Transgenic mice deficient in natural killer cells

Details Transgenic mice deficient in natural killer cells Transgenic mice deficient in natural killer cells

1999-05-14

2003-04-15

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C435S320100, C435S325000, C435S455000

Reexamination Certificate

active

06548737

ABSTRACT:

This invention was made with Government support under National Institutes of Health Grant RO1-AI33903. The Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
The present invention relates to non-human animals which have a natural killer (NK) cell immunodeficiency but have a normal complement of other lymphocytes.
Natural killer (NK) cells were initially described on the basis of their capacity to spontaneously kill certain tumor targets (
1
). This “natural killing” ability did not require prior deliberate immunization of the host with the tumor cells. This led to the hypothesis that NK cells are involved in tumor surveillance, whereby NK cells eliminate developing cancerous cells before they become established tumors. However, this hypothesis has not been rigorously tested because of the lack of a suitable animal model. Subsequent studies have also suggested that NK cells are involved in other aspects of the immune response. Current data suggest that NK cells have the capacity to respond to certain pathogenic organisms, particularly in the earliest phases (hours to days) of the immune response (
2
). This contrasts with a more delayed response (days to weeks) in an acquired, specific immune response by B and T cells, that is dependent on clonal expansion and proliferation for specific antibody and cell-mediated responses, respectively (
3
). On the other hand, NK cells do not appear to require the physical rearrangement of antigen receptor genes, such as such as is required for B (immunoglobulin) and T cell antigen receptors, and are present in mice that lack components of the recombination machinery (
4
). They must therefore utilize other mechanisms to express a repertoire of receptor molecules that can respond to the universe of tumors or pathogens.
However, NK cells may guide the development of the acquired, specific response, by regulating isotype switching of immunoglobulin isotypes, for example (
5
). This function appears to be dependent on production of cytokines by the NK cell. Another function attributed to NK cells is their apparent capacity to reject incompatible bone marrow transplants (
6
). Moreover, NK cells are apparently involved in rejecting solid tissues, such as cardiac allotransplants (
7
). Thus, NK cells appear to be significant components of the immune system.
All studies of NK cells to date, however, have been limited by the lack of a suitable animal model in which NK cells are selectively and chronically or developmentally absent. The treatment of animals with antibodies which bind to cell surface markers is often employed to deplete cells with those markers in the treated animal. In experiments involving mice, anti-asialoGM1 or anti-NK1.1 antibodies are routinely utilized (
8
,
9
). However, those antibodies react with molecules that are expressed on other cells as well as NK cells, such that misleading information can be obtained. For example, a small population of T cells, termed NK/T (or NK1.1+T or NK1 T) cells because they express the NK1.1 antigen otherwise expressed predominantly by NK cells, have been recently shown to be capable of mediating tumor clearance and cytokine production, previously attributed only to NK cells (
10
). Furthermore, antibody administration is plagued by inherent problems, e.g., that the effect is short lived and that antibody administration may produce other effects on the immune system, such as anti-immunoglobulin formation. Thus, better models for NK cell function in vivo are required.
Immunodeficient hosts can be developed for use as animal models which avoid the problems inherent with the administration of antibodies. Such animals have been used to study various aspects of the immune response. For example, severe combined immunodeficient (scid) mice have a mutation in DNA-dependent protein kinase (DNA-PK) with concomitant abnormalities in the recombination events involved in formation of B and T cell antigen receptors (
11
). Also, X-linked (xid) mice have a defect in a protein tyrosine kinase which results in a B cell deficiency and nude (nu) mice do not have a thymus and have a defect in T cell development (
12
). All of these mice have been invaluable in the initial identification and subsequent molecular characterization of B and T cells. As importantly, they have led to an understanding of the contributions of these cells to the normal and deranged immune system. Moreover, these immunodeficient mice have been exploited to carry human cells for the purposes of creating a humanized mouse with which to study infections, tumor formation, drug therapy, and toxicities of various agents (
13
). On the other hand, these animals have NK cells that can still reject transplanted tissue. Thus, with respect to the elimination of NK cell function, these animals have limitations.
Several mouse strains have been described which contain deficiencies in NK cell function. However, all of these strains also contain other immune deficiencies. These strains have arisen by the mechanisms of spontaneous mutation, gene targeting, or transgene technology.
Spontaneous NK cell deficient mice have been reported but all contain other immune system defects. One of the first described was the beige (Bg) mouse which contains a mutation in a lysosomal transport protein (Lyst) that is not NK cell specific (
14
). Although this mouse has defects in its capacity to kill tumor targets in vitro, it also has global defects in granule formation, altering the function of other granule-containing lymphocytes. Another mouse, the “motheaten” mouse has a defect in the intracellular tyrosine phosphatase SHP-1 and abnormalities in NK cell development, but also has abnormalities in nearly all hematopoietic lineages (
15
). Thus, no mouse strain has been reported which has a spontaneous mutation that selectively affects NK cells.
Gene targeting approaches, where the function of a particular gene is eliminated, have resulted in the creation of other mouse strains that have defects in the targeted gene and in NK cells. However, regardless of whether these mice are defective in NK cell function or have a relative absence of NK cells, other aspects of the immune system are invariably and significantly altered due to mutation of the targeted gene (Table 1). Mice with a targeted deficiency in the beta chain of the IL-2 receptor fail to develop NK cells and intestinal epithelial lymphocytes (IEL), and also have defective responses to IL-2 (
16
). Mice with a targeted deficiency of the common gamma chain of the IL-2 receptor fail to develop T and NK cells (
17
). Moreover, they are broadly deficient in immune responses because the gamma chain is the signal transduction component of several cytokine receptors, including IL-2, IL-4, IL-7, and IL-15. Mice lacking the Flt-3 molecule have an NK cell deficiency but also have broad defects in hematopoiesis (
18
). Mice with a targeted deficiency in the Ikaros gene have a defect in lymphopoiesis such that all lymphocytes (B,T, NK cells) fail to develop (
19
). These mice die in the perinatal period, apparently due to infections. Mice which lack the IRF-1 transcription factor have defects in interferon responses, and hematopoiesis, especially NK, NK/T, and T cell development (
20
). Moreover, these mice die in the neonatal period. Mice with a targeted deficiency in the Ets-1 transcription factor have an NK deficiency but also have defects in thymic T cell development and T cell antigen receptor-mediated T cell activation (
21
). Moreover, these mice display an increased perinatal mortality and surviving mice die before adulthood. Mice which lack the Id2 molecule have a defect in NK cell development but still possess some peripheral NK cells with the ability to kill tumor cells (
22
). Moreover, these mice display an increased neonatal mortality and retarded growth, and fail to develop peripheral lymphoid organs such as lymph nodes and Peyer's patches. Thus, among gene targeted mice, there are no mice with an absence of NK cells with a relative sparing of other lymphocyte lineages and wi

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