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It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms. One of these mechanisms is the NKG2D receptor that recognizes ligands whose expression is induced by cell transformation. In this study, we show that in NKG2D receptor-deficient mice, increasing numbers of B cells begin to express NKG2D ligands as they age. Their absence in wild-type mice suggests that these cells are normally cleared by NKG2D-expressing cells. NKG2D-deficient mice and mice constitutively expressing NKG2D ligands had increased incidence of B cell tumors, confirming that the inability to clear NKG2D ligand-expressing cells was important in tumor suppression and that NKG2D ligand expression is a marker of nascent tumors. Supporting a role for NKG2D ligand expression in controlling the progression of early-stage B cell lymphomas in humans, we found higher expression of a microRNA that inhibits human NKG2D ligand expression in tumor cells from high-grade compared with low-grade follicular lymphoma patients., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux after stimulation with an APC in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naive T cells in the lymph node occurs in distinct phases. In phase I, T cells make multiple transient contacts with dendritic cells before entering a phase II, where they exist in stable clusters with dendritic cells. It has been suggested that T cells signal during transient contacts of phase I, but this has never been shown directly. Because time-dependent loss of calcium dyes from cells hampers long-term imaging of cells in vivo after antigenic stimulation, we generated a knock-in mouse expressing a modified form of the Cameleon fluorescence resonance energy transfer reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro, we observed transient, oscillatory, and sustained calcium flux after contact with APC, but these behaviors were not affected by the type of APC or Ag quantity, but were, however, moderately dependent on Ag quality. In vivo, we found that during phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naive T cells signal during phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of phase II., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Plasmacytoid dendritic cells (pDCs) are a dendritic cell subset that secrete type I IFNs in response to microbial stimuli. The scaffold protein, CD2-associated protein (CD2AP), is a marker of human pDCs as it is highly expressed in this cell type. Recently, in human pDCs, decreased CD2AP expression appeared to enhance the production of type I IFNs via an inhibitory receptor-induced signaling cascade. In this study, we sought to determine the role of CD2AP in murine pDCs using CD2AP knockout (KO) mice. CD2AP was dispensable for the development of pDCs and for the upregulation of activation markers following stimulation. Loss of CD2AP expression did not affect the production of type I IFNs stimulated by TLR ligation, and only slightly impaired type I IFN production when inhibitory pathways were engaged in vitro. This was also confirmed by showing that CD2AP deficiency did not influence type I IFN production by pDCs in vivo. Because CD2AP plays a role in regulating actin dynamics, we examined the actin cytoskeleton in pDCs and found that activated CD2AP KO pDCs had significantly higher levels of actin polymerization than wild-type pDCs. Using two different inflammation models, we found that CD2AP KO pDCs have a defect in lymph node migration, correlating with the defects in actin dynamics. Our work excludes a role for CD2AP in the regulation of type I IFNs in pDCs, and suggests that the major function of CD2AP is on the actin cytoskeleton, affecting migration to local lymph nodes under conditions of inflammation.

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.

Engagement of a T cell to an APC induces the formation of an immunological synapse as well as reorientation of the microtubule-organizing center (MTOC) toward the APC. How signals emanating from the TCR induce MTOC polarization is not known. One group of proteins known to play a critical role in asymmetric cell division and cell polarization is the partitioning defective (Par) family of proteins. In this study we found that Par1b, a member of the Par family of proteins, was inducibly phosphorylated following TCR stimulation. This phosphorylation resulted in 14-3-3 protein binding and caused the relocalization of Par1b from the membrane into the cytoplasm. Because a dominant-negative form of Par1b blocked TCR-induced MTOC polarization, our data suggest that Par1b functions in the establishment of T cell polarity following engagement to an APC.

Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFbeta decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.

