Use of severe combined immunodeficient mice to measure developmental potential of B-cell precursors

MODELS OF LYMPHOID AND MYELOID RECONSTRUCTION IN SCID MICE and differentiation of human leukaemic cells. If the model can be extended to allow the development of large numbers of functional human B and T cells, it will become the model of choice. A major advance in the development of a human immune system or human haematopoietic system in SCID mice was the recent observation by several groups that manipulations to reduce the non-specific immune system in SCID mice markedly improves the engraftment of human cells. Thus, treatment of SCID recipients with anti-asialo-GM1 antibodies or transfer of the scid gene onto the NOD background both reduce the NK and macrophage activities, making the modified SCID mice much better recipients for human cells. Interestingly, the NOD SCID recipients appear to require the addition of less growth factor than for normal SCID recipients. The d e v e l o p m e n t o f various SCID models of human disease has opened the door for experimentation that was previously impossible. Although the S C I D m o u s e will u n d o u b t e d l y be r e p l a c e d by

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"designer" mice carrying a mixture of deleted genes and transgenes to produce specific desired phenotypes, the original SCID mouse was an unprecedented advance in x e n o t r a n s p l a n t a t i o n and has played a major role in the current development of new models. The papers in the SCID Forum will address several aspects of current models, including the strengths and weaknesses of each. Hopefully, this discussion will help the reader evaluate the current SCID models.

J. Reimann Institut fiir Mikrobiologie der Universitiit Ulm Albert-Einstein-Allee 11 D-89069 Ulm (Donau) (Germany) and R.A. Phillips Hospital for Sick Children hnmunology and Cancer Research 555 University Avenue Toronto, Ontario M5G 1X8 (Canada)

Use of severe combined immunodeficient mice to measure developmental potential of B-cell precursors E. M o n t e c i n o - R o d r i g u e z and K. D o r s h k i n d

Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121 (USA)

The SCID mouse has been used to measure the lymphoid developmental potential of haemopoietic cells, but the use of sophisticated in vitro culture systems may allow a more sensitive means to examine primary B-cell development. However, the SCID mouse still is an excellent recipient in which secondary differentiation events and regulatory controls operative during lymphopoiesis can be studied. The bone marrow is the site of primary B-lymphocyte development during postnatal life. Immature h a e m a t o p o i e t i c p r e c u r s o r s in that t i s s u e progress through a series of differentiative and proliferative steps that culminate in the production of virgin B cells (Osmond, 1986; Kincade, 1987). There is considerable interest in identifying and

isolating immature B-cell precursors in order to characterize them and define microenvironmental elements influencing their growth and development (Faust et al., 1993; Hardy et al., 1991). These studies depend upon the availability of assay systems that measure the developmental potential of putative p r o g e n i t o r populations. T r a d i t i o n a l l y , the only means to accomplish this was to inject the cells being analysed into syngeneic or congenic recipients. This approach has two important limitations. First, appropriate strains must be selected so that donor cells can be distinguished from those of the host. Second, recipient mice must be preconditioned with irradiation in order for efficient engraftment to occur. Survival of such animals depends on repopulation by cells that confer short-term radioprotection,

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and this is .not provided by purified lymphoid progenitors.

tify recipients that exhibit repopulation, and carrier cells must be coinjected to provide short-term radioprotection (Smith et al., 1991). The in vitro systems are sensitive enough in theory to analyse the developmental potential of a single progenitor (MullerSieburg et al., 1986). A final important advantage of using cloned stroma is that all cells that develop derive from the donor population.

The severe combined immunodeficient (SCID) mouse has a specific DNA repair defect that impairs the rearrangement of antigen receptor genes and development of mature T and B cells (Fulop and Phillips, 1990; Bosma and Carroll, 1991). This deficiency does not affect myelopoiesis, and the ability to efficiently engraft l y m p h o i d precursors in an apparently normal bone marrow microenvironment makes the S C I D mouse an appropriate model in which to study B-lymphoid development (Dorshkind, 1991). Preconditioning of SCID mice prior to transplantation of donor cells with low dose irradiation facilitates e n g r a f t m e n t (Fulop and Phillips, 1986). However, even in the latter instance, the sublethal dose administered does not affect short term survival of the mice. B-cell repopulation in SCID m o u s e r e c i p i e n t s is p r i m a r i l y d e r i v e d f r o m the grafted cells. However, since approximately 10-20 % of S C I D mice can b e c o m e leaky ( B o s m a et al., 1988) it is prudent to confirm the donor origin of the repopulating cells. This is particularly critical when low levels of repopulation are being measured.

