In the fight against non-small cell lung cancer, it has been observed that treatment with the epidermal growth factor receptor (EGFR) mAb cetuximab is more effective when EGFR expression levels are higher. Why this is true, and whether it is due to the Fab or Fc portion of the mAb, had not been determined until Derer et al. (p. 5230) demonstrated the mechanism of action (MoA) of cetuximab. Using the BHK-21 cell line transfected with varying levels of EGFR, the authors produced target cells that expressed EGFR at levels comparable with those found in clinical samples. They determined that Fab-mediated effector mechanisms and levels of EGFR expression were negatively correlated, as treatment with cetuximab caused inhibition of EGF-induced EGFR phosphorylation independent of EGFR expression. Fc-mediated MoA, including Ab-dependent cell-mediated cytotoxicity (ADCC) due to NK cells, monocytes, and polymorphonuclear cells (PMNs) correlated positively with EGFR expression levels. The same was found for complement-dependent cytotoxicity. Mononuclear cell ADCC required lower levels of EGFR expression and mAbs than PMN-mediated MoA. The authors confirmed their Fc MoA results by testing a diverse array of human tumor cell lines and using RNAi-induced knockdown of EGFR. Taken together, the data present a comprehensive picture indicating the mechanism behind cetuximab therapy efficacy in EGFR-expressing cancers.

The chronic inflammatory disease periodontitis results from disruption of homeostasis between the periodontal tissue and the microbiota. Bone loss and tissue damage are consequences of excessive host inflammation, in which TLRs and complement have been implicated. Additionally, the periodontal pathogen Porphyromonas gingivalis can subvert the C5a receptor (C5aR) during the initiation of disease. Abe et al. (p. 5442) therefore investigated the involvement of C5aR in periodontitis pathogenesis and its potential as a therapeutic target. Because mice lacking either C5aR or TLR2 are resistant to periodontitis, and complement activation can synergize with TLR signaling, the authors assayed for C5aR–TLR2 synergy in periodontitis. Indeed, activation of C5aR and TLR2 synergistically enhanced inflammatory cytokine production in the gingiva, compared with activation of either receptor alone. Local administration of a C5aR antagonist (C5aRA) efficiently blocked P. gingivalis-induced inflammatory responses and bone loss in both a preventative and therapeutic manner. In a second P. gingivalis-independent model of ligature-induced periodontitis, local C5aRA administration or C5aR deficiency again protected against inflammatory bone loss. Thus, C5aR signaling is necessary for periodontitis, even in the absence of microbial exploitation of this receptor, and local delivery of C5aRAs may find utility in both preventing and treating this disease.

During malaria infection, humans are more susceptible to bacterial pathogens, including non-Typhoid Salmonella, but the mechanism of this susceptibility has been unclear. Previous work by Cunnington et al. (p. 5336) showed that in a mouse model of malaria, the hemolysis induced by the parasite impaired the neutrophil oxidative burst and allowed a more permissive environment for bacterial infection. Heme oxygenase 1 (HO-1) is a heme-degrading enzyme with cytoprotective effects that is produced as a result of hemolysis. In murine malaria, neutrophilic impairment occurs in the bone marrow as a result of HO-1 induction in neutrophil precursors. In this article, Cunnington et al. demonstrated that a similar process occurs in humans. The authors studied the neutrophil function of 58 Gambian children infected with Plasmodium falciparum. They also examined erythrocyte count, haptoglobin, hemopexin, plasma heme, heme uptake receptor expression, and HO-1 induction. They found that the majority of neutrophils produced during malaria infection had a reduced oxidative burst and recovered only after 8 wk. HO-1 expression and hemolysis markers were correlated with neutrophil oxidative burst impairment. Thus, the data indicate that a similar mechanism is occurring in humans and mice during malarial infection, leading to dysfunctional neutrophils with a reduced ability to fight bacterial infection. The authors also suggest that these data point to a pathway that regulates hemolysis-induced HO-1 production as a potential therapeutic target to reduce bacterial susceptibility during human malarial infection.

