The C.B.17 scid mouse strain as a model for human disseminated leukaemia and myeloma in vivo

Pergamon

0145-2126(94)E0024-4

Leukemia Research Vol. 18, No. 7, pp. 513-522, 1994 Copyright © 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0145-212~/94 $7.(10 + 0.00

THE C.B.17 SCID MOUSE STRAIN AS A MODEL FOR HUMAN DISSEMINATED LEUKAEMIA AND MYELOMA IN VIVO

ALEX R. CATTAN and ELIZABETH DOUGLAS

Department of Haematology, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, U.K.

(Received 18 August 1993. Revision accepted 29 December 1993)

Abstract--Using the C.B.17 scid mouse strain, we have developed a model of disseminated leukaemia and myeloma using five human cell lines, CCRF-Cem, Molt-4, Raji, IM9 and HS- Sultan. Introduction of any of these cell lines by either an intravenous or an intraperitoneal route eventually kills the mouse due to leukaemia or myeloma cell load. Neoplastic cells can be found in the blood, liver and bone marrow. Intraperitoneal transfer produces a local solid tumour whereas intravenous transfer produces foci of neoplastic cells in the spine and brain. A single dose of melphalan is able to increase survival time from infection of a lethal dose of the T-cell leukaemia cell line, CCRF-Cem.

Key words: Chemotherapy, leukaemia, myeloma, scid.

Introduction

DURING INVESTIGATIONS into immunoglobulin levels in congenic inbred mice (Balb/c.C57BL/La), Bosma et al. [1] noted four littermates with low level IgG production in the C.B.17 strain. These mice were crossed and backcrossed to produce the homozygous congenic inbred strain, C.B.17 scid. Although this strain is not A D A (adenosine deaminase) deficient [11 it has a similar phenotype to the disease of severe combined immunodeficiency (scid) seen in man where there is no quantifiable (or extremely small) mature T- or B-cell compartment. The deficiency seen in mice although similar to the human disorder, has some differences at the DNA level [2].

In children with the scid disorder, where a suitable donor exists, an allogenic bone marrow transplant is performed after depletion of mature T-cells to reduce graft vs host disease (GVHD). It is a peculiarity of scid mice that it is often possible to transfer large numbers of allogenic mature T-cells without causing GVHD although GVHD occasionally may

Abbreviations: A DA, adenosine deaminase; ALL, acute lymphoblastic leukaemia; EBV, Epstein-Barr virus; vIFN, interferon gamma: GVHD, graft vs host disease; i.p., intraperitoneally; i.v., intravenously; LAK, lymphocyte activated killer; scid, severe combined immunodeficiency disease.

Correspondence to: Alex R, Cattan, Department of Haematology, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, U.K.

513

occur [3]. Transfer of human blood lymphocytes often results in the development of Epstein-Barr virus (EBV) driven lymphoblastoid tumours after a latency period of 10 or more weeks [3]. Although this could suggest a change in T-cell control it may also be that T-cells are not being renewed or that they are anergic [4]. The literature is confused as to how normal T- and B-cells are in scid mice as there are conflicting reports that transferred cells have a normal [5] or an abnormal phenotype [6].

There have been several attempts to produce haemopoietic human/scid mouse chimeras. At first, attempts were made to transfer bone marrow cells directly to mice with only transient success [7]. Nami- kawa et al. [8] have produced chimeric mice by the implantation of human foetal liver and thymus under the kidney capsule and recently Kyoizumi et al. [9] have extended this by using fragments of foetal bone only.

Recently, some groups have reported the successful use of immunotoxins with T- and B-cell models of human leukaemia [10-12] and one group has reported the prior treatment of a human B- lymphoma cell line with yIFN and human lymphocyte activated killer cells (LAK) [13] with some efficacy in scid mice. There have also been reports of the use of a single agent [10, 12, 14] and dual agent [15] chemotherapy using an immunotoxin plus cyclophosphamide to treat scid mice with disseminated leukaemia after the transfer of human cell lines,

514 A . R . CATTAN and E. DOUGLAS

As part of our studies into drug resistance development in leukaemia [16], we wished to develop in vit,o models to investigate the mechanisms of drug resistance and as models for the use of novel drug therapeutic protocols for leukaemia and myeloma. In this article, we report our findings using five human cell lines to develop a model of disseminated leukaemia and myeloma in scid mice. We also show that survival time after transfer of one of the cell lines can be increased by the use of a single dose of the alkylating cytotoxic agent, melphalan.