These same arguments apply when studying the B-cell developmental potential of human haematopoietic stem cells. A rare (0.05-0.1%) population of human Thy-1 ÷ CD34 ÷ bone marrow cells highly enriched for candidate haematopoietic stem cells has been isolated and SCID mice were used to measure their B-cell developmental potential. In some experiments, the lymphoid developmental potential of the highly purified human cells was tested by microinjecting them into human long bone fragments that were implanted into SCID mice (Baum et al., 1992). However, in vitro culture systems may represent a simpler alternative to study human B-cell differentiation. F o l l o w i n g seeding of the Thy-1 ÷ C D 3 4 + human cells onto a cloned murine stromal cell line, B cells were generated (Baum et al., 1992).

A) Analysis of primary lymphoid development/n

B) Analysis of secondary B-cell development in

vitro

vivo

The development of in vitro cultures in which p r i m a r y B-cell d i f f e r e n t i a t i o n can be m e a s u r e d makes it appropriate to ask whether or not these systems offer advantages over the use of SCID or other mouse strains. Indeed, most aspects of B-cell development can be duplicated in vitro using modifications of the culture conditions originally defined by Whitlock and Witte (Whitiock and Witte, 1982; Dorshkind and Witte, 1987; Kincade et al., 1989), and several laboratories have cloned bone marrow stromal cells that provide the conditions required to support the maturation of early B-cell precursors into B cells (Dorshkind, 1990).

While in vitro systems can be used to measure primary B-cell differentiation, they do not allow the assessment of how systemic influences might affect that process. Furthermore, the culture systems do not provide a reliable means to analyse normal and pathologic secondary B-cell differentiation events. It is in these instance that the SCID mouse has proven to be of particular value.

If the main experimental aim is to measure the ability of a particular murine progenitor to generate B lineage cells, then several considerations support the claim that in vitro systems are more efficient than in vivo repopulation studies in SCID mice or any other strain. First, there is evidence that pluripotent stem cells are maintained under long-term culture conditions (Wineman et al., 1993) and that they can develop into B lineage cells following culture over stromal cells in vitro (Muller-Sieburg et al., 1986). Second, numbers of early progenitors are often limiting, in which case their homing to appropriate niches in the bone marrow following injection may be an issue. While clonal analysis of haematopoietic stem cell differentiation in vivo has been reported, multiple mice must be examined to iden-

The characterization of cells present in the longterm B-cell cultures described by W h i t l o c k and Witte (1982) provides an e x a m p l e of how S C I D mice can be used to measure normal, secondary Bcell development. Although surface IgM expressing lymphocytes and their progenitors are present in the cultures, the failure to demonstrate the capacity of the cells to secrete immunoglobulin and proliferate in response to mitogenic stimulation raised questions about their functional potential (Dorshkind et al., 1986). Following reconstitution of SCID mice with cultured cells, surface IgM-expressing lymphocytes were detected in the spleen, and immunization with a T-cell-independent antigen resulted in secretion of immunoglobulins of multiple isotypes. These findings indicated that cells from the cultures could reconstitute normal humoral i m m u n i t y in S C I D mice (Dorshkind et al., 1986). While normal events were the focus of the a b o v e analysis, additional studies on repopulation of S C I D mice with bone marrow cells from NZB mice demonstrated that B-

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cell abnormalities, such as autoantibody secretion, can also be measured ( D o r s h k i n d et al., 1989). Taken together, these studies demonstrate that the SCID mouse provides an ideal model system in which secondary B-cell differentiation events can be analysed.

IN SCID MICE

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progenitors due to potential leakiness of the recipients. Whether or not mice in which the recombinase activating gene (Rag) has been deleted will circumvent this complication remains to be tested (Mombaerts et al., 1992; Shinkai et al., 1992).

