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This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named “L-DC” since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties. [ABSTRACT FROM AUTHOR]

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates. [ABSTRACT FROM AUTHOR]

Forkhead box P3 (Foxp3+) regulatory T (Treg)-cell function is controlled by environmental cues of which cytokine-mediated signaling is a dominant component. In vivo, interleukin-4 (IL-4)-mediated signaling via IL-4 receptor alpha (IL-4Rα) mediates Treg cell transdifferentiation into ex-Foxp3 T helper 2 (Th2) or T helper 17 (Th17) cells. However, IL-4-mediated signaling also reinforces the Foxp3 Treg compartment in vitro. We generated Foxp3-specific IL-4Rα-deficient mice and demonstrated differential efficiency of IL-4Rα deletion in male (approximately 90%) and female (approximately 40%) animals, because of cyclic recombinase (Cre)-mediated X-linked foxp3 inactivation. Irrespective of the degree of IL-4Rα deletion within the Foxp3+ Treg cell population, mice showed exacerbation of immune effector responses with aggravated tissue pathology in tissue-dwelling helminth infections (Schistosoma mansoni or Nippostrongylus brasiliensis). Mechanistically, IL-4Rα deletion in males and females led to a reduced expression of Foxp3 and subsequently an impaired accumulation of Foxp3+ Treg cells to inflamed tissues. In-depth cellular typing by flow cytometry revealed that the impairment of IL-4Rα-mediated signaling during helminth infections decreased the ability of central Treg cells to convert into effector Treg (eTreg) cells and caused a significant down-regulation of markers associated with Treg cell migration (C-X-C motif chemokine receptor 3 [CXCR3]) and accumulation in inflamed tissues (GATA binding protein 3 [GATA3]) as well as survival (B cell lymphoma 2 [Bcl-2]). These findings unprecedentedly, to our knowledge, uncover a role for IL-4Rα signaling in the positive regulation of Foxp3+ Treg cell function in vivo. Complementing our past knowledge on a widely reported role for IL-4Rα signaling in the negative regulation and transdifferentiation of Foxp3+ Treg cells in vivo, our present findings reveal the host requirement for an intact, but not reduced or potentiated, IL-4Rα-mediated signaling on Foxp3+ Treg cells to optimally control inflammation during helminth infections. [ABSTRACT FROM AUTHOR]

Programmed cell death-1 (PD-1) is an inhibitory immune checkpoint receptor that negatively regulates the functioning of T cell. Although the direct targets of PD-1 were not identified, its inhibitory action on the TCR signaling pathway was known much earlier. Recent experiments suggest that the PD-1 inhibits the TCR and CD28 signaling pathways at a very early stage ─ at the level of phosphorylation of the cytoplasmic domain of TCR and CD28 receptors. Here, we develop a mathematical model to investigate the influence of inhibitory effect of PD-1 on the activation of early TCR and CD28 signaling molecules. Proposed model recaptures several quantitative experimental observations of PD-1 mediated inhibition. Model simulations show that PD-1 imposes a net inhibitory effect on the Lck kinase. Further, the inhibitory effect of PD-1 on the activation of TCR signaling molecules such as Zap70 and SLP76 is significantly enhanced by the PD-1 mediated inhibition of Lck. These results suggest a critical role for Lck as a mediator for PD-1 induced inhibition of TCR signaling network. Multi parametric sensitivity analysis explores the effect of parameter uncertainty on model simulations. [ABSTRACT FROM AUTHOR]

High usage of progestin-only injectable contraceptives, which include the intramuscular injectables depo-medroxyprogesterone acetate (DMPA-IM, Depo-Provera) and norethisterone (NET) enanthate (NET-EN or Nur-Isterate), correlates worldwide with areas of high HIV-1 prevalence. Epidemiological data show a significant association between usage of DMPA-IM and increased HIV-1 acquisition but no such association from limited data for NET-EN. Whether MPA and NET have similar effects on HIV-1 acquisition and pathogenesis, and the relationship between these effects and the dose of MPA, are critical issues for women’s health and access to suitable and safe contraceptives. We show for the first time that MPA, unlike NET, significantly increases HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and a cervical cell line model. The results provide novel evidence for a biological mechanism whereby MPA, acting via the glucocorticoid receptor (GR), increases HIV-1 replication by at least in part increasing expression of the CCR5 HIV-1 coreceptor on target T-lymphocytes. MPA, unlike NET, also increases activation of T-cells and increases the CD4/CD8 ratio, suggesting that multiple mechanisms are involved in the MPA response. Our data offer strong support for different biological mechanisms for MPA versus NET, due to their differential GR activity. The dose-dependence of the MPA response suggests that significant effects are observed within the range of peak serum levels of progestins in DMPA-IM but not NET-EN users. Dose-response results further suggest that effects of contraceptives containing MPA on HIV-1 acquisition and disease progression may be critically dependent on dose, time after injection and intrinsic factors that affect serum concentrations in women. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection. [ABSTRACT FROM AUTHOR]