Materials and Methods

Cell lines Molt-4 (TdT +, CD5 +) and CCRF-Cem (CD5 +) are T-

lymphoblastoid cell lines derived from two patients with T-ALL. Raji (CD10 +, CD19 +) is an EBNA + lymphoblast- like cell line derived from a patient with Burkitt's lymphoma. IM9 (EBNA +) and HS-Sultan (IgGk, CD10 ÷, CD19 +, EBNA +) are cell lines derived from patients with multiple myeloma with HS-Sultan being a true plasma cell. All cell lines were obtained from the American Tissue Culture Collection repository. These cell lines were reseeded periodically from frozen stocks of the original received line.

Scid mice A breeding nucleus of 12 pairs was obtained from the

MRC, Mill Hill, London and maintained in a positive pressure, HEPA filtered isolator (Moredon Ltd, Edinburgh). Subsequent stock havc been maintained by brother/sister mating. A second isolator was used to main- tain the experimental mice. Both groups of mice were given autoclaved water and irradiated food ad libitum.

Melphalan Alkeran (Wellcome) was provided by Cytotoxic Phar-

macy, Royal Victoria Infirmary, Newcastle upon Tyne diluted according to the manufacturer's instructions at 9.2 mg/ml. This was further diluted to the concentrations indicated in the text in phosphate buffered saline containing 0.5% human serum albumin. All dilutions were kept on ice and were used within 1 h of primary dilution.

Immunoglobulin levels All mice, prior to entering the study, were checked for

serum IgM and IgG levels using a modified catchment ELISA developed in this department. Briefly, ELISA plates (Dynatech) were coated with rabbit anti-mouse IgM (Dako) or goat anti-mouse IgG (Pierce Labs), blocked with gelatin, followed by dilutions of mouse serum then rabbit anti-mouse IgM (Dako) or goat anti-mouse IgG (Pierce) coupled with horse radish peroxidase and finally visualized using the chromogen, O-phenyldiamine (Sigma). The assay has a lower level of sensitivity of 10 ng/ ml. Only mice with less than or equal to 1 ~tg/ml IgM entered the study.

Tissues Routine blood smears were made by taking a small drop

of blood from the tip of the tail. Other tissues (usually spleen, liver, kidney, bone marrow pooled from both femurs and blood by cardiac puncture) were taken at the time of death and a single cell suspension made for further analysis. Other tissues (for example, testes, heart, lungs and lymph nodes) were taken if they appeared to be involved during macroscopic examination. Blood was sep- arated using the micro-technique described by Potter & Potter [17]. Cytospins and smears were stained using Wright's stain. Sections (3 ~tm thick) were cut from for- malin-fixed, paraffin-embedded blocks of mouse tissues and stained with haematoxylin-eosin.

Flow cytometry Cell suspensions (2 x 107/ml) were stained using anti-

CD5, CD10 or CD19 (Becton Dickinson) and analysed using a Becton Dickinson FACScan following the manufacturer's instructions.

lmmunofluorescence Cytospin preparations of cell suspensions (1 x 105/slide)

were fixed with 5% (v/v) acetic acid in methanol and stained directly using fluoroscein labelled (Fab')z fragments of goat anti-isotype or light chain antisera (Kallestadt).

Results

Transfer of 1 × 107 cells of any of the five cell lines caused mortality due to leukaemia, independent of the sex of the host mouse, in 100% of the mice within

TABLE 1. CELL LINE, INJECTION ROUTE AND SURVIVAL TIME WITH TRANSFER OF 1 × 10 7 CELLS

Experiment Cell line Route Mortality range (days) Mice surviving/Total in group

1 CCRF-Cem i.v. 28-42 0/6 2 CCRF-Cem i.p. 28-46 0/6 3 Molt-4 i.v. 37-41 0/8 4 Molt-4 i.p. 49--60 0/4 5 Raji i.v. 17-19 0/7 6 IM9 i.v. 26-33 0/7 7 HS-Sultan i.v. 24-29 0/3

Cells (100 Ixl) were inoculated by the route indicated and mice were culled according to standard Home Office procedure.

i.v., intravenously; i.p., intraperitoneally.

