Roger Ferrini

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.

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.

Function of the TCRα Enhancer in αβ and γδ T Cells

Abstract We have used gene targeted mutational approaches to assess the role of the T cell receptor α (TCRα) enhancer (Eα) in the control of TCRα and TCRδ gene rearrangement and expression. We show that Eα functions in cis to promote Vα to Jα rearrangement across the entire Jα locus, a distance of greater than 70 kb. We also show that Eα is required for normal αβ T cell development; in this lineage, Eα is required for germline Jα expression, for normal expression levels of rearranged VαJα genes, and for expression of a diverse Vα repertoire. In γδ T cells, Eα is not required for VδDJδ rearrangement, but, surprisingly, is required for normal expression levels of mature VδDJδ transcripts and for expression of germline Jα transcripts. Our findings imply that Eα function is not limited to the TCRα components of the TCRα/δ locus or to the αβ lineage; rather, Eα function is important in both αβ and γδ lineage T cells.

RAG2 :GFP KNOCKIN MICE REVEAL NOVEL ASPECTS OF RAG2 EXPRESSION IN PRIMARY AND PERIPHERAL LYMPHOID TISSUES

We generated mice in which a functional RAG2:GFP fusion gene is knocked in to the endogenous RAG2 locus. In bone marrow and thymus, RAG2:GFP expression occurs in appropriate stages of developing B and T cells as well as in immature bone marrow IgM+ B cells. RAG2:GFP also is expressed in IgD+ B cells following cross-linking of IgM on immature IgM+ IgD+ B cells generated in vitro. RAG2:GFP expression is undetectable in most immature splenic B cells; however, in young RAG2:GFP mice, there are substantial numbers of splenic RAG2:GFP+ cells that mostly resemble pre-B cells. The latter population decreases in size with age but reappears following immunization of older RAG2:GFP mice. We discuss the implications of these findings for current models of receptor assembly and diversification.

Granzyme A Cleaves a Mitochondrial Complex I Protein to Initiate Caspase-Independent Cell Death

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.

SEK1/MKK4 is required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development.

SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.

Developmental Regulation of TCRδ Locus Accessibility and Expression by the TCRδ Enhancer

Abstract We have used gene-targeted mutation to assess the role of the T cell receptor δ (TCRδ) enhancer (Eδ) in αβ and γδ T cell development. Mice lacking Eδ exhibited no defects in αβ T cell development but had a severe reduction in thymic and peripheral γδ T cells and decreased VDJδ rearrangements. Simultaneous deletion of both Eδ and the TCRα enhancer (Eα) demonstrated that residual TCRδ rearrangements were not driven by Eα, implicating additional elements in TCRδ locus accessibility. Surprisingly, while deletion of Eδ severely impaired germline TCRδ expression in double-negative thymocytes, absence of Eδ did not affect expression of mature δ transcripts in γδ T cells. We conclude that Eδ has an important role in TCRδ locus regulation at early, but not late, stages of γδ T cell development.