Exhaustive Elucidation of HLA-Bound Peptides See article p. 2639

Finding Flexibility in the Formation of γδ T cells See article p. 2668

cPLA2α Controls Neutrophil Transit See article p. 2873

HVEM Helps T Cells Maintain Memory See article p. 2968

The highly polymorphic classical MHC molecule HLA-C and the less polymorphic nonclassical MHC molecules HLA-E and HLA-G can interact with NK cell receptors as well as TCRs, thus participating in both innate and adaptive immune responses. Di Marco et al. (p. 2639) have now comprehensively characterized peptides naturally presented by these broadly relevant MHC molecules. Lymphoblastoid C1R cells, which have a functional Ag presentation pathway but only express low levels of endogenous HLA molecules, were transfected with HLA-C (the 15 most common alleles), HLA-E, or HLA-G. HLA-presented peptides were then analyzed using liquid chromatography tandem mass spectrometry following immunoaffinity chromatography of HLA molecules from cell lysates. HLA-C peptide ligands were most commonly nonameric, but the HLA-C alleles showed different preferences for C-terminal peptide residues that bound to a hydrophobic anchor position, and variability in residues binding to a second anchor position (at either position 2 or 3). Contrary to previous data grouping HLA-C allotypes into two supertypes, the data collected here supported characterization into five distinct groups. Among the HLA-C peptide ligands, overlap between the peptides themselves and the proteins from which the peptides were derived was assessed for the 500 most abundant ligands for each allotype and overlap was found to be very low unless allotypes shared anchor residues. Peptide ligands identified in this study were clustered using GibbsCluster 1.1, which could be used to define binding motifs, and predictive SYFPEITHI matrices were established. Comparison of the predictive power of these matrices to that of the NetMHCpan-3.0 algorithm indicated that the SYFPEITHI matrices generally performed better in predicting HLA peptide ligands. Peptides bound to HLA-E and HLA-G were also analyzed, and whereas HLA-E ligands consisted of only a few MHC I leader peptides, 2258 peptide ligands were identified for HLA-G*01:01. The data and predictive tools generated in this study should provide a useful resource for future studies of the many immune processes involving HLA-C, HLA-E, and HLA-G.

An effective long-lived response to a viral infection requires the differentiation of CD8+ T cells into both short-lived effector cells (SLECs), which provide rapid protection, and memory precursor effector cells (MPECs), which can develop into memory cells that protect against future reinfection. To better understand how costimulatory molecules affect the differentiation of MPECs, Desai et al. (p. 2968) investigated the involvement of the TNFR superfamily member herpes virus entry mediator (HVEM) in the CD8+ T cell response to respiratory viral infections. Following intranasal infection of wild-type (WT) mice with a recombinant vaccinia virus Western Reserve strain expressing full-length OVA protein (rVacV-WR-OVA), adoptively transferred OVA-specific CD8+ T cells (OT-I cells) expanded during the effector phase of the antiviral response to a similar degree whether they expressed HVEM. However, accumulation of HVEM−/− OT-I memory cells, both circulating and lung-resident, was strongly reduced relative to WT OT-I cells. HVEM was found to be expressed on naive cells, downregulated on SLECs, and selectively retained on MPECs during infection of WT mice with either rVacV-WR-OVA or WT VacV-WR. Although mice receiving WT or HVEM−/− OT-I cells cleared the recombinant virus with similar efficacy, fewer MPECs and more SLECs developed from HVEM−/− than from WT OT-I cells, and data from an apoptotic gene array suggested that HVEM−/− MPECs had impaired survival. Transfer of WT OT-I cells into mice deficient in the HVEM ligands LIGHT or BTLA resulted in impaired memory cell accumulation only in the mice lacking LIGHT, suggesting its importance for HVEM activity on MPECs. To confirm the relevance of these results to protective memory responses, the authors showed that mice previously infected with rVacV-WR-OVA that had received WT, but not HVEM−/−, OT-I cells were able to survive a lethal dose of OVA-expressing influenza virus. With data highly relevant to vaccine design, this study suggests that HVEM may play an important role in CD8+ MPEC maintenance and subsequent immune responses to respiratory viral challenge.