Disseminated leukaemia in scid mice 515

17-60 days depending on the cell line transferred (Table 1; Fig. 1). If cells were transferred to mice with high levels of serum IgM (>1 ~g/ml), leukaemia developed in an unreliable fashion with on occasions mice receiving the high cell doses (1 x 107) showing no clinical symptoms of disease (data not shown). Where the two T-cell lines used were transferred to the mouse by an intraperitoneal route, vascularized solid tumours were also induced in these mice in the peritoneal cavity (Table 2).

The tissue distribution of the leukaemic cells was similar, independent of route of transfer, however, where tested intraperitoneal transfer of the cell lines did not produce spinal or brain foci of leukaemic cells and was never shown to induce hind leg paralysis (Table 2). In contrast, intravenous transfer of the cell lines routinely produces hind leg paralysis and invasion of the spine and brain without inducing solid tumour formation except with the two plasma cell

lines (Table 2; Fig. 1). Although the tissue distribution is generally similar using both routes of transfer, lower cell numbers were required to give a similar tumour load when cells were transferred intravenously compared with intraperitoneal transfer

(data not shown). When cells isolated from tissues of mice dying after inoculation with three of the cell lines (CCRF-Cem, Raji, HS-Sultan), were typed for CD antigen expression by flow cytometry and for cytoplastic immunoglobulins (HS-Sultan), they showed a similar pattern of expression of these markers as the parent cell lines (Tables 3 and 4). Some of these cell suspensions were seeded into tissue culture medium and allowed to establish as tissue culture populations. When these cells were checked for CD antigen expression and by morphology, they had a similar phenotype to the parent cell lines used to inoculate the mice from which the cells had been isolated (Table 3).

TABLE 2. TISSUE DISTRIBUTION OF TttE LEUKAEMIC CELL LINES

Mouse tissues involved Cell line Route Blood Spleen Liver Bone marrow Solid tumour Miscellaneous

CCRF-Cem i.v. + + + + - Brain, spine, P i.p. + + + + +

Molt-4 i.v. + + + + - Brain, spine, P i .p. + + + nd +

IM9 i.v. + + + + + P Raji i.v. + + + + - Ascites, brain, spine, P HS-Sultan i.v. + + + + + Ascites, kidney

Mice were inoculated with 1 x 10 7 (100 gl) of the cell lines and by the route indicated. A positive sign indicates in the case of a solid tumour that one was present or in the other tissues that there were >5% tumour cells by differential count. In brain and spine sections neoplastic cells were present (see also Fig. 1). nd, not done: P, paralysis in hind legs.

TABLE 3. IMMUNOPHENOTYPING BY FLOW CYTOMETRY OF

TUMOUR CELLS HARVESTED FROM SCID MOUSE TISSUES,

PRE- AND POST TISSUE CULTURE

CCRF-Cem Raji HS-Sultan (%CD5) (%CD19) (%CD10)

Cell source Pre Post Pre Post Pre Post

Ascites . . . . 46 87 Blood 20 >95 2 94 < 1 nd Bone marrow 31 74 57 >95 2 nd Kidney < 1 nd 5 89 nd nd Liver 93 nd 5 92 18 >95 Spleen 75 68 91 >95 < 1 nd

Immunophenotyping by flow cytometry of tumour cells harvested from scid mouse tissues, pre- and post tissue culture. Single cell suspensions made from the excised tissues from mice injected with the cell lines as indicated, were labelled for expression of CDS, CD10 or CD19. nd, not done; - , no ascites present.