The mechanism underlying the transient accumulation of CD4 at the immunological synapse (IS) and its significance for T cell activation are not understood. To investigate these issues, we mutated a serine phosphorylation site (S408) in the cytoplasmic tail of murine CD4. Preventing phosphorylation of S408 did not block CD4 recruitment to the IS; rather, it blocked the ability of CD4 to leave the IS. Surprisingly, enhanced and prolonged CD4 accumulation at the supramolecular activation cluster in the contact area had no functional consequence for T cell activation, cytokine production, or proliferation. Protein kinase C theta (PKCtheta)-deficient T cells also displayed enhanced and prolonged accumulation of wild-type CD4 at the IS, indicating that theta is the critical PKC isoform involved in CD4 movement. These findings suggest a model wherein recruitment of CD4 to the IS allows its phosphorylation by PKCtheta and subsequent removal from the IS. Thus, an important role for PKCtheta in T cell activation involves its recruitment to the IS, where it phosphorylates specific substrates that help to maintain the dynamism of protein turnover at the IS.

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.

The MAPK ERK is required for LPS-induced TNF production by macrophages. Although the scaffold kinase suppressor of Ras (KSR)1 is required for efficient Erk activation by mitogenic stimuli, the role of KSR1 in ERK activation by inflammatory and stress stimuli is unknown. In this study, we examined the effects of KSR deficiency on ERK activation by stress stimuli and show that ERK activation by TNF, IL-1, and sorbitol is attenuated in the absence of KSR1. To determine the significance of this defect in vivo, we tested KSR-deficient mice using a passive transfer model of arthritis. We found that the induction of arthritis is impaired in the absence of KSR. Thus, KSR plays a role in ERK activation during inflammatory and stress responses both in vitro and in vivo.

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-gamma. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1epsilon was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-gamma release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1epsilon or B7-1 compared with untransfected P815. In addition, both RAE1epsilon and B7-1 enhanced Ag-independent IFN-gamma secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.

Signals mediated by the p38alpha MAPK have been implicated in many processes required for the development and effector functions of innate and adaptive immune responses. As mice deficient in p38alpha exhibit embryonic lethality, most analyses of p38alpha function in lymphocytes have relied on the use of pharmacologic inhibitors and dominant-negative or constitutively active transgenes. In this study, we have generated a panel of low passage p38alpha(+/+), p38alpha(+/-), and p38alpha(-/-) embryonic stem (ES) cells through the intercrossing of p38alpha(+/-) mice. These ES cells were used to generate chimeric mice by RAG-deficient blastocyst complementation, with the lymphocytes in these mice being derived entirely from the ES cells. Surprisingly, B and T cell development were indistinguishable when comparing chimeric mice generated with p38alpha(+/+), p38alpha(+/-), and p38alpha(-/-) ES cell lines. Moreover, proliferation of p38alpha(-/-) B and T cells in response to Ag receptor and non-Ag receptor stimuli was intact. Thus, p38alpha is not an essential component of signaling pathways required for robust B and T lymphocyte developmental, nor is p38alpha essential for the proliferation of mature B and T cells.

The poliovirus receptor (PVR) belongs to a large family of Ig molecules called nectins and nectin-like proteins, which mediate cell-cell adhesion, cell migration, and serve as entry receptors for viruses. It has been recently shown that human NK cells recognize PVR through the receptor DNAM-1, which triggers NK cell stimulation in association with beta(2) integrin. In this study, we show that NK cells recognize PVR through an additional receptor, CD96, or T cell-activated increased late expression (Tactile). CD96 promotes NK cell adhesion to target cells expressing PVR, stimulates cytotoxicity of activated NK cells, and mediates acquisition of PVR from target cells. Thus, NK cells have evolved a dual receptor system that recognizes nectins and nectin-like molecules on target cells and mediates NK cell adhesion and triggering of effector functions. As PVR is highly expressed in certain tumors, this receptor system may be critical for NK cell recognition of tumors.

Although the accumulation of lipid rafts at the immunological synapse is now well accepted, the degree of the accumulation, the localization within the fine structure of the immunological synapse, and the region from which lipid rafts are recruited have not been defined. In this work we show that lipid rafts preferentially accumulate in the central zone of the immunological synapse, the central supramolecular activation complex (C-SMAC). However, quantitative analyses indicate that the level of recruitment of lipid rafts to the C-SMAC is relatively small and suggests that rearrangement of lipid rafts from the peripheral zone of the synapse into the C-SMAC can account for this accumulation. We also assessed the effects of CD28 deficiency on lipid raft recruitment to the immunological synapse. The accumulation of lipid occurred independently of the CD28/B7 system and was not measurably altered by CD28.