The maturation state of a T cell determines the nature of its response to weak vs strong ligands, but the molecular mechanism responsible for this regulation is not clear. Full TCR activation requires the induction of a conformational change in CD3 (CD3Δc), and Gil et al. (p. 3900 ) hypothesized that “developmental tuning” involving the CD8 coreceptor modulates CD3Δc to affect Ag recognition by the TCR. First, the authors observed that stimulation with both weak and strong peptide:MHC (pMHC) ligands induced CD3Δc in pre-selection double-positive thymocytes, but only strong ligands could induce CD3Δc after positive selection. Inhibiting CD8 interaction with pMHC, using either blocking Abs or mutant tetramers defective in CD8 binding, demonstrated that this interaction was required for CD3Δc induction, whereas Src kinase activity was not. This requirement was applicable to both pre-selection thymocytes and mature CD8+ T cells and was not due solely to CD8-mediated enhancement of TCR:pMHC affinity. CD8 is known to undergo increased sialylation after positive selection, so the authors next assessed the effect of removing this glycosylation. Neuraminidase treatment restored CD3Δc induction and functional responses induced by weak pMHC ligands in both mature T cells and post-selection thymocytes. This elegant study thus identifies a role for glycosylation in the developmental regulation of TCR stimulation.

The HIV-1 accessory protein Nef has been hypothesized to promote viral pathogenesis through several mechanisms, including evasion of CTL attack via down-regulation of MHC I on infected cells. Although this function of Nef has been demonstrated in vitro and in acute infection, its in vivo relevance during chronic HIV infection has not been elucidated. Lewis et al. (p. 4075 ) therefore analyzed Nef-mediated MHC I down-regulation in 11 chronically infected individuals representing a broad range of clinical disease states. Sequence analysis identified genetically distinct nef quasispecies in most subjects that demonstrated a wide range of abilities to down-regulate MHC I in vitro, suggesting that Nef function varies widely in vivo. The ability of Nef variants from different subjects to down-regulate MHC I on HIV-infected cells directly correlated with the resistance of these cells to CTL killing, indicating that viral isolates that fail to down-regulate MHC I become vulnerable to CTL attack. Additionally, Nef function correlated with CD4+ T cell counts and CTL response breadth, suggesting that Nef activity is strongest when CTL pressure is greatest. These data indicate that Nef down-regulation of MHC I regulates HIV pathogenesis in vivo, and modulation of this activity could therefore be clinically useful.

Complicated interactions between interconnected signaling pathways regulate T cell differentiation into Th1, Th2, and Th17 cells. To begin to sort out the pathways that modulate Th17 differentiation, Tanaka et al. (p. 3746 ) developed conditional knockout mice lacking suppressor of cytokine signaling 1 (SOCS1) in T cells (SOCS1-cKO) and analyzed the effects of this deletion on T cell differentiation. SOCS1-cKO mice were resistant to experimental autoimmune encephalomyelitis (EAE) due to greatly increased levels of IFN-γ and coordinately reduced induction of Th17 cells. Reduced Th17 differentiation was dependent upon enhanced IFN-γ/STAT1 signaling, indicating that SOCS1 inhibits IFN-γ activity to allow Th17 development. The authors next assessed the signaling cascades responsible for IFN-γ-mediated inhibition of Th17 differentiation and found that SOCS1-deficient T cells showed increased SOCS3, which suppresses IL-6 signaling, and reduced activation of STAT3, which is induced by IL-6 and TGF-β. SOCS3 could be shown to suppress STAT3 phosphorylation, leading to reduced Th17 differentiation and EAE development. The increase in Th1 development in SOCS1-cKO mice was traced to impairment in the TGF-β-mediated inhibition of IFN-γ. Enhanced IFN-γ signaling in T cells lacking SOCS1 then led to impaired TGF-β-mediated RORγt induction and Smad activity, reducing Th17 differentiation. Thus, proper SOCS1 activity is vital to the cross-regulation of Th1 and Th17 differentiation.

The intracellular chaperone Hsp70 is up-regulated by cellular stress and has been observed extracellularly, where it activates macrophages and other immune cells. Hsp70 contains no clear secretory signal, so Vega et al. (p. 4299 ) set out to determine how this molecule manages to exit the cell. Experiments using artificial membranes showed that Hsp70 could integrate into the lipid bilayer and allow cation conduction. Moving to studies of intact cells, the authors found that Hsp70, positioned with its C terminus exposed, could be observed on the surface of stressed cells. Further analysis localized Hsp70 to GM1-rich, detergent resistant membrane domains and suggested that cholesterol could enhance the interaction of Hsp70 with the membrane. Differential centrifugation then identified the presence of Hsp70, but not other Hsps, in the extracellular membrane, or exosome, fraction, and these exosomes were shown to be derived from the cellular plasma membrane. These Hsp70-containing extracellular membranes were then incubated with macrophages and were demonstrated to strongly induce TNF-α production. In vivo, stressed cells may therefore release exosomes containing Hsp70 to induce macrophage activation.