TABLE 4. PHENOTYPE OF CELLS HARVESTED FROM A

MOUSE INOCULATED WITH HS-SULTAN

Percentage of cells labelled Cell source kappa lambda gamma morphology

Ascites 90 < 1 nd >95 Blood 15 <1 nd nd* Bone marrow 5 < 1 nd 9 Kidneyt 35 < 1 30 60 Liver~ 25 < 1 20 51 Spleen 9 < 1 6 7

Phenotype of cells harvested from a mouse inoculated with HS-Sultan. Tissues were excised from mice, labelled with fluorescein conjugated (Fab')2 fragments of goat anti- human A, i¢ or y antisera and the percentage of cells labelled determined. The same cell populations were stained (Wright's) and the percentage blast cells determined.

* Too few cells for morphology. ? A tumour focus was excised from these tissues.

nd, not done.

516 A, R. CATI'AN and E. DOUGLAS

FIG. 1.


FIG, 1. Cont inued .

518 A, R. CATrAN and E. DOUGLAS

irn

IT

FIG. 1. (a) Peripheral blood cytospin from a mouse with the T-cell leukaemia, Molt-4 showing two blast cells and three mouse neutrophils, stained with Wright's: bar = 10 ~tm. (b) Cytospin preparation of ascites from a mouse with the lymphoblastoid leukaemia, Raji, stained with Wright's; bar = 15 gm. (c) Peripheral blood cytospin from a mouse with the plasma cell leukaemia, HS-Sultan, showing six blast cells, stained with Wright's; bar = 20 ~tm. (d) A 3 gm paraffin-embedded section of the frontal lobe of brain taken from a mouse with the T-cell leukaemia, CCRF-Cem, showing a turnout focal point (T) stained with haematoxylin-eosin: bar = 100 ~tm. (e) A 3 mm paraffin-embedded, oblique-transverse section of the spinal column taken from a mouse with the T-cell leukaemia, CCRF-Cem, showing massive marrow tumour infiltration stained with haematoxylin-eosin: bar = 100 #m. (f) Flow cytometric analysis of tissues taken from a mouse with the T-cell leukaemia, CCRF-Cem, using CD5 or an isotype matched control; PBMC, peripheral blood mononuclear cells; BM, femur

bone marrow cells; all tissues were prepared as a single cell suspension.

Disseminated leukaemia in scid mice

TABLE 5. SEX DIFFERENCES IN MORTALITY WITH CELL LINE AND DOSE

519

Experiment Cell line Mouse sex Cell dose (×105 i.v.) Mortality range (days) Mice surviving/Total in group

la CCRF-Cem M 100 28-38 0/4 V 38 42 0/2

lb M 10 42 0/2 F 42-50 0/2

lc M 1 49 0/2 F 46-62 0/2

ld M 0.1 51 61 0/3 F >82 2/2

2a Molt-4 M l(/0 37 41 (1/4 F 39-4l 0/4

2b M 10 37-47 (1/4 F nd nd

2c M 1 49-58 0/4 F 97 >1(14 2/3

2d M 0.1 45-79 0/4 F > 104 3/3

3a Raji M 100 19 0/3 F 17-19 0/4

3b M ll) 18-21 0/2 F 19-32 0/2

3c M 1 21 0/2 F s-22 0/2

3d M 0.1 24-26 0/2 F 32 0/2

4 IM9 Only 1 x 107 cells transferred in both sexes, see Table 1

5a HS-Sultan M 100 29 0//2 F 24 0/1

5b M 10 23-29 0/2 F 26-29 0/2

5c M l 29 0/2 F 36 0/1

5d M 0.1 38 >63 1/2 F >63 2/2

Male (M) and female (F) mice were inoculated i.v. with 100 ~tl of the cell type and number as indicated.

In all the cell lines tested, there is a sex difference in mortality compared with cell dose with male mice being generally more susceptible to death with lower numbers of cells transferred (Table 5). This is most marked when Molt-4 cells are used with a 2 log- arithmic difference in the lethal dose. The least difference is seen when Raji cells are used which also cause the most rapid onset of leukaemic death in both sexes.

