David A. Ferrick

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.

Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4

Certain IFN regulatory factor (IRF) transcription factors indirectly influence T helper (Th) cell differentiation by regulating the production of IL-12. Here, we show that IRF4 directly regulates Th cell differentiation in vitro and in vivo during murine leishmaniasis. In the absence of IRF4, IL-12-induced Th1 cell differentiation was compromised, while IL-4 failed to induce Th2 cell differentiation. Instead, IL-4 tended to induce Th1 cells, defined by production of IFN-γ and TNF. Although early IL-4 signaling was normal in IRF4−/− Th cells, the protein GATA-3, a transcription factor critical for Th2 development, was not up-regulated following IL-4 treatment. Retroviral overexpression of GATA-3 rescued Th2 differentiation. Therefore, IRF4 deficiency manifests itself as severely dysregulated Th cell differentiation.

Posttranscriptional downregulation of c-IAP2 by the ubiquitin protein ligase c-IAP1 in vivo.

ABSTRACT Inhibitor of apoptosis proteins (IAPs) c-IAP1 and c-IAP2 were identified as part of the tumor necrosis factor receptor 2 (TNFR2) signaling complex and have been implicated as intermediaries in tumor necrosis factor alpha signaling. Like all RING domain-containing IAPs, c-IAP1 and c-IAP2 have ubiquitin protein ligase (E3) activity. To explore the function of c-IAP1 in a physiologic setting, c-IAP1-deficient mice were generated by homologous gene recombination. These animals are viable and have no obvious sensitization to proapoptotic stimuli. Cells from c-IAP1−/− mice do, however, express markedly elevated levels of c-IAP2 protein in the absence of increased c-IAP2 mRNA. In contrast to reports implicating c-IAPs in the activation of NF-κB, resting and cytokine-induced NF-κB activation was not impaired in c-IAP1-deficient cells. Transient transfection studies with wild-type and E3-defective c-IAP1 revealed that c-IAP2 is a direct target for c-IAP1-mediated ubiquitination and subsequent degradation, which are potentiated by the adaptor function of TRAF2. Thus, the c-IAPs represent a pair of TNFR-associated ubiquitin protein ligases in which one regulates the expression of the other by a posttranscriptional and E3-dependent mechanism.

T cell function and expression are dramatically altered in T cell receptor Vγ1.1Jγ4Cγ4 transgenic mice

Abstract We have characterized transgenic mice carrying a functional T cell receptor (TCR) Cγ4 (Vγ1.1Jγ4Cγ4) gene. Results indicate that active transcription of the Cγ4 transgene can influence expression of the endogenous Cγ4, Cγ1 (Vγ3-, Vγ4-, Vγ2-, or Vγ5Jγ1Cγ1) and Cγ2 (Vγ1.2Jγ2Cγ2) genes, while the ultimate expression of other TCR δ, α, and β chain genes, as well as the adult T cell response, are relatively unaltered. Cells expressing transgenic Cγ4 and endogenous δ TCR transcripts can migrate to the skin as dendritic epithelial cells (DEC) even though Cγ4 cells are rarely, if at all, found in the skin. Transgenic and control mice were compared at 2 weeks, 6–7 weeks, and older. At 2 weeks, the thymus of transgenic mice, particularly the medulla, was much larger than control. Moreover, peripheral lymphoid tissues of younger mice were markedly (as much as 100-fold) more immunoreactive (both Con A response and alloreactivity). These differences, although persistent, became smaller in older mice. The data suggest that transgene expression has a major effect on T cell development and reactivity.

Enhanced TCR-induced Apoptosis in Interferon Regulatory Factor 4-deficient CD4 Th Cells

Transcription factors of the interferon regulatory factor (IRF) family contribute to the regulation of cell proliferation and apoptosis. Here, we show that CD4+ T helper (Th) cells lacking IRF4 (IRF4−/−) are highly sensitive to apoptosis. After infection of IRF4−/− mice with the protozoan parasite Leishmania major, the lesion-draining lymph nodes developed the prototypic lymphadenopathy of wild-type mice after 4 wk, but demonstrated almost total loss of cellularity and enhanced apoptosis after 7 wk. In vitro, activation of IRF4−/− CD4+ Th cells led to greatly increased apoptosis compared with wild-type cells. Coculture of IRF4−/− and IRF4+/+ CD4+ cells did not increase survival of IRF4−/− CD4+ cells, indicating that the enhanced rate of IRF4−/− Th cell apoptosis was neither transferable nor due to lack of a cytokine. Enhanced CD4+ cell apoptosis was also observed after anti-CD95 mAb treatment, despite normal CD95 expression. Removal of endogenous cytokines, notably interleukin (IL)-4, led to increased and equally high levels of IRF4−/− and IRF4+/+ cell apoptosis, whereas the protective activity of exogenous IL-4 was reduced in IRF4−/− CD4+ cells despite normal expression of the IL-4 receptor. Therefore, IRF4 is central in protecting CD4+ cells against proapoptotic stimuli.