Human Ig was detected in the serum of reconstituted SCID mice already 1week after reconstitution. The highest levels of human IgG, as well as of IgM and IgA, were obtained at 8-12 weeks post-reconstitution (Fig. 1). The serum IgG levels reached 1-4 mg/ml (approximately 10 YO-40% of the normal human serum concentration), while IgM and IgA levels, at their peaks, were usually