When melphalan was used as a single agent, it was able to prolong the survival of mice after an intravenous transfer of a lethal dose of CCRF-Cem cells (Table 6). This survival was dependent on cytotoxic drug concentration. High doses of melphalan resulted in toxic death of the mice which was dependent on the route of drug administration. At the end of the experiment, the surviving mice were culled. In all these mice using morphology and flow cyto- merry, no leukaemic cells were found in the peripheral blood ( <1% CD5), however, leukaemic cells

were found in small numbers in the spleen (2-5% CD5) and in leukaemic foci in the liver. Spleens were not particularly enlarged nor did the mice show any obvious clinical signs at this stage (ruffled fur, hypo- thermia, hind leg paralysis) which were found both in control mice and in those dying from the disease with the lower doses of melphalan.

Discussion

Although several groups have reported the growth of primary leukaemic cells in scid mice [18-21], the general ability of scid mice to support the outgrowth of these tumour cell populations is restricted. Often other cellular components such as skin [22] or bone marrow matrix [20] are required to support the growth of these tumour cell populations, arguing that other components such as adhesion molecules and/or growth factors play a supportive role. Since primary tumour populations are often contaminated with

520 A. R. CAa-rAN and E. DOUGLAS

TABLE 6. SURVIVAL OF MICE INJECTED INTRAVENOUSLY WITH C C R F - C E M AND TREATED WITH MELPHALAN

Group Melphalan dose (~tg) Drug route Mortality (days) Mice surviving/Total in group

a 300 i.v. 12, 13" 0/2 b 300 oral >84 2/2 c 150 i.v. >84 2/2 d 150 oral >84 2/2 e 75.50 i.v. >84 2/2 f 75.50 oral 33, 70 0/2 g 37.75 i.v. 56, 65 0/2 h 37.75 oral 56, 65 0/2 i 0 i.v. 33, 69 0/2 k 0 oral 35, 69 0/2

Female mice were injected i.v. with 1 × 106 CCRF-Cem cells on day 0 and treated days later by the route and doses indicated. Melphalan was prepared as outlined in the

* Toxic death.

with melphalan 3 Methods section.

other cell types, EBV-driven lymphoproliferation, particularly in the absence of T-cell mediated sup- pression, frequently results in the outgrowth of a non-desired tumour phenotype. This is especially relevant when peripheral blood is used as a source of material for transfer to scid mice [23, 24]. Others have reported that natural killer cells which are of normal levels in scid mice [25], may influence the outgrowth of transferred human cells such that many groups use pretreatment of the mice with radiation [26] or cyclophosphamide [21] in an attempt to make the mouse more susceptible to in vivo turnout pro- liferation. As a consequence although the scid mouse offers an attractive model for the study of both normal and abnormal haemopoiesis, there are several practical difficulties, not least the inability of these mice to support many primary tumour populations (vide supra). For these reasons, we believe that a reliable model capable of supporting disseminated human haemopoietic tumour cell lines offers an appealing alternative for the study of biological par- ameters in vivo, for example, drug pharmacok- inetics, novel drug combinations and the role of natural killer cells in an immuno-incompetent environment.

The initial breeding nucleus of mice showed high levels of IgM. It is unknown if these mice were pauciclonal for immunoglobulin as has been reported by others [27]. Since scid mice may become spon- taneously "leaky" for immunoglobulin and it is not possible to breed in this "leakiness" [27], it would seem that other factors such as stress or environ- mental, may have been responsible for these initially high levels of immunoglobulin in the serum. These mice continued to produce high levels of IgM until they were culled. During this period, we transferred human leukaemic cell lines and found that only mice with low IgM levels were capable of expansion of

these lines in vivo reliably as occasional mice with high IgM levels did not always support the tumour lines. The levels of serum IgM in leaky mice could be extremely high with some animals as a high as 1.5 mg/ml (the normal IgM level in our BALB/c colony is <500 ~tg/ml). For this reason, only mice with serum IgM of less than 1 ~tg/ml were used. Originally both serum IgM and IgG were monitored but since a high level of IgG in the absence of a high level of IgM has never been found in over a 500 individual sera measurements, IgG levels are no longer measured. Gordon et al. [28] have reported that animals with a high percentage of polymorphonuclear cells in the peripheral blood correlate with low level serum immunoglobulins in scid mice. Although this is generally true, we have found that in an occasional mouse there is unfortunately a high percentage of polymorphonuclear cells and a high level of IgM. The converse also occurs occasionally, however, false negatives are not as important as false positives. As the authors reported, this may be a useful first analysis but may only be used in con- junction with an assay for serum immunoglobulins.