Viruses such as HIV and hepatitis C virus are able to escape immune attack by mutating immunodominant CTL epitopes. However, only a fraction of the mutations predicted to allow CTL escape occur in vivo. Such selective mutation has been suggested to relate to maintenance of viral fitness, but the inability to directly test this hypothesis in humans has prevented its in vivo analysis. Butler et al. (p. 3926 ) conducted an in-depth structural and biological analysis of the immunodominant S510–518 (S510) epitope of the JHM strain of mouse hepatitis virus (JHMV) and determined that CTL escape is a much more complex process than previously appreciated. The authors first determined the crystal structures of S510 and W513S, which bears a mutation in a TCR contact residue, complexed to H-2Db. These structures provided a model on which to base the experiments that followed. Although mutations at either position 4 or 8 of the S510 peptide would be predicted to allow CTL escape, only mutations in Trp4 were observed in vivo, and these almost exclusively involved changes to Arg. Other substitutions were found to allow CTL escape without altering viral fitness both in vitro and in vivo, but these mutations were not naturally selected. The authors were unable to trace this selectivity to de novo CTL responses, viral fitness, or holes in the TCR repertoire and concluded that selection of viral escape mutants is a complex process modulated by forces beyond those previously postulated.

Autoreactive B cells and autoantibodies have been shown to be involved in the pathogenesis of the complex autoimmune disease multiple sclerosis (MS), but the targets recognized by these autoantibodies are not clear. Somers et al. (p. 3957 ) employed the technique known as serological antigen selection to identify a panel of novel Ags recognized by autoantibodies in MS patients. The authors made a phage display library of cDNA products expressed in active MS brain plaques and screened this library with the pooled cerebrospinal fluid (CSF) of 10 MS patients. Analysis of the clones enriched by this procedure identified eight target Ags that were then used to screen a large panel of CSF specimens from MS patients and patients with other inflammatory or noninflammatory neurological disorders. These eight Ags showed 86% specificity and 45% sensitivity in discriminating MS patient samples vs controls, indicating the diagnostic potential of these Ags as markers of MS. Preliminary data indicated that autoantibodies specific for these Ags might also be found in the sera of some MS patients, further supporting the clinical applicability of this study. These data open the door for future studies that may identify useful disease markers of autoimmune diseases.

Anti-carbohydrate natural Abs, particularly those specific for αGal, play a protective role in the primate immune response. Developmental regulation of B cells that produce these natural Abs is not well understood, so Benatuil et al. (p. 3839 ) developed an Ig knock-in mouse model expressing an αGal-specific BCR and studied the development of B cells in mice expressing or lacking αGal as a self-Ag. Mice lacking αGal (M86VHVLGT0/0) had high serum titers of functional αGal-specific Abs, and B cells expressing these Abs differentiated into splenic marginal zone B cells. In contrast, mice expressing αGal (M86VHVLGT+) lacked these αGal-specific Abs. Up-regulation of RAG-2 expression was observed in Ig knock-in pre-B cells cultured on stromal cells expressing αGal, suggesting that B cells in M86VHVLGT+ mice underwent tolerance via receptor editing. The authors also developed mice bearing an extra copy of the knock-in BCR on the αGal-expressing background and found, to their surprise, that B cells expressing αGal-specific Abs were able to develop in these mice. Binding experiments suggested that these Abs were of lower affinity than those in M86VHVLGT0/0 mice. Taken together, these data clarify some features of the development of natural Ab-expressing B cells and suggest that receptor editing can lower the affinity of self-reactive Abs.

Costimulation is partially mediated through B7.2 (CD86) in T cells. Kapsogeorgou et al. (p. 3815 ) have identified a new splice variant of this molecule in humans, termed B7.2C, that lacks exon 4 of the full-length transcript. The full-length transcript B7.2A contains an IgV-like counter receptor-binding domain that is encoded by exon 4 and lacking in the newly-described B7.2C splice variant. B7.2C mRNA and protein surface expression were detected only in peripheral blood monocytes and non-neoplastic salivary gland epithelial cells. The authors found that monocytes down-regualted B7.2C upon activation and that when expressed in isolation on Chinese hamster ovary (CHO) cells (CHO-B7.2C) the newly discovered molecule could not provide costimulation to T cells. However, when CHO cells were transfected with B7.2C and B7.2A, the presence of B7.2C inhibited the ability of B7.2A to provide costimulation within a range of 23–69%. Inhibition of costimulation was directly correlated to the amount of B7.2A vs B7.2C expression. Thus, the authors present a new B7.2 molecule that can modulate the strength of costimulatory signals, potentially through obstructing the formation of B7.2A clusters necessary for signaling.

Summaries written by Jennifer Hartt Meyers, Ph.D.