Gamma delta T17 (γδT17) cells, which express IL-23R and reside in secondary lymphoid organs and at epithelial surfaces, are commonly thought to develop in the thymus shortly before birth and to be maintained in the lymphoid periphery through self-renewal in response to homeostatic cytokine signals. Given that γδT17 cells have been shown to participate in the immunopathology of a variety of inflammatory diseases of epithelial barriers, Muschaweckh et al. (p. 2668) investigated the regulation of γδT17 cell populations during homeostasis and inflammation. Under homeostatic conditions, adult irradiated Rag1−/− mice grafted with either T cell depleted adult bone marrow or fetal liver from IL-23R reporter (Il23rgfp/+) mice lacked IL-23R+γδT17 cells in the thymus, lymph nodes, and spleen. Despite the absence of γδT17 cells in these mice under homeostatic conditions, induction of skin inflammation promoted formation of skin lesions and development of IL-23R+ γδT17 cells that expressed ROR-γt and produced IL-17 in the lymph nodes. TCR stimulation of naive IL-23R γδ T cells from IL-23R reporter mice in the presence of various cytokines revealed that only the combination of IL-1β and IL-23 could induce de novo generation of γδT17 cells from IL-23R γδ cells. Whereas IL-1β appeared to be sufficient to induce low level IL-23R expression in precursor γδT17 cells, IL-23 was indispensable for the expansion of extrathymically generated γδT17 cells in vivo. Analysis of cells from secondary lymphoid tissues of unmanipulated Rag1−/−/Il23rgfp/+ bone marrow chimeric mice revealed that TCR-Vγ4+ chain–expressing CD122IL-23R γδ T cells, but not TCR-Vγ1+ γδ T cells, could be induced to express IL-23R, ROR-γt, and IL-17 in response to IL-1β and IL-23. Finally, whereas analysis of TCR triggering in Nur77 reporter mice revealed no differences between Vγ4+ and Vγ1+ γδ T cells in the peripheral immune compartment, significant TCR engagement in Vγ1+ γδ T cells was observed in the thymus, which would be expected to prevent expression of ROR-γt and favor commitment to the γδT1 lineage. In conclusion, this study demonstrates that although the adult thymus ceases production of γδT17 cells under homeostasis, γδT17 cells can be induced in the periphery from IL-23RCD122Vγ4+ γδ T cell precursors in response to IL-1β under inflammatory conditions. Furthermore, identification of cytokines that differentially induce populations of γδT17 cells under homeostatic versus inflammatory conditions may be relevant in the design of strategies to limit immunopathology in the setting of autoimmunity and chronic inflammation in non-lymphoid tissues.

The generation of eicosanoids, lipids known to promote inflammatory processes including neutrophil chemotaxis and transepithelial migration, is regulated by phospholipase A2 (PLA2), which has several isoforms with distinct activities in different physiological contexts. A better understanding of the specific ways in which PLA2 isoforms regulate production of specific eicosanoids could lead to the development of strategies for more precise therapeutic targeting of inflammatory processes in diseases such as cystic fibrosis. Cytosolic PLA2α (cPLA2α), which is expressed by multiple cell types in the lung, has been shown to promote production of leukotriene B4 (LTB4), an eicosanoid that amplifies neutrophil trans-epithelial migration into the lung. Yonker et al. (p. 2873) therefore sought to determine how cPLA2α might affect bacterial infection-induced neutrophil migration across the lung epithelium. Specific inhibition of cPLA2α in human neutrophils, but not epithelial cells, reduced neutrophil transepithelial migration across cells infected with the pathogenic Pseudomonas aeruginosa strain PAO1. PAO1-induced transepithelial migration was also reduced by pharmacologic inhibition in neutrophils of ERK, which posttranslationally regulates cPLA2α activity, but not of calcium-independent PLA2 (iPLA2) isoforms. Inhibition of either cPLA2α or ERK, but not iPLA2, in neutrophils also reduced neutrophil LTB4 production during PAO1-induced transepithelial migration, suggesting a role for LTB4 in cPLA2α enhancement of neutrophil migration. Similar experiments using mouse cpla2α−/− neutrophils showed reduced migration of these cells across lung epithelial monolayers infected with PAO1 and reduced LTB4 production, relative to wild-type (WT) cells. Interestingly, cpla2α−/− neutrophils retained the ability to respond to LTB4 signals, and normal migration was restored if these cells were mixed with WT neutrophils that were able to produce normal levels of LTB4. As a more physiologic model, human airway basal stem cells were cultured at an air–liquid interface, and migration of primary human neutrophils across the resultant airway epithelial barrier following PAO1 infection was monitored using micro-optical coherence tomography. Migration and LTB4 production were again reduced if neutrophils were treated with a cPLA2α inhibitor. This identification of the specific involvement of cPLA2α in LTB4 production during bacterial infection–induced neutrophil transepithelial migration may lead to strategies to specifically target this enzyme in diseases involving neutrophilic inflammation.