Whereas disseminated leukaemia and myeloma could be induced by cells transferred either intraperitoneally or intravenously, the latter induced disease more dependably with fewer cell numbers and never induced a solid tumour when the T-cell and B-lymphoblastoid lines were used (Table 1). The myeloma lines, however, often induced a solid intraperitoneal tumour and were usually accompanied by the production of ascitic fluids irres- pective of the route of transfer used. Intravenous transfer of the cell lines routinely induces foci of neoplastic cells in the liver carrying a similar CD antigen profile and morphology to the parent cells (Tables 3 and 4, Fig. 1) which has also been reported by others [10]. Most leukaemic patients at pres-


entation are likely to have hepatosplenomegaly indi- cating neoplastic involvement in both liver and spleen [29]. In leukaemia and myeloma, it is not unusual to find intrathecal and brain involvement especially latterly in the disease, however, solid tumours in the peri toneum as have been found after intraperitoneal transfer are most unusual in human disease and would be diagnosed as a different disease. Isolates taken from any of the tissues where disease was evident were capable of establishing tissue culture lines which were identical in phenotype to the cell line transferred to the mouse (Table 3). Even though disease was not evident macroscopically, we have managed to establish cells in culture from various other tissues, such as kidney, lung, salivary glands, thymus. From the data available it is unknown whether these tissues were involved directly or if the cultured cells were isolated from circulating blood in these tissues.

At high cell numbers both female and male mice succumb to disease within the same time scale and with a similar disease profile. Within an experiment the mice were age-matched. Although there is no large difference in the survival time seen for three of the cell lines (Table 5), all three induce a slightly more rapid disease in males than in females. In the fourth line, Molt-4, disease onset and susceptibility is much greater in males than in females and shows a 2 log cell dose change in males (Table 5). Recent experiments with this cell line show that this difference is as much as 3 logs with male mice dying after transfer of 1000 cells (data not shown). It was felt because of the low number of animals that these data do not currently allow statistical analysis, however, this does warrant further research. This difference is seen if the cells are transferred intravenously or intraperitoneally although the difference is more marked if the cells are transferred intravenously. In humans, males are generally more likely to die from leukaemia and follow a more pro- gressive path of disease than females [30]. Although the reason for this difference in susceptibility in mice is unknown, we are currently investigating the role androgens may play in this model or if natural killer cells which are known to be present in scid mice, may account for this sex difference [25].

The length of survival of mice treated with a lethal dose of the T-cell leukaemia cell line, CCRF-Cem, can be increased by a single administration of the DNA alkylating agent, melphalan (Table 6). The doses of melphalan used are similar to the phar- macological doses of the drug used with leukaemic patients prior to transplantation (100~tg/mouse approximates to a dose of 3 mg/kg). The higher doses of melphalan proved to be toxic to the mice. The

cause of death was presumed to be due to lethal ablation of the bone marrow (mice died during the night and were not suitable for autopsy). The absol- ute amounts of drug delivered were dependent on the route of administration with intravenous more toxic than oral administration (Table 6). Although melphalan was able to increase the survival of the mice and protect against death due to leukaemia, at the end of the experiment, the surviving mice harboured leukaemic cells in the liver and spleen but not in the peripheral blood. In all cases there were very few leukaemic cells present but since these were in discrete foci it would seem probable that these mice would have succumbed to the disease at a later date.

In conclusion, we have shown that it is possible to produce a human disseminated T- and B-leukaemia and myeloma model in scid mice where if the cells are introduced intravenously the pattern of disease is similar to that seen in the human counterpart. This route of transfer produces lesions in the brain and spinal column of the mice as is often found in late stage leukaemia and myeloma or after prior chemotherapy in man. On the other hand, intraperitoneal cell transfer also induces solid tumours, similar to the situation when leukaemic cell lines are transferred to nude mice. Finally, we have shown that in this model it is possible to extend survival time to a T-cell leukaemia cell line using a single dose of the cytotoxic alkylating agent, melphalan. This animal model should prove invaluable for the study of new chemo- therapeutic regimens.

