Cell Co-receptor Research Articles

Naïve helper T cells with high CD5 expression have increased calcium signaling.

The adaptive immune response is orchestrated by T helper cells and their function is dependent upon interactions between the T cell receptor (TCR), peptide MHC (pMHC) and co-receptors. TCR-pMHC interactions initiate calcium signaling cascades which determine T cell activation, survival, proliferation and differentiation. CD5 is a co-receptor that plays an important role in regulating T cell signaling and fate during thymocyte education. CD5 surface expression on mature single positive thymocytes correlates with the TCR signal strength for positive selecting self-ligands. CD5 also plays a role in T cell function after thymic development is complete. Peripheral T cells with higher CD5 expression respond better to foreign antigen than those with lower CD5 expression and CD5-high T cells are enriched in memory populations. In our study, we examined the role of CD5 expression and calcium signaling in the primary response of T cells using two Listeria monocytogenes specific T helper cells (LLO118 and LLO56). These T cells recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have divergent primary and secondary responses and different levels of CD5 expression. We found that each T cell has unique calcium mobilization in response to in vitro stimulation with LLO190-205 and that CD5 expression levels in these cells changed over time following stimulation. LLO56 naïve T helper cells, which expresses higher levels of CD5, have higher calcium mobilization than naïve LLO118 T cells. Three days after in vitro stimulation, LLO118 T cells had more robust calcium mobilization than LLO56 and there were no differences in calcium mobilization 8 days after in vitro stimulation. To further evaluate the role of CD5, we measured calcium signaling in CD5 knockout LLO118 and LLO56 T cells at these three time points and found that CD5 plays a significant role in promoting the calcium signaling of naïve CD5-high LLO56 T cells.

Role of the Wnt/β-Catenin Pathway in the Pathogenesis of Alcoholic Liver Disease.

The Wnt pathway is a highly conserved signal transduction pathway that plays an important role in diverse aspects of hepatic physiology. The Wnt pathway, consisting of canonical and noncanonical arms, is composed of secreted glycoproteins, cell surface receptors and co-receptors, and complex intracellular regulatory machinery that regulate a large number of cellular functions. β-Catenin is the main effector of the canonical Wnt pathway and hepatocyte-specific loss of the protein leads to increased susceptibility to alcoholic steatohepatitis. Hepatocytes with disrupted β-catenin demonstrate mitochondrial dysfunction, defective oxidative phosphorylation, and increased oxidative stress. β- Catenin knockout mice have decreased expression of alcohol metabolizing enzymes and increased blood alcohol levels that along with hypoglycemia and hyperammonemia, lead to increased mortality upon alcohol exposure. Disruption of hepatic β-catenin affects fatty acid oxidation and fasting ketogenesis and thereby profoundly affects systemic energy homeostasis. Given the combined roles of Wnt/β-catenin signaling in hepatocellular bioenergetics and regeneration, the Wnt pathway also contributes to alcohol-induced hepatic fibrogenesis and hepatocarcinogenesis. Targeting the Wnt/β-catenin pathway represents an attractive strategy for the treatment of alcohol-induced liver disease.

Modulation of CD4+ T cell polarization using non-canonical co-receptors

Abstract CD4+ T helper cells are a diverse group of cells promoting target cell death and production of antibodies. These cells acquire one of several fates following signals through the T cell receptor, co-receptors, and cytokines. The cytokine requirements inducing the polarization of T cell fate has been heavily evaluated and reasonably well defined. However, the influence provided by co-receptors has not been completely elucidated. Traditionally, in vitro T cell polarization assays utilize CD28 stimulation as the primary co-receptor signal required for differentiation. Others have observed the importance of supplementary co-receptors in activation of T helper cells, though little is known about their function in early stage polarization. We hypothesized that the addition of signaling through supplemental co-receptors would alter T helper cell polarization. To conduct these studies, we isolated naïve T cells and polarized them towards Th1 or Tfh fates in the presence or absence of recombinant B7-DC Fc chimera or recombinant B7-H4 Fc chimera. We observed a significant and selective alteration in T helper cell polarization in the presence of these non-canonical co-receptors. These data suggest a complex interplay between cytokine and co-receptor signals that cooperate for robust Th cell polarization. Furthermore, these findings illustrate the potential for novel mechanisms of T cell manipulation for vaccine design and treatment of T cell-mediated diseases.

Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1

SummaryEndoglin (ENG)/CD105 is an essential endothelial cell co-receptor of the transforming growth factor β (TGF-β) superfamily, mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1) and involved in tumor angiogenesis and preeclampsia. Here, we present crystal structures of the ectodomain of human ENG and its complex with the ligand bone morphogenetic protein 9 (BMP9). BMP9 interacts with a hydrophobic surface of the N-terminal orphan domain of ENG, which adopts a new duplicated fold generated by circular permutation. The interface involves residues mutated in HHT1 and overlaps with the epitope of tumor-suppressing anti-ENG monoclonal TRC105. The structure of the C-terminal zona pellucida module suggests how two copies of ENG embrace homodimeric BMP9, whose binding is compatible with ligand recognition by type I but not type II receptors. These findings shed light on the molecular basis of the BMP signaling cascade, with implications for future therapeutic interventions in this fundamental pathway.

CD3/Bars: A Novel Bispecific Format for the Treatment of B-Cell Lymphomas

BackgroundAutoantigens are suspected to play an important role in the pathogenesis of different types of B cell neoplasia. Suggestive of this hypothesis is the restricted usage of a stereotyped IgHv repertoire in CLL, MCL and DLBCL. Further evidence supporting this notion is the identification of specific autoantigens as the target of the B-cell receptor from malignant lymphomas and myelomas, such as paratarg-7 as antigenic target for 15% of paraproteins of MGUS and MM patients. ARS2 was previously identified as the autoantigenic target for the B-cell receptor of approximately 25% of DLBCLs of the ABC type, here termed ARS2 reactive lymphomas. We had recently shown that the B-cell receptor antigens can be used to target B-cell lymphoma cells in vitro and in vivo in an approach designated as BARs (B-cell receptor antigens for reverse targeting), the first therapeutic strategy in oncology with absolute and exclusive specificity for the malignant clone. To test whether BARs can substitute the B-cell binding antibody (e.g. CD19) in T-cell engaging bispecific antibodies, we designed a bispecific CD3/BAR product consisting of a recombinant single chain fragment (scFv) against CD3 linked to ARS2 (CD3-ARS2). One arm of this construct should engage the T cell co-receptor CD3 of human T cells, and the other site should bind to the B cell receptor of ARS2 reactive lymphomas thus specifically redirecting and activating T cells against lymphoma cells.Material and methodsVL and VH genes of the CD3 - OKT3 hybridoma and the DNA sequence of the 33 amino acids containing the B-cell receptor binding epitope of ARS2 were cloned into a pcDNA 3.1 vector by standard cloning techniques. VH and VL were linked by a glycine-serine linker, as was VL to the ARS2 epitope resulting in VH-(Gly4Ser1)n-VL-(Gly4Ser1)n-ARS2 peptide chain. The final cloning product was transfected in HEK cells for production of the bispecific construct. Binding capacity to lymphoma cell lines (OCI-Ly3, U2932, HBL-1) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of CD3-ARS2 after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay.ResultsThe CD3 - ARS2 bispecific construct bound to CD3 on T cells and the B-cell receptor of ARS2 reactive lymphoma cells. CD3/ARS2 induced rapid cytotoxicity exclusively in ARS2 reactive lymphoma cell lines at concentrations as low as 250 ng/ml with an effector - target ratio of 10:1. Specific T-cell mediated cytotoxicity reached 40% after 4 hours. Lymphoma cell lines with BCRs of a specificity other than ARS2 were not affected.ConclusionThe CD3/BAR construct is a novel therapeutic principle for the treatment of B-cell lymphomas, suggesting that BARs might also be useful as part of CAR-T cells. Compared to CD3/CD19 bispecific antibodies the CD3/BARs are exclusively cytotoxic against the malignant clone and spare normal B-cells. This should considerably reduce the acute toxicity of T-cell engaging bispecific constructs and circumvent long-term B cell depletion. Experiments comparing the cytotoxic capacity of CD3/BARs with CD3/CD19 bispecific antibodies are underway, as are analyses evaluating possible synergisms of these constructs. DisclosuresNo relevant conflicts of interest to declare.

IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression.Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice.In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through Filipin based cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of lipid rafts formation with decreased levels of ganglioside GM1. Thereby, Inadequate CD19/BCR co-receptor signaling caused by disruption of lipid rafts homeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo.Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining lipid rafts and CD19 surface expression. IFITM3-dependent lipid raft stability and CD19 surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP.

Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP. Moreover, the CTLD of CD72c, a lupus-susceptible allele, binds to Sm/RNP less strongly than that of lupus-resistant CD72a Reduced binding of CD72c is supported by x-ray crystallographic analysis that reveals a considerable alteration in charge at the putative ligand-binding site. Thus, CD72 appears to specifically inhibit B cell response to the endogenous TLR7 ligand Sm/RNP through CTLD-mediated recognition of Sm/RNP, thereby preventing production of anti-Sm/RNP antibody crucial for development of lupus.

Abstract 2225: Image analysis-based PD-L1 companion and complementary diagnostics

Abstract The PD-1 pathway, comprised of the immune cell co-receptor Programmed Death 1 (PD-1) and its ligands PD-L1 and PD-L2, mediates local immunosuppression in the tumor microenvironment. Immune checkpoint modulators are designed to block the local immunosuppression caused by this pathway. The FDA approved anti-PD-1 antibody therapies Opdivo® (nivolumab; Bristol-Myers Squibb) and Keytruda® (prembrolizumab; Merck &amp; Co) rely on PD-L1 immunohistochemistry (IHC) in vitro diagnostic (IVD) tests to determine the PD-L1 status in patients in non-small cell lung cancer (NSCLC), in order to predict response to these drugs. The current complementary diagnostic for Opdivo® (Dako 28-8 PharmDx®) relies on a pathologist scoring paradigm which considers any patient with ≥1% positive tumor cells an optimal candidate for Opdivo® treatment. However, overall survival (OS) is further increased when patients have ≥5% or ≥10% PD-L1 positive tumor cells. This scoring approach is vastly different than the PD-L1 scoring approach used in the Keytruda® companion diagnostic (Dako 22C3 PharmDx®), which utilizes a ≥50% positive tumor cells value to predict a positive Overall Response Rate (ORR; OS not yet determined). Thus, the 28-8 test for Opdivo® utilizes a more precise approach than the 22C3 test for Keytruda®, and requires a more calibrated scoring approach. This calibrated approach for Opdivo® requires the difficult challenge of pathologists reliably distinguishing membrane staining to define the fine gradations of 1%, 5% and 10% PD-L1 positive neoplastic cells. To best meet this challenge, we developed a digital Tissue Image Analysis (TIA) solution which enabled accurate, unbiased quantification of PD-L1 on a cell-by-cell basis to classify the percentage positive tumor cells in patients with high granularity. Using Flagship's proprietary CellMapTM algorithm, we evaluated 40 formalin-fixed paraffin-embedded (FFPE) NS-NSCLC samples which were stained using the Dako 28-8 PharmDx® PD-L1 IHC test. The TIA strategy digitally separated tumor cells from other cell types, and quantified membrane staining intensity according to a consistent threshold. The performance of the resulting IHC-TIA assay was evaluated in the context of a CLIA validation study performed by Flagship. The results demonstrated equivalency to the manually scored IVD reference standard; however, the TIA scoring of this assay provided consistent, unbiased, and more detailed scoring of PD-L1 stained tissues for determining the patients with ≥1, ≥5, and ≥10% PD-L1 positive tumor cells with greater confidence than a manual scoring approach. Moving forward, these TIA tools can be utilized to assess PD-L1 positive cell frequencies with greater reliability and granularity to identify optimal treatment cutpoints for these and other PD-L1 IHC tests used to predict response to PD-L1/PD-1 inhibitors. Citation Format: Joseph S. Krueger, Roberto Gianani, Brooke Hirsch, Stefan Pieterse, Famke Aeffner, David Young. Image analysis-based PD-L1 companion and complementary diagnostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2225.

Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment.

In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.

Quantifying the Sensitivity of HIV-1 Viral Entry to Receptor and Coreceptor Expression.

Infection by many viruses begins with fusion of viral and cellular lipid membranes, followed by entry of viral contents into the target cell and ultimately, after many biochemical steps, integration of viral DNA into that of the host cell. The early steps of membrane fusion and viral capsid entry are mediated by adsorption to the cell surface, and receptor and coreceptor binding. HIV-1 specifically targets CD4+ helper T-cells of the human immune system and binds to the receptor CD4 and coreceptor CCR5 before fusion is initiated. Previous experiments have been performed using a cell line (293-Affinofile) in which the expressions of CD4 and CCR5 concentration were independently controlled. After exposure to HIV-1 of various strains, the resulting infectivity was measured through the fraction of infected cells. To design and evaluate the effectiveness of drug therapies that target the inhibition of the entry processes, an accurate functional relationship between the CD4/CCR5 concentrations and infectivity is desired in order to more quantitatively analyze experimental data. We propose three kinetic models describing the possible mechanistic processes involved in HIV entry and fit their predictions to infectivity measurements, contrasting and comparing different outcomes. Our approach allows interpretation of the clustering of infectivity of different strains of HIV-1 in the space of mechanistic kinetic parameters. Our model fitting also allows inference of nontrivial stoichiometries of receptor and coreceptor binding and provides a framework through which to quantitatively investigate the effectiveness of fusion inhibitors and neutralizing antibodies.

CD4 Expression on Splenic DCs Coordinates their Positioning in a Cell-Intrinsic Manner and is Necessary for Optimal CD4+ T cell Activation

Abstract The immunological dogma of MHC-class restriction states helper T cells expressing the co-receptor CD4 perceive antigen in the context of MHC-class II while cytotoxic CD8+ T cells recognize antigen presented on MHC-class I. Paradoxically, dendritic cells (DCs) which express high levels of MHC molecules (yet lack recombined antigen receptors) differentially express CD4 and CD8α, which have classically been used to subset DC populations within secondary lymphoid tissues. Why DCs concurrently express MHC molecules and T cell co-receptors has puzzled immunologists since their discovery on DCs in the mid 1990’s. However, studying the function of these molecules on DCs is confounded by the fact that their T cell counterparts are absent in CD4 or CD8α-deficient mice. Here using a mixed chimera system in which CD4 deficiency can be restricted specifically within the DC compartment, we show CD4-expression on DCs is necessary to drive optimal antigen-specific CD4+ T cell proliferation in vivo. Impaired T cell proliferation which occurs when DCs lack CD4 appears to result from their inability to efficiently access the T cell follicle. Therefore, DC-intrinsic CD4 expression coordinates the micro-anatomical positioning of DCs within the spleen and is necessary for optimal CD4+ T cell activation.

Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody–Mediated Operational Tolerance Induction

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor.

ABSTRACTEntry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable.IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.

Complex formation among transforming growth factor-β (TGF-β) receptors and its modulation by coreceptors represent an important level of regulation for TGF-β signaling. Oligomerization of ALK5 and the type II TGF-β receptor (TβRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G-mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TβRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

The Src tyrosine kinase Lck binds to CD2, CD4-1, and CD4-2 T cell co-receptors in channel catfish, Ictalurus punctatus

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4+ and CD4−CD8− CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8+ CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8β. These data provide one possible explanation for the apparent low numbers of CD8+ CTL and the presence of CD4+ and CD4−CD8−CD2+ CTL in catfish.

