Laurie Davidson

Interleukin-2 receptor α chain regulates the size and content of the peripheral lymphoid compartment

Interleukin-2 receptor alpha chain (IL-2R alpha) expression occurs at specific stages of early T and B lymphocyte development and is induced upon activation of mature lymphocytes. Young mice that lack IL-2R alpha have phenotypically normal development of T and B cells. However, as adults, these mice develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion, which, for T cells, is correlated with impaired activation-induced cell death in vivo. Older IL-2R alpha-deficient mice also develop autoimmune disorders, including hemolytic anemia and inflammatory bowel disease. Thus, IL-2R alpha is essential for regulation of both the size and content of the peripheral lymphoid compartment, probably by influencing the balance between clonal expansion and cell death following lymphocyte activation.

Defective B cell development and function in Btk-deficient mice

Mutations in the Brutons tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV

The DNA-end-joining reactions used for repair of double-strand breaks in DNA and for V (D)J recombination, the process by which immunoglobulin and T-cell antigen-receptor genes are assembled from multiple gene segments, use common factors. These factors include components of DNA-dependent protein kinase (DNA-PK), namely DNA-PKcs and the Ku heterodimer, Ku70–Ku80, and XRCC4 (ref. 1). The precise function of XRCC4 is unknown, but it interacts with DNA ligase IV. Ligase IV is one of the three known mammalian DNA ligases; however, the in vivo functions of these ligases have not been determined unequivocally. Here we show that inactivation of the ligase IV gene in mice leads to late embryonic lethality. Lymphopoiesis in these mice is blocked and V (D)J joining does not occur. Ligase IV-deficient embryonic fibroblasts also show marked sensitivity to ionizing radiation, growth defects and premature senescence. All of these phenotypic characteristics, except embryonic lethality, resemble those associated with Ku70 and Ku80 deficiencies, indicating that they may result from an impaired end-joining process that involves both Ku subunits and ligase IV. However, Ku-deficient mice are viable, so ligase IV must also be required for processes and/or in cell types in which Ku is dispensable.

Wiskott-Aldrich Syndrome Protein-Deficient Mice Reveal a Role for WASP in T but Not B Cell Activation

The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.

Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction

BACKGROUND Antigen-receptor interactions on lymphocytes result in local clustering of actin, receptors and signaling molecules into an asymmetric membrane structure termed a cap. Although actin polymerization is known to be required, the mechanisms underlying cap formation are unclear. We have studied the events underlying cap formation using mice bearing a null mutation in vav (vav-/-), a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac. RESULTS Lymphocytes from vav-/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton. The vav-/- T cells were deficient in interleukin-2 (IL-2) production and proliferation, and the peak of Ca2+ mobilization was reduced although of normal duration. Activation of Jun N-terminal kinase or stress-activated kinase (JNK or SAPK) and mitogen-activated protein kinase (MAPK) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal. Despite the reduced Ca2+ mobilization, translocation of cytoplasmic NF-ATc to the nucleus was normal, reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin. Treatment of lymphocytes with cytochalasin D, which blocks actin polymerization, inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells. In transfection studies, either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription. CONCLUSIONS These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.

Growth retardation and leaky SCID phenotype of Ku70-deficient mice

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

A Targeted DNA-PKcs-Null Mutation Reveals DNA-PK-Independent Functions for KU in V(D)J Recombination

The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.

Analysis of C-MYC Function in Normal Cells via Conditional Gene-Targeted Mutation

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.

Delayed Lymphoid Repopulation with Defects in IL-4–Driven Responses Produced by Inactivation of NF-ATc

The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.