Acknowledgements--The support of the Tyneside Leu- kaemia Research Fund and Marrow Transplant 2000 are gratefully acknowledged. We wish also to thank Mr Rob Stewart for excellent technical assistance with the scid mouse colony, Ms Margorie Cummings for supplying melphalan, Mr Gordon Harrison for paraffin embedding and sectioning the mouse tissues, Drs Zor Maung and Penny Taylor for reviewing the manuscript and Ms Christine Redden for secretarial assistance.

References

1. Bosma G. C., Custer R. P. & Bosma M. J. (1983) A severe combined immunodeficiency mutation in the mouse. Nature 301,527.

2. Fulop G. M. & Phillips R. A. (1990) The scid mutation in mice causes a general defect in DNA repair. Nature 347, 479.

3. Mosier D. E., Gulizia R. J., Baird S. M. & Wilson D. B. (1988) Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 335, 256.

4. Tary-Lehmann M. & Saxon A. (1992) Human mature T-cells that are anergic in vivo prevail in SC1D mice reconstituted with human peripheral blood. J. exp. Med. 175, 503.

522 A.R. CATTAN and E. DOUGLAS

5. Krowka J. F., Sarin S., Namikawa R., McCune J. M. & Kaneshima H. (1991) Human T-cells in the SCID- hu mouse are phenotypically normal and functionally competent. J. Immun. 146, 3751.

6. Saxon A., Macy E., Denis K.~ Tary-Lehmann M., Witte O. & Braun J. (1991) Limited B-cell repertoire in severe combined immunodeficient mice engrafted with peripheral blood mononuclear cells derived from immunodeficient or normal humans. J. clin. Invest. 87, 658.

7. Kamel-Reid S. & Dick J. E. (1988) Engraftment of immune-deficient mice with human hematopoietic stem cells. Science 242, 1706.

8. Namikawa R., Weilbaecher K. N., Kaneshima H., Yee E. J. & McCune J. M. (1990) Long-term human haematopoieis in the SCID-hu mouse. J. exp. Med. 172, 1055.

9. Kyoizumi S., Baum C. M., Kaneshima H., McCune J. M., Yee E. J, & Namikawa R. (1992) Implantation and maintenance of functional human bone marrow in SCID-hu mice. Blood 79, 1704.

10. Jansen B., Uckun F. M., Jaszcz W. B. & Kersey J. H. (1992) Establishment of a human t(4;11) leukemia in severe combined immunodeficient mice and successful treatment using anti-CD19 (B43)-pokeweed antiviral protein immunotoxin. Cancer Res. 52, 406.

11. Uckun F. M., Chelstrom L. M., Irvin J. D., Finnegan D., Gunther R., Young J., Kuebelbeck V., Myers D. E. & Houston L. L. (1992) In vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin against BCL-1 murine B-cell leukemia. Blood 79, 2649.

12. Jansen B., Vallera D. A., Jaszcz W. B., Nguyen D. & Kersey J. H. (1992) Successful treatment of human acute T-cell leukemia in SCID mice using the anti- CD7-deglycosylated ricin A-chain immunotoxin DA7. Cancer Res. 52, 1314.

13. Schmidt-Wolf I. G. H., Negrin R. S., Kiem H.-P., Blume K. G. & Weissman I. L. (1991) Use of a SCID mouse/human lymphoma model to evaluate cytokine- induced killer cells with potent antitumor cell activity. J. exp. Med. 174, 139.

14. Ghetie M.-A., Richardson J., Tucker T., Jones D., Uhr J. W. & Vitetta E. S. (1991) Antitumor activity of Fab' and IgG-anti-CD22 immunotoxins in disseminated human B-lymphoma grown in mice with severe combined immunodeficiency disease: effect on tumor cells in extranodal sites. Cancer Res. 51, 5876.