Processes that increase Ig gene diversity, including somatic hypermutation (SHM) and gene conversion, rely upon the activity of the activation-induced deaminase (AID). Although capable of acting throughout the genome, AID is preferentially targeted to Ig genes, avoiding general genomic instability. To address the mechanism of AID targeting, Kohler et al. (p. 5314) analyzed a region downstream of the chicken IgL C region exon that was previously found to contain a diversification activator (DIVAC) element. Using an SHM reporter cassette inserted in the IgL locus in the DT40 chicken B cell line, the authors localized specific sequences important for AID-mediated SHM, first identifying a 1928-bp region containing strong DIVAC function. Deletion analysis revealed that DIVAC function was conferred by a complicated network of additive and synergistic actions of a large number of broadly dispersed DNA elements. Within the 1928-bp region, two core regions, designated F2 and F3, were identified as essential, but not completely sufficient, for full DIVAC activity. No DIVAC-dependent effects on epigenetic modifications in the mutation target region were observed. However, DIVAC was associated with enhanced levels of the “stalled” form of RNA polymerase II in this region, suggesting involvement in transcriptional elongation/pausing. The data generated in this study implicate a complex interplay of multiple DNA elements in the regulation of AID targeting to Ig genes, a full understanding of which awaits future exploration.

Although much is known about the development of CD1d-restricted NKT cells, the factors that drive their differentiation into functional subsets are not well understood. The transcription factor ZBTB7B acts indirectly to maintain CD4 expression in MHC class II-restricted thymocytes and has been suggested to be necessary for NKT cell maturation and activation. In this issue, Enders et al. (p. 5240) identified a Leu-to-Arg missense mutation in the BTB-POZ domain of ZBTB7B and comprehensively characterized the NKT cells of mice bearing this mutation (Zbtb7bhpls/hpls). Whereas the majority of wild-type splenic NKT cells are CD4+CD8, the splenic NKT cells of Zbtb7bhpls/hpls mice lacked CD4 expression, and most instead expressed CD8. The bias toward CD4CD8+ NKT cell development in Zbtb7bhpls/hpls mice occurred through a cell-intrinsic mechanism beginning after positive selection, and these cells maintained a competitive advantage in the periphery. Although NKT cells in Zbtb7bhpls/hpls mice produced very low levels of cytokines in general, many of them did produce IL-17 and expressed the transcription factor retinoic acid-related orphan receptor-γt in a cell-intrinsic manner. In contrast, a relatively small proportion of wild-type NKT cells expressed IL-17. Zbtb7bhpls/+ heterozygous mice demonstrated a reduction in CD4+ NKT cells and an increase in IL-17 production without an increase in CD8+ NKT cells, suggesting that ZBTB7B acts at multiple stages of NKT cell differentiation. Taken together, these data reveal that ZBTB7B tightly regulates CD4 expression in NKT cells and controls the development of the IL-17–expressing NKT cell subset.

As moderately self-reactive B cells are released into the periphery, anergizing these cells is important for protection against autoimmunity. These anergic B cells are maintained through the action of B cell-activating factor (BAFF) on both the canonical and noncanonical NF-κB signaling pathways. Yu et al. (p. 5185) determined that BAFF acts through B cell lymphoma 10 (Bcl10), an adaptor protein with an N-terminal caspase recruitment domain and a C-terminal Ser/Thr-rich region. In the absence of Bcl10, BAFF was unable to support immature B cell survival. BAFF-mediated survival in mature B cells deficient in Bcl10 was reduced when compared with wild-type B cells. Bcl10-deficient mice had a drastic reduction in the number of anergic B cells due to increased apoptosis. The authors confirmed that self-reactive anergic B cells were dependent on Bcl10 for survival by crossing their Bcl10-deficient mice with IgHEL transgenic mice and using soluble HEL to induce anergy. Additional experiments determined that stimulation of BAFF caused an IκB kinase β–Bcl10 association, implicating utilization of the canonical NF-κB pathway. Compared with wild-type B cells, Bcl10-deficient B cells had reduced IκBα phosphorylation and nuclear p50/c-Rel complex formation. B cells lacking Bcl10 stimulated with BAFF also had a reduced number of p52/RelB complexes compared with wild-type cells. This was due to a reduced expression of NF-κB2/p100 and indicated that the noncanonical NF-κB signaling pathway was also involved. In line with these data was the observation that B cells lacking Bcl10 were also deficient in Bcl-xL, an NF-κB target gene that is induced by BAFF. Thus, BAFF regulates the survival of self-reactive anergic B cells through Bcl10 and the effects this adaptor protein has on the canonical and noncanonical NF-κB pathways.