Christy A. Tarleton

Characterization of Myeloid and Plasmacytoid Dendritic Cells in Human Lung

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c+CD11c+CD14−HLA-DR+ cells and comprised ∼2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123+CD11c−CD14−HLA-DR+ cells and comprised ∼1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c+CD14− lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c+CD14+ lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-α than MDC-enriched cells. MDC-enriched cells were either CD123+ or CD123−, but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-α upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.

Histamine Release from the Basophils of Control and Asthmatic Subjects and a Comparison of Gene Expression between “Releaser” and “Nonreleaser” Basophils

Most human blood basophils respond to FcεRI cross-linking by releasing histamine and other inflammatory mediators. Basophils that do not degranulate after anti-IgE challenge, known as “nonreleaser” basophils, characteristically have no or barely detectable levels of the Syk tyrosine kinase. The true incidence of the nonreleaser phenotype, its relationship (if any) to allergic asthma, and its molecular mechanism are not well understood. In this study, we report statistical analyses of degranulation assays performed in 68 control and 61 asthmatic subjects that establish higher basal and anti-IgE-stimulated basophil degranulation among the asthmatics. Remarkably, 28% of the control group and 13% of the asthmatic group were nonreleasers for all or part of our 4-year long study and cycling between the releaser and nonreleaser phenotypes occurred at least once in blood basophils from 8 (of 8) asthmatic and 16 (of 23) control donors. Microarray analysis showed that basal gene expression was generally lower in nonreleaser than releaser basophils. In releaser cells, FcεRI cross-linking up-regulated >200 genes, including genes encoding receptors (the FcεRI α and β subunits, the histamine 4 receptor, the chemokine (C-C motif) receptor 1), signaling proteins (Lyn), chemokines (IL-8, RANTES, MIP-1α, and MIP-1β) and transcription factors (early growth response-1, early growth response-3, and AP-1). FcεRI cross-linking induced fewer, and quite distinct, transcriptional responses in nonreleaser cells. We conclude that “nonreleaser” and “cycler” basophils represent a distinct and reversible natural phenotype. Although histamine is more readily released from basophils isolated from asthmatics than controls, the presence of nonreleaser basophils does not rule out the diagnosis of asthma.

Reduced Fc RI-Mediated Release of Asthma-Promoting Cytokines and Chemokines from Human Basophils during Omalizumab Therapy

Background: Treating asthmatics with the humanized IgE-scavenging antibody, omalizumab (rhuMAb-E25, Xolair®), reduces airways inflammation and asthma symptoms. Previously, omalizumab was shown to cause a dramatic and reversible loss of cell surface high-affinity IgE receptors, FcΕRI, from the peripheral blood basophils of asthmatics. The consequences of receptor loss for the FcΕRI-mediated synthesis and release of cytokines implicated in allergic asthma have not been examined. Methods: Fifteen asthmatic volunteers each received omalizumab for 12 weeks. Peripheral blood basophils were isolated before, during, 2 weeks after and 6 months after omalizumab. Basophils were assayed for the basal and anti-IgE-stimulated release of cytokines, chemokines and histamine. Pooled data were analyzed by repeated measures ANOVA and by paired t tests. Results: Anti-IgE-stimulated human basophils synthesize and release Th2 cytokines (IL-4, IL-13) and chemokines (IL-8, RANTES). The anti-IgE-stimulated release of IL-4, IL-13 and IL-8 was reduced during omalizumab treatment and returned to pretreatment levels after omalizumab withdrawal. Omalizumab did not alter basophil histamine levels or basal and anti-IgE-stimulated histamine release. Conclusions: Omalizumab may reduce asthma symptoms in part by suppressing the FcΕRI-mediated production by basophils of Th2 cytokines and selected chemokines. Anti-IgE-stimulated basophil cytokine synthesis appears more sensitive than histamine release to the loss of FcΕRI caused by omalizumab treatment.

Increased Expression of Genes Linked to FcεRI Signaling and to Cytokine and Chemokine Production in Lyn-Deficient Mast Cells

Cross-linking the high-affinity IgE receptor, FcεRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn−/−) mice are hyperresponsive to FcεRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn−/− BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn−/− BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn−/− cells. Among genes implicated in the positive regulation of FcεRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn−/− BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcγRIIB), implicated in negative regulation of FcεRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn−/− BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1β)are all more highly expressed in Ag-stimulated Lyn−/− mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcεRI signaling and also to the response pathways that lead to allergy and asthma.