15. Ucken F. M., Chelstrom L. M., Finnegan D., Tuel- Ahlgren L., Manivel C., Irvin J. D., Myers D. E. & Gunther R. (1992) Effective immunochemotherapy of CALLA +Cu + human pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency using B43 (anti-CD19) pokeweed antiviral protein immunotoxin plus cyclophosphamide. Blood 79, 3116.

16. Hall A. G. & Cattan A. R. (1991) Drug resistant mechanisms in leukaemia. In Minimal Residual Disease in Leukaemia (Proctor S. J., Ed.), p. 655. Balliere, London.

17. Potter C. G. & Potter A. C. (1988) A rapid and ultra- simplified method for separating lymphocytes from blood. J. immun. Meth. 112, 143.

18. Cesano A., O'Connor R., Lange B., Finan J., Rovera G. & Santoli D. (1991) Homing and progression pat- terns of childhood acute lymphoblastic leukemias in severe combined immunodeficiency mice. Blood 77, 2463.

19. De Lord C., Clutterbuck R., Titley J., Ormerod M., Gordon-Smith T., Millar J. & Powles R. (1991) Growth of primary human acute leukemia in severe combined immunodeficient mice. Expl Hemat. 19, 991.

20. Waller E. K., Kamel O. W., Cleary M. L., Majumdar A. S., Schick M. R., Lieberman M. & Weissman I. L. (1991) Growth of primary T-cell non-Hodgkin's lymphomata in SCID-hu mice: requirement for a human lymphoid microenvironment. Blood 78, 2650.

21. Cesano A., Hoxie J. A., Lange B., Nowell P. C., Bishop J. & Santoli D. (1992) The severe combined immunodeficient (SCID) mouse as a model for human myeloid leukemias. Oncogene 7, 827.

22. Charley M. R., Tharp M., Locker J., Deng J.-S., Goslen J. B., Mauro T., McCoy P., Abell E. & Jega- sothy B. (1990) Establishment of a human cutaneous T-cell lymphoma in C.B-17 SCID mice. J. invest. Der- mat. 94, 381.

23. Amadori A., Veronese M. L., Mazza M. R., Zamarchi R., Mion M., D'Andrea E., Menin C., Del Mistro A., Leszl A., Calderazzo F., Panozzo M. & Chieco-Bianchi L. (1992) Lymphoma induction by human B-cells in scid mice. Leukemia 6 (Suppl. 3), 23S.

24. Duchosal M. A., Eming S, A., McConahey P. J. & Dixon F. J. (1992) The hu-PBL-SCID mouse model. Long-term human serologic evolution associated with the xenogeneic transfer of human peripheral blood leukocytes into SCID mice, Cell. Immun. 139, 468.

25. Barry T. S., Jones D. M., Richter C. B. & Haynes B. F. (1991) Successful engraftment of human postnatal thymus in severe combined immune deficient (SCID) mice: differential engraftment of thymic components with irradiation versus anti-asialo GM-1 immuno- suppressive regimens. J. exp. Med. 173, 167.

26. Uckun F. M., Downing J. R., Gunther R., Chelstrom L. M., Finnegan D., Land V. J., Borowitz M. J., Carroll A. J. & Crist W. M. (1993) Human t(1;19)(q23;p13) pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency. Blood 81, 3052.

27. Bosma G. C., Fried M., Custer R. P., Carroll A., Gibson D. M. & Bosma M. J. (1988) Evidence of functional lymphocytes in some (leaky) SCID mice. J. exp. Med. 167, 1016.

28. Gordon B. E., Durfee W. J., Bennet M, & Pakes S. P. (1991) Differential white blood cell counts as a preliminary screen for severe combined immunodeficient congenic mice. Laboratory Animal Science 41, 255.

29. Poplack D. G. (1991) Clinical manifestations of acute lymphoblastic leukaemia. In Hematology: Basic Prin- ciples and Practice (Hoffman R., Benz E. J., Shattil S. J., Furie B. & Cohen H. J., Eds), p. 776. Churchill Livingston, New York.

30. Ross J. R. Y. (1993) Oxford Region Leukaemia Regis- ter. Br. J. Haemat. 84 (Suppl. 1), 37.

Documents

The C.B.17 scid mouse strain as a model for human disseminated leukaemia and myeloma in vivo