BackgroundHuman memory CD4+ T cells can be either CD300a/c+ or CD300a/c- and subsequent analyses showed that CD4+ effector memory T (TEM) cells are mostly CD300a/c+, whereas CD4+ central memory T (TCM) cells have similar frequencies of CD300a/c+ and CD300a/c- cells.ResultsExtensive phenotypical and functional characterization showed that in both TCM and TEM cells, the CD300a/c+ subset contained a higher number of TH1 (IFN-γ producing) cells. Alternatively, TH17 (IL-17a producing) cells tend to be CD300a/c-, especially in the TEM subset. Further characterization of the IL-17a+ cells showed that cells that produce only this cytokine are mostly CD300a/c-, while cells that produce IL-17a in combination with other cytokines, especially IFN-γ, are mostly CD300a/c+, indicating that the expression of this receptor is associated with cells that produce IFN-γ. Co-ligation of the TCR and CD300a/c in CD4+ T cells inhibited Ca2+ mobilization evoked by TCR ligation alone and modulated IFN-γ production on TH1 polarized cells.ConclusionWe conclude that the CD300a/c receptors are differentially expressed on human TH1 and TH17 cells and that their ligation is capable of modulating TCR mediated signals.
The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.
Human CD300 molecules comprise a family of receptors that regulate many immune cell processes. They are mostly expressed on myeloid cells, although expression of two members, CD300a and CD300c, has also been described on lymphocytes. However, due to the lack of specific antibodies that distinguish between these two receptors, it has been difficult to determine the expression pattern and function of CD300a and CD300c in primary cells. Here, we have identified a specific monoclonal antibody, clone TX45, that recognizes only CD300c and show that within freshly isolated blood leukocytes, monocytes are the only cells that express CD300c on the cell surface. In vitro differentiation experiments revealed that CD300c is differentially expressed on different monocyte-derived cells, including macrophages and dendritic cells. Furthermore, TLR ligands LPS and flagellin dynamically regulate the expression of CD300c. Cross-linking of this receptor with clone TX45 monoclonal antibody induced calcium mobilization, upregulation of the costimulatory molecule CD86 and the production of inflammatory cytokines. Importantly, LPS-mediated production of inflammatory cytokines by monocytes was further enhanced if CD300c was simultaneously engaged by the agonist antibody. Altogether, our results show that human CD300c is an activating receptor expressed on monocytes and that it has a potential role in inflammatory responses.
BackgroundActivation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes.ResultsWe have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5’ phosphatase SHIP, was utilized by CD300a for its inhibitory activity.ConclusionThese studies provide new insights into the function of CD300a in tuning T and B cell responses.
B-acute lymphoblastic leukemia (ALL) is a malignancy of bone marrow-derived precursor-B cells and represents the leading cause of pediatric cancer death. Suppressive signals induced in the marrow of ALL patients may explain both the failure of host CD8 surveillance and inconsistent responses to adoptive CD8 therapy. We hypothesize that ALL induces a caspase-1 driven inflammasome in bone marrow monocyte/macrophages (BM-MM) that modulates CD8 function and contributes to relapse. To determine whether BM-MM could produce soluble factors that suppress CD8 function in the setting of ALL, we treated CFSE-labeled CD8 from healthy donor PBMC with supernatants from cocultures of primary human ALL and either CD14+ BM-MM or stromal fibroblast controls. CD8 exposed to supernatants from ALL cultured alone or with stromal fibroblasts maintained 100% proliferation, while those exposed to BM-MM cocultures failed to proliferate, regardless of whether ALL was cultured directly or in transwell with BM-MM. Cleaved caspase-1 was detected, both by qPCR (2.2-fold expression over endogenous control) and Western blot from BM-MM cultured with ALL, but not stromal fibroblasts Similar levels of soluble IL1B were detected from stromal fibroblast and BM-MM cocultures. Current studies are determining whether the reduced proliferative capacity observed in CD8 corresponds to impaired antileukemia killing and identifying specific signals downstream of caspase-1 that may be targeted to reverse this phenomenon.
High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment. We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models. To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1. Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion. These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. Disclosures No relevant conflicts of interest to declare.
