The αβ TCR complex is comprised of heterodimers, of which the αβ heterodimer binds to peptide–MHC complexes and interacts with CD3 heterodimers that subsequently trigger intracellular signaling cascades. Kim et al. (p. 2951) analyzed mammalian CD3εγ and CD3εδ heterodimers through structure-based mutagenesis to reveal geometric features of their ectodomains that are required for effective TCR assembly and activation. Previous studies showed that hydrophobic regions in the ectodomains of CD3εγ and CD3εδ promoted dimerization through hydrogen bonds between G-strands. In this study, structural comparisons between CD3εγ and CD3εδ showed strikingly different geometries between the CD3 ectodomains of each heterodimer, likely due to differences in amino acid sequences and hydrophobic bonding. Mutagenesis studies in which a portion of the CD3γ G-strand was replaced with amino acids from the CD3δ G-strand confirmed that the CD3γ G-strand formed a kinked structure that was important for optimal positioning of TCRβ and CD3εγ upon T cell activation. Sequence analysis confirmed coevolution in mammals of CD3γ and the TCR Cβ FG loop, which interacts with CD3γ, such that the unique quaternary structure formed by these interacting domains was preserved in different jawed vertebrates. These results reinforce the importance of evolutionary conservation of the TCR complex structural features to maintain biological function.

Zymosan is a fungal cell wall component that has been shown to promote or restrain autoimmunity in different mouse models of autoimmune disease. Burton et al. (p. 2754) have examined the mechanisms by which zymosan suppresses diabetes development in NOD mice. A single injection of zymosan was sufficient to dramatically delay the onset of diabetes in prediabetic NOD mice. This delay was dependent on zymosan-induced production of TGF-β and upregulation of programmed death ligand 1 (PD-L1) on CD11b+ macrophages. In addition, zymosan induced TGF-β secretion from macrophages through a mechanism involving TLR2 ligation and p38 and ERK MAPK phosphorylation. CD11b+TGFβ+PD-L1+ APCs from zymosan-treated mice promoted the expansion of FoxP3+ regulatory T cells (Tregs) in vitro. In addition, zymosan treatment caused an increase in FoxP3+ Tregs in the pancreatic infiltrate of NOD mice. These results demonstrate that zymosan can suppress diabetes by promoting Treg expansion via upregulation of TGF-β and PD-L1 and provide an intriguing approach for therapeutic interventions against diabetes.

West Nile virus (WNV) can cause severe acute fever and encephalitis in humans, but no Food and Drug Administration-approved vaccines against WNV are available. Demento et al. (p. 2989) tested a novel approach to vaccination against WNV using biodegradable nanoparticles containing rWNV envelope (WNVE) protein. In vitro stimulation of bone marrow-derived dendritic cells with WNVE-loaded nanoparticles tethered with immunostimulatory TLR9-specific CpG oligodeoxynucleotides (ODNs) promoted activation and induced secretion of IL-6 and IL-12 compared with cells stimulated with WNVE-loaded particles lacking CpG ODNs or rWNVE adsorbed onto aluminum hydroxide (alum) adjuvant. High titers of IgG1 were induced by the alum formulation, indicative of a Th2-biased response. In contrast, CpG ODN-modified rWNVE-loaded particles induced a Th1-biased response, indicated by high levels of IgG2a and IgG2b. Splenocytes from mice immunized with CpG ODN-modified rWNVE-loaded particles released greater amounts of IL-2 and IFN-γ upon ex vivo stimulation compared with splenocytes from mice immunized with unmodified WNVE particles or alum-adsorbed rWNVE. Moreover, immunization with CpG ODN-modified rWNV-loaded particles induced a greater frequency of circulating effector T cells and provided superior protection against WNV encephalitis in infected mice compared with other vaccine formulations. These results provide insight into a potentially effective approach for vaccination against WNV.

Interactions between the αLβ2 integrin LFA-1 on NK cells and ICAMs on target cells initiate adhesion events essential to immunological synapse formation and NK cell cytoxicity. Gross et al. (p. 2918) have examined the effect of ICAM mobility on the cell surface with respect to LFA-1–dependent NK cell functions. Pharmacological disruption of the actin cytoskeleton in target cells inhibited LFA-1–dependent conjugate formation with NK cells and prevented NK cell cytotoxic granule polarization. ICAM-1 and ICAM-2 appeared to be organized into immobile clusters, based on fluorescence recovery after photobleaching and total internal reflection fluorescence microscopy. These ICAM clusters readily diffused throughout the plasma membrane upon actin disruption. Conversely, immobilization of ICAM-2 via the expression of the actin-adaptor molecule ezrin on otherwise NK cell-resistant target cells increased LFA-1–dependent adhesion and polarization of cytotoxic granules. Furthermore, freely diffusible ICAM-1 in lipid bilayers did not promote NK cell adhesion, but NK cells readily adhered to immobile ICAM-1. These findings support a model in which ICAM-1 clusters that have limited mobility due to interactions with the actin cytoskeleton are fundamental to NK cell adhesion and granule polarization.

