Phosphatidylserine (PtdSer), which is reshuffled from the inner to the outer membrane of apoptotic cells, has been identified previously as a ligand of T cell/transmembrane, Ig, and mucin (TIM) family members TIM-1 and TIM-4. DeKruyff et al. (p. 1918) confirmed that both human and murine TIM-3 also bind PtdSer. Resolution of a cocrystal of PtdSer with an N-terminal portion of murine TIM-3 revealed a binding pocket for PtdSer in the IgV domain that required coordination of a Ca2+ ion. TIM-3 alleles from BALB/c and HBA mice differ by seven amino acids, and HBA TIM-3 had a lower binding affinity for liposome-associated PtdSer compared with BALB/c TIM-3, consistent with polymorphisms in the BC loop of the IgV domain. Fibroblasts transfected with BALB/c TIM-3 were more effective at binding to and taking up apoptotic cells by phagocytosis than HBA TIM-3–transfected cells. In addition, human TIM-3–transfected cells recognized and engulfed apoptotic cells. Lymphoid cells stably transfected with TIM-3 formed conjugates with apoptotic cells but did not phagocytose these cells, suggesting that TIM-3 recognition of apoptotic cells activates proapoptotic responses by B and T cells. These structural insights into TIM-3 and PtdSer interactions better define conserved functions of TIM-3 that may influence autoimmunity and tolerance.

Alloreactive T cells are responsible for graft rejection, and novel strategies are needed to specifically delete these problematic cells. Tanriver et al. (p. 1757) addressed this challenge in a murine model of transplantation by targeting alloreactive MHC class I molecules to dendritic cells. Conventional dendritic cells with a CD11chighCD4+CD8DC inhibitory receptor-2 (DCIR2)+ phenotype, which were shown previously to target alloreactive CD4+ T cells, were the focus of this unique targeting approach. Treatment of mice with an anti-DCIR2 Ab conjugated to either an MHC class I Kd allopeptide (33D1-Kd peptide) or a full-length Kd monomer (33D1-Kd monomer) successfully deleted alloreactive Ag-specific CD4+ T cells with indirect specificity. Treatment with either 33D1-Kd peptide or 33D1-Kd monomer alone did not prolong graft survival in mice. However, Ab-mediated depletion of CD8+ T cells coupled with 33D1-Kd monomer, but not 33D1-Kd peptide, treatment did support indefinite graft survival. Thus, blockade of both CD4+ T cells with indirect specificity and CD8+ T cells with direct specificity was necessary for graft tolerance. These results support development of similar strategies for transplantation tolerance that target T cells with indirect specificity.

Limbal stem cells (LSCs) are known for their roles in the repair and renewal of corneal tissue, and Holan et al. (p. 2124) describe their immunoregulatory capabilities. An LSC-enriched fraction was isolated from murine limbal tissues and identified by expression of the markers p63 and ATP-binding cassette superfamily G member 2. The presence of this LSC fraction inhibited proliferation of splenic lymphocytes stimulated with anti-CD3 together with either the T cell mitogen concavalin A or the B cell mitogen LPS. Inhibition was mediated by a soluble factor, as LSCs separated from lymphocytes by a semipermeable membrane or LSC supernatants still inhibited lymphocyte proliferation. Moreover, LSCs significantly inhibited secretion of IL-2, IL-6, and IFN-γ from concavalin A-stimulated lymphocytes compared with non-LSC limbal cells. Significantly elevated expression levels of Fas ligand and the antiapoptotic genes myeloid cell leukemia sequence 1, survivin, and X-linked inhibitor of apoptosis protein were observed in LSCs, and these cells were more resistant to apoptotic cell death compared with non-LSC cells. LSCs display immunoregulatory functions that can quell inflammatory cytokine responses and provide protection from apoptosis, thus buffering LSCs from damage caused by excess inflammation.

MicroRNAs (miRNAs) have been shown to regulate inflammation in a number of diseases. Oglesby et al. (p. 1702) observed that microRNA-126 (miR-126) is differentially expressed in cystic fibrosis (CF) airway epithelial cells compared with similar non-CF cells. Differential miRNA expression analysis revealed significantly lower levels of miR-126 in CF samples in comparison with non-CF samples. In silico analysis identified a sequence in the 3′-untranslated region of the gene Target of Myb1 (TOM1) as an miR-126 target, supported by evidence from CF and non-CF epithelial cells in which decreased miR-126 expression correlated with a significant increase in TOM1 mRNA expression. Downregulation of TOM1 by miR-126 was validated by cotransfection of pre–miR-126 with a luciferase reporter vector carrying the TOM1 gene and its 3′-untranslated region. TOM1 was previously shown to negatively regulate the IL-1β– and TNF-α–induced signaling pathways through inhibition of NF-κB and AP-1 transcription factors. In CF epithelial cells, TOM1 overexpression inhibited NF-κB responses to LPS or IL-1β and TOM1 knockdown increased NF-κB–mediated IL-8 production induced by LPS or IL-1β. These observations provide a novel link between miR-126 expression and CF airway inflammation that can be explored further for the development of better CF therapies.