References C) T-cell development Although this discussion has focused on B lymphopoiesis, it is appropriate to comment on the use of the SCID mouse for studies of T-cell development. h~ vitro systems that measure T-cell development, comparable to those described for B lymphopoiesis, have not been developed. Thus, T-cell development has primarily been studied in vivo. The SCID mouse has been useful for the study of human T-cell differentiation (McCune et al., 1988): for example, T-cell production results following injection of human T cell progenitors into human thymic fragments which are grafted under the kidney capsule of SCID mice. For murine studies of T-cell differentiation, the specific experimental question addressed dictates whether or not SCID mice are the appropriate model. If immature bone marrow cells are the source of reconstituting cells, then SCID mice are useful recipients because intravenous injection results in colonization of their thymus by T-cell precursors and initiation of T lymphopoiesis. However, characterization of intrathymic precursors necessitates that cells be reinjected into a thymus in w h i c h all m i c r o e n v i r o n m e n t a l c o m p o n e n t s are present (Wu et al., 1991). The small size of the SCID thymus makes intrathymic injection technically difficult, and microenvironmental components necessary to support d e v e l o p m e n t of particular thymic precursors may be absent (Shores et al., 1991 ). Therefore, studies of murine T lymphopoiesis will continue to rely on the use of normal mice in which donor and recipient cells can be distinguished on the basis of allotypic markers.

D) Conclusion The relative facility by which the B-cell developmental pathway can be duplicated in vitro has obviated the need to inject candidate progenitor cell populations in vivo. However, there are clear instances when the SCID mouse is the appropriate model. If the aim of the experimental question is to address homing potential of cells, the ability of isolated haemopoietic progenitors to stably repopulate lymphopoiesis, or various normal and pathologic secondary differentiation events, the SCID mouse is a valuable tool for studies of human and murine cells. However, it is important to confirm the donor origin of cells following repopulation with murine lymphoid

Baum, C.M., Weissman, I.L., Tsukamoto, A.S., Buckle, A.M. & Peault, B. (1992), Isolation of a candidate human hematopoietic stem-cell population. Proc. Natl. Acad. Sci. USA, 89, 2804-2808. Bosma, M.J. & Carroll, A.M. (1991), The SCID mouse mutant: definition, characterization, and potential uses. Ann. Rev. ImmunoL, 9, 323-350. Bosma, G.C., Fried, M., Custer, R.P., Carroll, A., Gilson, D.M. & Bosma, M.J. (1988), Evidence of finding lymphocytes in some (leaky) .SCID mice. J. Exp. Med., 167, 1016-1033. Dorshkind, K. (1990), Regulation of hemopoiesis by bone marrow stromal cells and their products. Ann. Rev. Immunol., 8, 111-137. Dorshkind, K. (1991), The severe combined immunodeficient (SCID) mouse, in "Immunological Disorders in Mice" (B. Rihova & V. Vetvicka) (pp. 1-21). CRC Press, Boca Raton, FL. Dorshkind, K., Denis, K.A. & Witte, O.N. (1986), Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice. J. bnmunol., 137, 3457-3463. Dorshkind, K. & Witte, O.N. (1987), Long-term murine hemopoietic cultures as model systems for analysis of B lymphocyte differentiation. Curr. Top. Microbiol. lmmunol., 135, 24-41. Dorshkind, K., Yoshida, S. & Gershwin, M.E. (1989), Bone marrow cells from young and old New Zealand black mice can reconstitute B lymphocytes in severe combined immunodeficient recipients. J. Autoimmun., 2, 173-186. Faust, E.A., Saffran, D.C., Toksoz, D., Williams, D.A. & Witte, O.N. (1993), Distinctive growth requirements and gene expression patterns distinguish progenitor B cells from pre-B cells. J. Exp. Med., 177, 915-923. Fulop, G.M. & Phillips, R.A. (1986), Full reconstitution of the immune deficiency in SCID mice with normal stem cells required low-dose irradiation of the recipients. J. Immunol., 136, 4438-4443. Fulop, G.M. & Phillips, R.A. (1990), The SCID mutation in mice causes a general defect in DNA repair. Nature (Lond.), 347, 479-482. Hardy, R.R., Carmack, E.E., Shinton, S.A., Kemp, J.D. & Hayakawa, K. (1991), Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med., 173, 1213-1225. Kincade, P.W. (1987), Experimental models for understanding B lymphocyte formation. Adv. lmmunol.. 41, 181-267. Kincade, P.W., Lee, G., Pietrangeli, C.E., Hayashi, S.L. & Gimble, J.M. (1989), Cells and molecules that regulate B lymphopoiesis in bone marrow. Ann. Rev. lmmunol., 7, 111-143.