Classical or M1 activity of microglia/macrophages has been described in several neurodegenerative and brain inflammatory conditions and has also been linked to expansion of ischemic injury in post-stroke brain. While different pathways of M1 polarization have been suggested to occur in the post-stroke brain, the precise underlying mechanisms remain undefined. Using a transient middle cerebral artery occlusion (MCAO) rat model, we showed a progressive M2 to M1 polarization in the perilesional brain region with M1 cells becoming one of the dominant subsets by day 4 post-stroke. Comparing key receptors involved in M1 polarization (CD8, IFNγR, Clec4, FcγR, TLR3 and TLR4) and their signal transducers (Syk, Stat1, Irf3, and Traf6) at the day 4 time point, we showed a strong upregulation of CD8 along with SYK transducer in dissected perilesional brain tissue. We further showed that CD8 expression in the post-stroke brain was associated with activated (CD68+) macrophages and that progressive accumulation of CD8+CD68+ cells in the post-stroke brain coincided with increased iNOS (M1 marker) and reduced Arg1 (M2 marker) expression on these cells. In vitro ligand-based stimulation of the CD8 receptor caused increased iNOS expression and an enhanced capacity to phagocytose E. coli particles; and interestingly, CD8 stimulation was also able to repolarize IL4-treated M2 cells to an M1 phenotype. Our data suggest that increased CD8 signaling in the post-stroke brain is primarily associated with microglia/macrophages and can independently drive M1 polarization, and that modulation of CD8 signaling could be a potential target to limit secondary post-stroke brain damage. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are promising candidates for skin wound repair due to their capabilities of accumulating at wounds and differentiating into multiple types of skin cells. However, the underlying mechanisms responsible for these processes remain unclear. In this study, we found that osteopontin (OPN) stimulated the migration of MSCs in vitro, and observed the recruitment of endogenous MSCs to a skin wound and their differentiation into keratinocytes and endothelial cells. In OPN knock-out mice, the recruitment of MSCs to the skin wound was significantly inhibited, and wound closure was hampered after an intradermal injection of exogenous MSCs compared to wild-type mice. Consistent with these observations, the expressions of adhesion molecule CD44 and its receptor E-selectin were significantly decreased in the lesions of OPN knock-out mice compared with wild-type mice suggesting that OPN may regulate the migration of MSCs through its interactions with CD44 during skin wound recovery. In summary, our data demonstrated that OPN played a critical role in activating the migration of MSCs to injured sites and their differentiation into specific skin cell types during skin wound healing. [ABSTRACT FROM AUTHOR]

Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. [ABSTRACT FROM AUTHOR]

Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. [ABSTRACT FROM AUTHOR]

Context: Fibrocytes appear to participate in inflammation and tissue remodeling in patients with thyroid-associated ophthalmopathy (TAO). These patients have increased frequencies of circulating TSH receptor (TSHR)- and CD40-positive fibrocytes, suggesting TSHR and CD40 may play roles in proinflammatory cytokine production, which ultimately leads to orbital inflammation and tissue remodeling. Objective: To investigate the potential interactions between the TSHR and CD40 signaling pathways and their roles in IL-6 and TNF-α production. Design and Outcome Measures: CD40 expression on fibrocytes was assessed using flow cytometry; IL-6 and TNF-α protein release using Luminex technology; increased IL-6 and TNF-α mRNA abundance, using real-time PCR; TSH- and CD40 ligand (CD40L)-stimulated Akt phosphorylation in fibrocytes, by western blot analysis; TSHR-CD40 protein-protein interaction, using co-immunoprecipitation, and CD40-TSHR co-localization, using immunocytochemistry. Results: TSH enhances CD40 expression at a pre-translational level in fibrocytes. Production of IL-6 and TNF-α after costimulation with TSH and CD40L was greater than that after TSH or CD40L stimulation alone. TSH and CD40L costimulation also resulted in greater Akt phosphorylation. Akt and nuclear factor (NF)-κB inhibitors significantly reduced cytokine production after TSH and CD40L costimulation. TSHR and CD40L are colocalized on the cell surface and form a complex. Conclusions: TSHR and CD40 in fibrocytes appear to be physically and functionally related. TSH stimulates CD40 production on the fibrocyte surface. Cytokine expression upon simultaneous stimulation of TSHR and CD40 is greater than levels achieved with TSH or CD40L alone. Increased expression of CD40 by TSH is a potential mechanism for this process. [ABSTRACT FROM AUTHOR]

Objective: To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production. Methods: RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. Results: Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways. Conclusions: Disruptive changes in CD44-HA interactions by HAoligos enhanced MMP-1 and MMP-3 production via activation of NF-κB and p38 MAPK signaling pathways in RSFs. [ABSTRACT FROM AUTHOR]

Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability. [ABSTRACT FROM AUTHOR]

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability. [ABSTRACT FROM AUTHOR]