IL-23 has been linked to autoimmune diseases by its involvement in the differentiation and expansion of IL-17–producing cells, but less is known about the role of IL-23 during intracellular pathogen infection. Riol-Blanco et al. (p. 1710) now describe a critical function for IL-23R–expressing cells in protecting mice during the transition between innate and adaptive responses against Listeria monocytogenes. IL-23R expression was observed on cells recruited to the peritoneal cavity of mice shortly after i.p. infection with L. monocytogenes. The majority of these cells produced IL-17, and cells were either γδ T cells or CD4CD8 double-negative (DN) αβ T cells. IL-23R expression was not required for recruitment to the site of infection but was needed to induce IL-17 production and promote expansion of these subsets. IL-23R–expressing DN T cells promoted antibacterial responses through production of IFN-γ and TNF-α in addition to IL-17. Furthermore, adoptive transfer of DN T cells from L. monocytogenes-infected wild-type mice into RAG2−/− mice prior to bacterial challenge significantly reduced bacterial replication in otherwise immunocompromised mice. Together, these results suggest that IL-23R–mediated production of IL-17 from γδ and DN T cells helps bridge gaps in protection during the transition between innate and adaptive immune responses to intracellular bacteria.

T cell development is governed by encounters with various cell types during movement from the thymic cortex to the medulla. Subsequent egress from the medulla to the periphery involves changes in the expression of multiple adhesion molecules on the surface of single-positive (SP) CD4+ and CD8+ T cells. Qiu et al. (p. 1681) have identified a new role for the adhesion receptor CD155 in SP T cell retention in the medulla prior to emigration. CD155-deficient mice or wild-type adult mice treated with anti-CD155 Ab had significantly fewer mature SP CD8+ T cells in their thymi compared with untreated wild-type mice, but the frequency of SP CD4+ T cells was unaffected. Wild-type mice showed an age-dependent accumulation of mature SP CD8+ T cells in the thymus from birth to maturity at 8–10 wk of age, but the frequency of these cells was chronically low in the thymi of CD155-deficient mice throughout this period. The lack of mature SP CD8+ T cells in the thymi of mutant mice was linked to premature egress of these cells from the medulla. In addition, a shift in the TCR repertoire toward autoantigen-specific CD8+ T cells was observed in CD155-deficient mice compared with wild-type mice, indicating a defect in negative selection. These results define a novel role for CD155 in CD8+ T cell development during negative selection and thymic egress.

Little is known about the function of forkhead box P3+CD25+CD8+ T cells in vivo during pathogenic SIV infection. Nigam et al. (p. 1690) present evidence that these cells suppress antiviral SIV immune responses. Typically, very low frequencies of CD8+ T cells expressing CD25 and forkhead box P3 were observed in the blood and colorectal mucosa of uninfected rhesus macaques. SIV infection induced rapid expansion of CD8+ regulatory T cells (Tregs) in the blood and even greater expansion in the colorectal mucosa, a preferred tissue for viral replication. CD8+ Tregs from chronically SIV-infected macaques expressed markers associated with regulatory functions, including CTLA-4 and CD39. These cells suppressed proliferation of SIV-specific T cells in vitro, and the frequency of CD8+ Tregs corresponded directly to viremia and inversely to the levels of SIV-specific T cell responses in vivo. Hyperimmune activation was implicated as a mechanism of CD8+ Treg activation because these cells also proliferated in HIV-infected humans but not in SIV-infected sooty mangebeys, a natural SIV host that is typically asymptomatic. Thus, expansion of CD8+ Tregs is associated with suppression of anti-SIV T cell responses, suggesting that therapeutic intervention targeting these cells may improve antiviral immunity.

Coligation of BCR and FcγRIIB molecules by IgG immune complexes (IgG-ICs) blocks B cell signaling through inhibition of BCR phosphorylation. Liu et al. (p. 1977) have used state-of-the-art live cell imaging to more precisely define the mechanisms and kinetics of this FcγRIIB-mediated inhibition. Nearly all BCR and FcγRIIB molecules on the surface of primary B cells exposed to membrane-associated IgG-ICs colocalized into microclusters compared with untreated cells or cells exposed to F(ab′)2-ICs to crosslink BCRs. Despite this colocalization, IgG-IC treatment limited the accumulation of BCRs in microclusters and slowed the process of B cell spreading and contraction on these APC-like membranes. Further live-cell single-molecule analyses showed that the rapid and stable incorporation of FcγRIIB into lipid rafts blocked the formation of immobile BCR oligomers with cytoplasmic domains equipped for BCR signaling. This inhibition was dependent on intact FcγRIIB signaling. These observations describe an earlier role for FcγRIIB in limiting B cell activation that was not previously appreciated by biochemical analyses and may aid in the development of novel therapeutic interventions for autoimmune diseases.

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