A difference in Ca2+ signaling patterns is one feature that distinguishes Th1 from Th2 cells and contributes to their unique phenotypes. Using microarray analysis, Weber et al. (p. 2836) identified candidate genes related to Ca2+ signaling that were differentially expressed in Th1 and Th2 cells. One gene, which displayed higher expression levels in Th2 cells compared with Th1 cells, was Trpm4. Trpm4 is a cation channel involved in membrane depolarization. Inhibition of functional Trpm4 expression via small-interfering RNA-mediated silencing or dominant-negative mutant expression caused an increase in Ca2+ levels in Th2 cells compared with control cells. Conversely, inhibition of Trpm4 in Th1 cells in the same manner decreased Ca2+ levels relative to control cells. Trpm4 expression inhibition significantly altered cellular motility and production of IL-2, IL-4, and IFN-γ in Th1 and Th2 cells. Trpm4 silencing significantly increased the nuclear localization of the transcription factor NFATc1 in Th2 cells and resulted in decreased nuclear localization in Th1 cells. Interestingly, NFATc1 is known to be influenced by Ca2+ signaling and involved in the expression of various cytokines in Th1 and Th2 cells. Overall, differential Trpm4 expression in Th subsets may be an important factor in skewing T cell functions by affecting Ca2+ signaling and NFATc1 nuclear localization.

The secretory machinery of a Th cell is rapidly repositioned toward its immunological synapse with a cognate APC, but the molecular events essential for Th cell polarization and the effect of polarization on APC function remain uncertain. In this issue, Bertrand et al. (p. 2887) identified a distinct role for protein kinase C (PKC) ζ, which has been shown to affect cell polarity and asymmetry during Th cell polarization. TCR engagement was associated with rapid activation of Th cell-expressed PKCζ at the immunological synapse with cognate Ag-pulsed dendritic cells (DCs). Activated PKCζ appeared to be essential to Th cell polarization, as Th cells treated with a PKCζ inhibitor or transfected with an inactive form of PKCζ showed a significant reduction in polarization of secretory machinery toward DCs compared with Th cells containing activated PKCζ. The PKCζ pathway also affected DC function such that inhibition of PKCζ in Th cells significantly reduced the release of IL-12 by cognate DCs relative to IL-12 release by DCs interacting with activated PKCζ-intact Th cells. In addition, PKCζ signaling was required for the polarization of Th cell-expressed IFN-γ and CD40L toward DCs, suggesting a link between PKCζ-driven Th cell polarization and IL-12 production by cognate DCs. These observations support a unique role for PKCζ signaling in Th cell polarization and better distinguish the molecular events surrounding Th cell activation of DCs.

B cells initiate innate immune responses in part through recognition of pathogens by TLRs. These innate B cell responses lead to enhanced proliferation, differentiation, and cytokine secretion. In this issue, two papers highlight novel functions of B cells during Salmonella enterica serovar Typhimurium infection. Morrison et al. (p. 2737) showed that TLR-mediated activation of B cells caused a transient decrease in CD62L surface expression and redirected B cell trafficking. Murine B cells treated with the TLR2 ligand PAM3CSK4 or the TLR9 ligand CpG DNA showed a rapid and significant shedding of CD62L from the cell surface compared with unstimulated cells. This shedding process was both TLR and MyD88 dependent. CpG DNA-stimulated CD62Llow donor B cells migrated to the spleen but were excluded from lymph nodes and Peyer's patches upon transfer into recipient mice, which directly contrasted with the migration patterns of untreated CD62Lhi B cells in different recipients. Treatment of mice with CpG DNA or infection with S. enterica also caused rapid CD62L shedding from B cells and preferential trafficking to the spleen. Thus, TLR- and MyD88-dependent shedding of CD62L expression can rapidly alter B cell trafficking in mice, which may lead to enhanced B cell responses in the spleen in the face of microbial infections.

In the second paper, Barr et al. (p. 2783) characterized the contributions of TLR and BCR signaling in B cells toward generating effective T cell responses during S. enterica serovar Typhimurium infection. Intact BCR and TLR signaling pathways were each required for B cell cytokine secretion following primary S. enterica infection. Compared with wild-type mice, transgenic mice expressing an anti-HEL BCR, which cannot recognize S. enterica, or MyD88-deficient mice showed diminished B cell secretion of IL-6, IL-10, and IFN-γ following infection. In response to primary infection, chimeric mice that lacked MyD88 in B cells also showed impaired cytokine production as well as compromised Th1 responses compared with wild-type chimera controls, but memory Th1 responses were not affected. In contrast, B cells expressing an anti-HEL BCR had significantly reduced memory Th1 responses following secondary infection compared with control mice, thus affirming a role for BCR recognition of S. enterica for memory responses. Chimeric mice in which B cells had defects in Ag presentation also had compromised memory, but normal primary Th1 responses. These results indicate that BCR and TLR signaling play unique roles in B cell help to T cells. Overall, these two reports illuminate new roles for B cells during S. enterica infection.

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