In This Issue

Engagement of FcγRIIa by IgG complexes sets off signaling events that can either provide protection against infection or cause damaging inflammation, as is the case with several autoimmune disorders. One particular genetic polymorphism of FcγRIIa, coined the high responder/low responder (HR/LR) polymorphism, is associated with differences in binding of FcγRIIa to various IgG classes and altered signaling responses. Ramsland et al. (p. 3208) have defined the three-dimensional structure of FcγRIIa–Fc complexes to better understand the effects of FcγRIIa polymorphisms on IgG binding. Analysis of crystal structures of FcγRIIa-HR and FcγRIIa-LR dimers revealed that the dimer interactions occur at different interfaces, which affect accessibility to IgG binding sites. The structure of FcγRIIa bound to the Fc of human IgG1 was also resolved and helped clarify how different IgG classes and IgG glycosylation patterns influence FcγRIIa binding to IgG. Mutagenesis studies of FcγRIIa pinpointed distinct but overlapping epitopes of three different FcγRIIa-blocking mAbs that affect IgG binding. The authors also suggested a model in which FcγRIIa-HR dimers bind to Ag–IgG complexes and form signaling complexes at the cell surface. Thus, these findings provide insight into the influence of polymorphisms on FcγRIIa structure and function.

During T cell development, the double negative (DN) 3 thymocyte subset passes through the β-selection checkpoint, which involves rearrangement of the TCR β-chain locus and formation of the pre-TCR. Miz-1 is a BTB/POZ transcription factor that has been shown, through studies with a conditional knockout mouse expressing a form of Miz-1 lacking a functional BTB/POZ domain (Miz-1^ΔPOZ), to influence transcription in lymphoid precursors. In this issue, Saba et al. (p. 2982) observed that Miz-1^ΔPOZ mice had a block at the β-selection checkpoint and had significantly fewer DN3b and DN4 cells and reduced pre-TCR surface expression compared with wild-type (WT) controls. Miz-1^ΔPOZ mice had no defects in V(D)J recombination or expression of T cell-related genes, including CD3ε, TCRβ, pTα, Rag1, Rag2, and Tdt. Nonetheless, a significantly higher rate of apoptosis was observed in DN3 cells from Miz-1^ΔPOZ mice, which coincided with increased expression of p53 target genes, including PUMA, Noxa, Cdkn1a, and Bax. The block in the β-selection checkpoint in Miz-1^ΔPOZ mice could be overcome by coexpression of transgenes encoding the antiapoptotic molecule Bcl2 and a rearranged TCRαβ. These results suggest that Miz-1 is critical to the β-selection checkpoint by modulating expression of the pre-TCR and regulating apoptosis, thus assuring survival and expansion of DN3 thymocytes.

The cytokine IL-10 is well known for its tolerogenic effects on both myeloid and lymphoid cells, although the study of IL-10–producing cells has been hindered by a lack of tools and mouse models that enable accurate detection. Bouabe et al. (p. 3165) describe the development of a reporter mouse that facilitates highly sensitive measurements of IL-10. This model was based on detection of IL-10–driven expression of the reporter enzyme TEM-1 β-lactamase (Bla), which can be measured in live cells by fluorescence resonance energy transfer in the presence of the Bla substrate coumarin-cephalosporin-fluorescein-(4). IL-10 expression was detected in all myeloid and lymphoid cells of naive IL-10–Bla reporter (ITIB) mice, and interestingly, macrophages were the main IL-10 producers in the spleen, liver, and lungs. Infiltrating macrophages were also the primary source of IL-10 in the tumors of ITIB mice injected with melanoma cells. In contrast, IL-10 was produced predominantly by neutrophils in ITIB mice infected with Yersinia enterocolitica. Additional studies demonstrated that ITIB mice could be used to track the kinetics of IL-10 production, as well as the effects of IL-10–modulating molecules. Thus, the ITIB mouse offers an innovative tool for elucidating and tracking the expression of IL-10 during a host of diverse immune responses.

Immunological synapse formation between T cells and APCs prompts downstream signaling events that lead to T cell activation. This process is accompanied by membrane raft microdomain formation and actin polymerization. Tomassian et al. (p. 2993) have shown that the scaffold protein caveolin-1 is essential to coordinating these events in CD8⁺ T cells. Compared with wild-type (WT) controls, CD8⁺ T cells, but not CD4⁺ T cells, from caveolin-1–deficient (cav-1^−/−) mice had defects in T cell proliferation and effector functions following TCR/CD28 stimulation. In addition, actin polymerization and membrane raft polarization at the immunological synapse were both compromised in CD8⁺ T cells from cav-1^−/− mice relative to WT controls. Caveolin-1 was required for NFATc1-driven cytokine expression following TCR engagement but was not needed for NF-κB–dependent gene expression. Cav-1^−/− mice developed suboptimal CD8⁺ T cell responses to lymphocytic choriomeningitis virus infection, and ovalbumin-specific cav-1^−/− CD8⁺ T cells transferred into WT hosts responded poorly to OVA restimulation. Taken together, these findings support a role for caveolin-1 as a key mediator of TCR-driven signaling events in CD8⁺ T cells following immunological synapse formation.