Background: Hypothetically, psychotic disorders could be caused or conditioned by immunological mechanisms. If so, one might expect there to be peripheral immune system phenotypes that are measurable in blood cells as biomarkers of psychotic states. Methods: We used multi-parameter flow cytometry of venous blood to quantify and determine the activation state of 73 immune cell subsets for 18 patients with chronic schizophrenia (17 treated with clozapine), and 18 healthy volunteers matched for age, sex, BMI and smoking. We used multivariate methods (partial least squares) to reduce dimensionality and define populations of differentially co-expressed cell counts in the cases compared to controls. Results: Schizophrenia cases had increased relative numbers of NK cells, naïve B cells, CXCR5+ memory T cells and classical monocytes; and decreased numbers of dendritic cells (DC), HLA-DR+ regulatory T-cells (Tregs), and CD4+ memory T cells. Likewise, within the patient group, more severe negative and cognitive symptoms were associated with decreased relative numbers of dendritic cells, HLA-DR+ Tregs, and CD4+ memory T cells. Motivated by the importance of central nervous system dopamine signalling for psychosis, we measured dopamine receptor gene expression in separated CD4+ cells. Expression of the dopamine D3 (DRD3) receptor was significantly increased in clozapine-treated schizophrenia and covaried significantly with differentiated T cell classes in the CD4+ lineage. Conclusions: Peripheral immune cell populations and dopaminergic signalling are disrupted in clozapine-treated schizophrenia. Immuno-phenotypes may provide peripherally accessible and mechanistically specific biomarkers of residual cognitive and negative symptoms in this treatment-resistant subgroup of patients. [ABSTRACT FROM AUTHOR]

SAP (SH2D1A) is required intrinsically in CD4 T cells to generate germinal center responses and long-term humoral immunity. SAP binds to SLAM family receptors, including SLAM, CD84, and Ly108 to enhance cytokine secretion and sustained T cell:B cell adhesion, which both improve T follicular helper (Tfh) cell aid to germinal center (GC) B cells. To understand the overlapping roles of multiple SLAM family receptors in germinal center responses, Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption (Slamf1,5,6Δ/Δ) mice were generated using CRISPR-Cas9 gene editing to eliminate expression of SLAM (CD150), CD84, and Ly108, respectively. Gene targeting was highly efficient, with 6 of 6 alleles disrupted in 14 of 23 pups and the majority of alleles disrupted in the remaining pups. NKT cell differentiation in Slamf1,5,6Δ/Δ mice was defective, but not completely absent. The remaining NKT cells exhibited substantially increased 2B4 (SLAMF4) expression. Surprisingly, there were no overt defects in germinal center responses to acute viral infections or protein immunizations in Slamf1,5,6Δ/Δ mice, unlike Sh2d1a-/- mice. Similarly, in the context of a competitive environment, SLAM family receptor expressing GC Tfh cell, GC B cell, and plasma cell responses exhibited no advantages over Slamf1,5,6Δ/Δ cells. [ABSTRACT FROM AUTHOR]

Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases. [ABSTRACT FROM AUTHOR]

Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells’ ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated “missing-self” reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors. [ABSTRACT FROM AUTHOR]

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide. [ABSTRACT FROM AUTHOR]

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis. [ABSTRACT FROM AUTHOR]

HIV-1 fusion leading to productive entry has long been thought to occur at the plasma membrane. However, our previous single virus imaging data imply that, after Env engagement of CD4 and coreceptors at the cell surface, the virus enters into and fuses with intracellular compartments. We were unable to reliably detect viral fusion at the plasma membrane. Here, we implement a novel virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment—at the plasma membrane or, possibly, in early pH-neutral vesicles. Virus particles are co-labeled with an intra-viral content marker, which is released upon fusion, and an extra-viral pH sensor consisting of ecliptic pHluorin fused to the transmembrane domain of ICAM-1. This sensor fully quenches upon virus trafficking to a mildly acidic compartment, thus precluding subsequent detection of viral content release. As an interesting secondary observation, the incorporation of the pH-sensor revealed that HIV-1 particles occasionally shuttle between neutral and acidic compartments in target cells expressing CD4, suggesting a small fraction of viral particles is recycled to the plasma membrane and re-internalized. By imaging viruses bound to living cells, we found that HIV-1 content release in neutral-pH environment was a rare event (~0.4% particles). Surprisingly, viral content release was not significantly reduced by fusion inhibitors, implying that content release was due to spontaneous formation of viral membrane defects occurring at the cell surface. We did not measure a significant occurrence of HIV-1 fusion at neutral pH above this defect-mediated background loss of content, suggesting that the pH sensor may destabilize the membrane of the HIV-1 pseudovirus and, thus, preclude reliable detection of single virus fusion events at neutral pH. [ABSTRACT FROM AUTHOR]

Background: CD8+ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8+ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated. Methods: Circulating CD8+ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8+ Tc/Tregs. Results: While COPD patients have elevated circulating percentage of CD8+ T cells, healthy smokers have higher frequency of CD8+ Tregs. Elevated percentages of CD8+ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8+ T cells, while facilitated both the proliferation and apoptosis of CD8+ Tregs. Notably, the effects of CSE on CD8+ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8+ Tregs. Conclusion: The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation. [ABSTRACT FROM AUTHOR]