A Comparison of Inflammatory Mediators Released by Basophils of Asthmatic and Control Subjects in Response to High-Affinity IgE Receptor Aggregation

Background: In human blood basophils, cross-linking the high-affinity IgE receptor FcΕRI with multivalent antigen activates a signaling pathway leading to secretion of inflammatory mediators and cytokine production. Basophils are known to play an important role in the pathogenesis of asthma but there has been no comprehensive examination of the effectors these cells produce. Here a study of the transcription and release of a selection of chemokines and cytokines from basophils was undertaken. Methods: A Cartesian antibody array provided an effective method of assaying for multiple cytokines and chemokines simultaneously. Results were verified by RT-PCR and ELISA assays. This allowed the comparison of freshly prepared peripheral blood basophil responses to cross-linking of the high-affinity IgE receptor, with and without preincubation with IL-3. Results: Evidence that human blood basophils produce the chemokines MIP-5, eotaxin and GM-CSF was provided by antibody array and RT-PCR analyses. Preincubation with IL-3 enhanced the expression and release of IL-13, IL-8 and mRNA transcripts encoding MIP-5 and GATA2 in basophils from both asthmatic and control subjects. Leptin mRNA transcription, storage and release in basophils are described for the first time. Conclusions: Surveying cytokine and chemokines stored and released by peripheral blood basophils shows that asthmatic and control subjects share similar profiles even when their degranulation responses are distinct. Evidence is provided for the production of leptin, GM-CSF, eotaxin and MIP-5 by peripheral blood basophils. IL-3 preincubation enhances the production and release of IL-8 upon IgE receptor cross-linking.

Interaction between tumor cell surface receptor RAGE and proteinase 3 mediates prostate cancer metastasis to bone

Human prostate cancer often metastasizes to bone, but the biological basis for such site-specific tropism remains largely unresolved. Recent work led us to hypothesize that this tropism may reflect pathogenic interactions between RAGE, a cell surface receptor expressed on malignant cells in advanced prostate cancer, and proteinase 3 (PR3), a serine protease present in inflammatory neutrophils and hematopoietic cells within the bone marrow microenvironment. In this study, we establish that RAGE-PR3 interaction mediates homing of prostate cancer cells to the bone marrow. PR3 bound to RAGE on the surface of prostate cancer cells in vitro, inducing tumor cell motility through a nonproteolytic signal transduction cascade involving activation and phosphorylation of ERK1/2 and JNK1. In preclinical models of experimental metastasis, ectopic expression of RAGE on human prostate cancer cells was sufficient to promote bone marrow homing within a short timeframe. Our findings demonstrate how RAGE-PR3 interactions between human prostate cancer cells and the bone marrow microenvironment mediate bone metastasis during prostate cancer progression, with potential implications for prognosis and therapeutic intervention. Cancer Res; 77(12); 3144-50. ©2017 AACR.

Differential expression of Ikaros isoforms in monozygotic twins with MLL-rearranged precursor-B acute lymphoblastic leukemia.

Infant leukemia associated with rearrangement of the MLL gene typically presents with high-risk clinical features. Relapse is common despite aggressive therapy and perturbations in signaling pathways may contribute to disease resistance. We evaluated twin 4-month-old monozygotic baby boys who presented with MLL-rearranged precursor-B acute lymphoblastic leukemia. Two different MLL/AF4 variants were found in both the twins, the first involving MLL intron 8 and AF4 intron 3 and the second stemming from translocations of MLL exon 10 and AF4 exon 4. We detected expression of the DNA-binding Ikaros isoforms, Ik1, Ikx+, Ik2 and the dominant-negative Ik4 Ikaros isoform in both patients. However, the dominant-negative Ik8 isoform was detected in only 1 boy, suggesting a common genetic ontogeny that was modulated by leukemic evolution. Further exploration of Ikaros expression in the background of MLL rearrangements may provide new insights into disease pathogenesis and could offer targets for novel chemotherapeutic agents.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Selection of phage-displayed accessible recombinant targeted antibodies (SPARTA): methodology and applications

We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.