The experimental autoimmune uveitis (EAU) model in mice has been used to better understand human uveitis and other autoimmune diseases affecting the CNS. Previous studies have suggested that EAU is an acute disease that differs from progressive uveitis in humans. Oh et al. (p. 3338) carried out a longitudinal study in mice over a 6-mo period that revealed aspects of EAU consistent with chronic uveitis. Mice immunized with an ocular autoantigen, interphotoreceptor retinoid-binding protein (IRBP), resolved symptoms of acute EAU by 30 days postimmunization. However, signs of chronic retinal inflammation emerged during the 225-d observation period, resulting in retinal degeneration and vision loss by the end of the study. IRBP-specific CD4⁺ memory T cells were detected in the bone marrow (BM) during the chronic phase, and reimmunization with IRBP triggered these cells to convert to effector T cells that could cause uveitis. Interestingly, STAT3-deficient mice did not develop chronic uveitis following IRBP immunization. This discrepancy was linked to a defect in trafficking of IRBP-specific CD4⁺ memory T cells to the BM, and was most likely related to decreased T cell expression of osteopontin and α4β1 integrin. Moreover, uveitis could be transferred to naive mice with IRBP-stimulated BM cells from WT, but not STAT3-deficient, mice with chronic uveitis. These data define a chronic phase of EAU and indicate that autoantigen-specific memory T cells in the BM may be a potential target for therapies against chronic autoimmune conditions.

Several processes drive secondary Ig gene diversification, including somatic hypermutation, gene conversion, and class switch recombination. Activation-induced cytidine deaminase (AID) is the key enzyme involved in generating DNA mutations for these processes, but its activity is tightly regulated to avoid off-target mutations. Kothapalli et al. (p. 3247) identified two cis-regulatory mutational enhancer elements (MEEs) in the chicken IGL locus that facilitate AID-driven diversification. Using the endogenous IGL promoter and gene in the chicken DT40 cell line, two different but functionally redundant MEEs were identified as being required for AID-mediated mutagenesis. These MEEs functioned as sequence elements separate from transcriptional enhancer elements to recruit AID to this locus, and a 222-bp fragment was identified as a key component of one of the MEEs. Sequence analysis confirmed that both the sequences and function of MEEs are conserved across different bird species, suggesting the preservation of binding sites for trans-acting factors involved in AID functions. These results indicate that MEEs act as unique cis elements that are essential to regulating AID activity and preventing undesirable mutagenesis.

Type 1 diabetes (T1D) develops from the destruction of pancreatic islet β-cells by autoreactive T cells. Previous studies in NOD mice have shown that T1D can be prevented by rapid activation of NK cells upon treatment with CFA. Lee et al. (p. 2898) now show that this protective NK cell response requires CD1d expression. Activation of both NK and NKT cells was triggered in response to the inactivated Mycobacterium tuberculosis component of CFA in wild-type, but not CD1d^−/−, mice. The NKT cells also produced IFN-γ, but not IL-4, upon activation. Interestingly, CFA activation of NKT cells was not dependent on MyD88 signaling. In addition, protection of NOD mice from T1D by CFA was CD1d dependent. These results suggest that lipid components of M. tuberculosis recognized by CD1d can activate NKT cells and induce NK cell and inflammatory responses that prevent T1D. Future studies are needed to identify the components that may be considered as potential therapeutics for treating T1D.

Human IgG4 molecules are uniquely characterized by their ability to form half-molecules that can recombine with other half-molecules to form bispecific Abs through a process called Fab-arm exchange. Labrijn et al. (p. 3238) examined the molecular determinants essential to Fab-arm exchange in human IgG4 molecules and investigated the existence of this phenomenon in other species. Previous studies have shown that Fab-arm exchange requires interactions between the CPSCP amino acid sequence in the core-hinge region and the IgG4 CH3 domain. Mutagenesis analysis carried out in this study identified the arginine at position 409 as the essential amino acid within the human IgG4 CH3 domain for driving Fab-arm exchange. Evidence of IgG Fab-arm exchange was observed in vitro from a variety of species, including rabbit, monkey, horse, and pig. A conserved amino acid sequence within the CH3 domain was not defined, but leucine 405 was identified as the key residue in the CH3 domain for mediating Fab-arm exchange in IgG4 molecules from Macaca mulatta. Analysis of different IgG subclasses by native mass spectrometry revealed that the CH3–CH3 interaction strength correlated with the ability of a particular IgG subclass to undergo Fab-arm exchange. These observations provide unique insight into a conserved immunological mechanism that may be applied toward optimizing therapeutic Abs.

Summaries written by Christiana N. Fogg, Ph.D.