Arda Shahinian

Lymphoproliferative Disorders with Early Lethality in Mice Deficient in Ctla-4

The role of the cell-surface molecule CTLA-4 in the regulation of T cell activation has been controversial. Here, lymph nodes and spleens of CTLA-4-deficient mice accumulated T cell blasts with up-regulated activation markers. These blast cells also infiltrated liver, heart, lung, and pancreas tissue, and amounts of serum immunoglobulin were elevated. The mice invariably became moribund by 3 to 4 weeks of age. Although CTLA-4-deficient T cells proliferated spontaneously and strongly when stimulated through the T cell receptor, they were sensitive to cell death induced by cross-linking of the Fas receptor and by gamma irradiation. Thus, CTLA-4 acts as a negative regulator of T cell activation and is vital for the control of lymphocyte homeostasis.

Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.

The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the hosts defense against microorganisms and their pathogenic factors.

Early Lethality, Functional NF-κB Activation, and Increased Sensitivity to TNF-Induced Cell Death in TRAF2-Deficient Mice

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 (refs 1,2,3) and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine co-stimulatory receptor–ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-1 do not interact with proteins in the CD28–B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1–Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyers patches. Presensitized mice treated with B7RP-1–Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor–ligand pair that is structurally related to CD28–B7 and is involved in the adaptive immune response.

Duration of TCR stimulation determines costimulatory requirement of T cells.

Current models suggest that T cells that receive only signal-1 through antigenic stimulation of the T cell receptor (TCR) become anergic, but will mount an immune response when a costimulatory signal-2 is provided. Using mice deficient for an important costimulatory molecule, CD28, we show that a transient signal-1 alone, either through infection with an abortively replicating virus, or through injection of viral peptide, anergizes CD8+ T cells, demonstrating the biological relevance of T cell anergy in vivo. However, in the absence of CD28, continued presence of signal-1 alone, either through prolonged viral replication or repeated injection of peptide, prevents the induction of anergy and generates a functional T cell response in vivo.

The Transcription Factor NF-ATc1 Regulates Lymphocyte Proliferation and Th2 Cytokine Production

NF-ATc1 is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In activated T cells, nuclear NF-AT binds to the promoter regions of multiple cytokine genes and induces their transcription. The role of NF-ATc1 was investigated in recombination activating gene-1 (RAG-1)-deficient blastocyst complementation assays using homozygous NF-ATc1-/- mutant ES cell lines. NF-ATc1-/-/RAG-1-/- chimeric mice showed reduced numbers of thymocytes and impaired proliferation of peripheral lymphocytes, but normal production of IL-2. Induction in vitro of Th2 responses, as demonstrated by a decrease in IL-4 and IL-6 production, was impaired in mutant T cells. These data indicate that NF-ATc1 plays roles in the development of T lymphocytes and in the differentiation of the Th2 response.

Impaired Negative Selection of T Cells in Hodgkin's Disease Antigen CD30–Deficient Mice

CD30 is found on Reed-Sternberg cells of Hodgkins disease and on a variety of non-Hodgkins lymphoma cells and is up-regulated on cells after Epstein-Barr virus, human T cell leukemia virus, and HIV infections. We report here that the thymus in CD30-deficient mice contains elevated numbers of thymocytes. Activation-induced death of thymocytes after CD3 cross-linking is impaired both in vitro and in vivo. Breeding the CD30 mutation separately into alpha beta TCR-or gamma delta TCR-transgenic mice revealed a gross defect in negative but not positive selection. Thus, like TNF-receptors and Fas/Apo-1, the CD30 receptor is involved in cell death signaling. It is also an important coreceptor that participates in thymic deletion.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF‐κB‐dependent gene transcription

Induction of NF‐κB‐dependent transcription requires phosphorylation and subsequent degradation of I‐κB, an inhibitor of NF‐κB, followed by nuclear translocation and DNA binding of NF‐κB. Tumor necrosis factor receptor‐associated factor 2 (TRAF2) plays a role in NF‐κB activation in response to cytokines such as tumor necrosis factor α (TNFα). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I‐κBα in vitro. The physiological function of T2K was investigated using T2K‐deficient mice. Heterozygotes appear normal, but t2k−/− animals die at ∼E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K‐deficient fetal liver support normal lymphocyte development. Furthermore, t2k−/− embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFα‐induced apoptosis. In response to either TNFα or IL‐1 induction, t2k−/− embryonic fibroblasts exhibit normal degradation of I‐κB and κB‐binding activity. However, NF‐κB‐directed transcription is dramatically reduced. These results demonstrate that, like I‐κB kinase β and the RelA subunit of NF‐κB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF‐κB‐directed transcription, apparently independent of I‐κB degradation and NF‐κB DNA binding.

IL-7 Engages Multiple Mechanisms to Overcome Chronic Viral Infection and Limit Organ Pathology

Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases.

Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses.

Costimulation through the inducible costimulator (ICOS) and its ligand (ICOSL) is essential for T cell–dependent B cell responses, but the cellular and temporal dynamics underlying its in vivo effects are poorly defined. Here we have shown that Icosl−/− and Icos−/− mice had similar phenotypes and that ICOS-ICOSL costimulation modulated the early but not late phases of IgG1 affinity maturation. Exploiting the adoptive transfer of T or B cells from primed Icosl−/− mice, we provided genetic evidence that costimulation through ICOSL was essential for primary but not secondary helper T cell responses and for the control of both T and B cell activities, resulting in T cell–dependent IgG1 production.