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McCune, J'.M., Namikawa, R., Kaneshima, H., Shultz, L.D., Lieberman, M. & Weissman, I.L. (1988), The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science, 241, 1632-1639. Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S., Papaionannou, V.C. et al. (1992), RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68, 869-877. Muller-Sieburg, C.E., Whitlock, C.A. & Weissman, I.L. (1986), Isolation of two early B lymphocyte progenitors from mouse marrow: a committed pre-pre-B cell and a clonogenic Thy-I 1o hematopoietic stem cell. Cell. 44, 653-662. Osmond, D.G. (1986), Population dynamics of bone marrow B lymphocytes, hnmunol. Rev., 93. 103-124. Shinkai, Y., Rathburn, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charmin, J., Rath, M., Young, F., Stall, A.M. et al. (1992), RAG-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell, 68, 858-867.

Shores, E.W., Van Ewijk, W. & Singer, A. (1991), Disorganization and restoration of thymic medullary epithelial cells in T cell receptor-negative SCID mice: evidence that receptor-bearing lymphocytes influence maturation of the thymic microenvironment. Eur. J. bnmunol., 21, 1657-1661. Smith, L.G., Weissman, I.L. & Heimfeld, S. (1991), Clonal analysis of hematopoietic stem-cell differentiation in vivo. Proc. Natl. AcYM. Sci. USA, 88. 2788-2792. Wineman, J.P., Nishikawa, S. & Muller-Sieburg, C.E. (1993), Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit. Blood, 81,365-372. Whitlock, C.A. & Witte, O.N. (1982), Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc. Natl. Acad. Sci. USA, 79, 36083612. Wu, L., Antica, M., Johnson, G.R., Scollay, R. & Shortman, K. (1991), Developmental potential of the earliest precursor cells from the adult mouse thymus. J. Exp. Med., 174, 1617-1627.

Fate of T and B cells transferred to SCID mice J. Sprent, C.D. Surh and D. T o u g h Department o f hnmunology, IMM4, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037 (USA)

Tracing the fate of lymphocytes and stem cells after transfer to SCID mice is a useful approach for determining the potential lifespan of T and B cells and their precursors. A summary of our recent work in this area is given below.

Stem cell reconstitution of S C I D mice It is well established that the immunodeficiency in SCID mice can be overcome by reconstitution with normal bone marrow (BM) or foetal liver (FL) cells (Bosma and Carrol, 1991). According to the literature, stem cell reconstitution of SC1D mice is poor unless the mice are conditioned with irradiation (Fulop and Phillips, 1986). In our experience, however, SCID mice can be reconstituted without prior irradiation. In fact, untreated C.B-17 SCID mice from our colony can be completely reconstituted in

terms o f T- and B-cell function with as few as 3 × 105 T-depleted BALB/c BM cells (Sprent et al., 1991 ). Although reconstitution is minimal within the first 5 weeks post-transfer, by 8 weeks, the thymus is near-normal size, and the lymphoid organs contain large numbers of functional T and B cells. The efficient reconstitution we see in unirradiated SCID mice may reflect that our SCID colony is unusually "clean" (though it is now less clean than it was). Stem cell reconstitution of unirradiated adult SCID mice only applies to H-2-compatible stem cells. To obtain engraftment of H-2-incompatible stem cells, prior irradiation is essential. Provided that the conditioning dose of irradiation is kept below 400 cGy, SCID mice reconstituted with allogeneic BM or FL cells survive well in our hands and show no signs of graft-versus-host disease (GVHD). Interestingly, this is not the case when irradiated SCID mice are reconstituted with xenogeneic rat