Quercetin is an antioxidant previously shown to inhibit acute lymphoblastic leukemia (ALL) growth and induce both apoptosis and autophagy in malignant hematologic cell lines derived from AML and T-ALL. Since autophagy is a critical mechanism in the response to cellular stress, we hypothesized that high-risk ALL cells had higher resting autophagy than standard risk ALL cells. To test our hypothesis, we quercetin-treated stable cell cultures of ALL isolated from the diagnostic marrow specimens of one clinically high-risk (HR-ALL, an infant carrying the t(4;11) MLL-rearrangement) and one cytogenetically normal standard-risk pediatric patient (SR-ALL), and compared viability and the expression of molecular markers of programmed cell death and autophagy by Western Blot. First, we established a dose-response curve that identified 50 uM quercetin as the target dose for potency and efficacy in HR- and SR-ALL. Next, we compared viability between HR-and SR-ALL treated with 50 uM quercetin at 3, 12, 24 and 48 hours. HR-ALL treated with quercetin exhibited a significantly greater reduction in viability measured by Annexin V/PI staining and MTT reagent conversion (2-way ANOVA p = 0.04 and p = 0.005). HR-ALL cells also exhibited greater catalytic cleavage and activation programmed death markers, Caspase 3 and PARP1 by Western Blot, compared to SR-ALL. Because high levels of autophagy can induce cell death, we asked whether HR-ALL cells had a higher resting autophagy rate. We compared expression of the prototypic autophagy protein, Beclin-1 at 30min, 1hr, 2hr, 3hr, and 4hr after treatment with 10mM of the autophagy inhibitor 3-methyladenine (3-MA). Because 3-MA inhibits autophagy at the early stage of nucleation, the rate of breakdown of downstream proteins such as Beclin-1 is a proxy measure for the rate of resting, non-stress induced autophagy. HR-ALL cells exhibited a significantly greater rate of Beclin-1 breakdown after 3-MA treatment as compared to SR-ALL cells (slope -0.235 vs. 0.131, p = 0.003) consistent with a higher resting autophagy rate in HR-ALL. To determine whether quercetin treatment impacts this higher rate of autophagy, we treated HR-ALL and SR-ALL cells with 50uM of quercetin for 24hrs and compared, by Western blot, the expression of regulatory proteins at each stage of autophagy: nucleation (ATG7, Beclin-1), expansion (ATG5, ATG16L1) and maturation (LC3A/B). Unexpectedly, quercetin inhibited expression of all autophagy proteins, with a greater decrease in HR-ALL. We next parsed whether selective autophagy inhibition at formation of the autophagosome, using 3MA, or later endosome acidification, using chloroquine, could explain the differential cytotoxicity of quercetin in HR-ALL. 3MA and chloroquine treatment alone resulted in significantly fewer Annexin+/PI+ HR- and SR-ALL cells and less MTT conversation compared to quercetin treatment alone (p<0.05) suggesting that selective inhibition of the autophagy complex was insufficient to explain quercetin cytotoxicity. Finally, we asked whether differences in an upstream regulator of the autophagy/apoptotic balance contribute to higher quercetin toxicity in HR-ALL cells. The regulatory protein high mobility group box-1 (HMGB1) has been shown to induce autophagy by interacting with Beclin-1, and we have previously demonstrated overexpression of HMGB1 in HR-ALL. We verified interaction of HMGB1 with Beclin-1 by immunoprecipitation, and observed a substantial reduction in complex formation after 50 uM quercetin treatment in HR-ALL cells. (1511153 vs. 70442 Beclin-1 band volumes after IP p<0.001), along with decreased expression of the anti-apoptotic proteins, Bcl-2 and Mcl-1, factors proposed to be regulated by the Beclin-1/HMGB1 complex. Taken together, these data indicate that regulation of the autophagy-apoptotic balance is a mechanism used by ALL cells to mitigate cellular stress, and is the first to describe the effects of quercetin in the context of autophagy in ALL. Future studies will compare the expression profile of critical autophagy proteins following chemotherapy treatment in a larger cohort of high-risk ALL cells, and delineate the role of HMGB1 as an autophagy regulator using our established knockdown model. Disclosures No relevant conflicts of interest to declare.
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