The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

Association between co-inhibitory molecule gene tagging single nucleotide polymorphisms and the risk of colorectal cancer in Chinese.

T lymphocyte immune responses are controlled by both co-stimulatory and co-inhibitory signaling through T cell co-receptors. Cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death 1 (PD-1) and B and T lymphocyte attenuator (BTLA) are all co-inhibitory molecules that negatively regulate the activation of T cells. In this study, we investigated the relationship between ten tagging SNPs in three co-inhibitory molecule genes and colorectal cancer (CRC). We conducted a hospital-based case-control study consisting of 601 cases with CRC and 627 CRC-free individuals from the Heilongjiang Province of China. The rs7421861 CT genotype was significantly associated with the risk of colorectal cancer compared to the wild-type TT genotype (adjusted OR 1.314, 95% CI 1.012-1.706, P = 0.041). The rs2705535 TT genotype was associated with the risk of rectal cancer [OR 1.819 (1.093-3.027), P = 0.021]. There was statistical interaction between the PD-1/rs2227982 (CT + TT) genotypes and high seafood intake (>once/week), as well as the CTLA-4/rs231777 variant and high pungent food intake (>3 times/week). The AG + AA genotypes of CTLA-4/rs3087243 statistically and antagonistically interacted with soybeans, pork and alcohol intake and were associated with CRC risk. Analogously, BTLA/rs1844089 interacted with pork intake, PD-1/rs7421861 with beef and lamb consumption and PD-1/rs6710479 with barbecue consumption. Haplotype G-C-G-A-T-T-A was significantly associated with CRC risk (OR 1.221 P = 0.034). These data indicate potential associations between BTLA and PD-1 polymorphisms and CRC susceptibility. Additionally, the three co-inhibitory molecule gene SNPs have environmental interactions associated with CRC risk.

Abstract A56: Characterization of the tumor microenvironment of a humanized mouse model of ovarian cancer after treatment with anti-B7-H4 recombinant antibodies

Abstract Ovarian cancer is the deadliest gynecological malignancy. Disease recurrence occurs in 70% of patients diagnosed with stage III or IV epithelial ovarian cancer, in part due to immune evasion and suppression in the tumor microenvironment. Tumor infiltration with tumor-associated macrophages (TAMs) has been associated with poor patient survival and TAMs express B7-H4, an orphan ligand that modulates T cell responses. Common features observed in tumor microenvironments include hypoxic stress and other environmental cues that prompt the expression of immunomodulators such as CTLA4 and PD1, leading to increased tumor cell ability to escape immune recognition. We showed that B7-H4 is also expressed on ovarian cancer cells in vivo, suggesting that tumor cells may also hamper cell-mediated anti-tumor immune responses via inhibition of T cell co-receptors. We thus sought to determine whether targeting functional cell subsets expressing B7-H4 could restore an anti-tumor immune response and/or promote tumor cell death. We isolated novel recombinant anti-B7-H4 antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library that was derived from B cells of ovarian cancer patients. We demonstrated that B7-H4 scFv-mediated inhibition could rescue activation of tumor antigen-TCR-specific T cells in vitro, and we also showed that anti-B7-H4 scFv administration could delay tumor growth in a novel humanized NSG mouse model of ovarian cancer (Dangaj, D. et al., Novel recombinant human B7-H4 antibodies overcome tumoral immune escape to potentiate T cell anti-tumor responses, Cancer Research, 2013). We hypothesize that use of anti-B7-H4 scFv in vivo targeted functionally diverse tumor mass components (TAMs and tumor cells) leading to the reprogramming of the tumor microenvironment towards tumor rejection. Here we present a preliminary ex vivo analysis of the human tumors infiltrated with human leukocytes harvested from the B7-H4-treated humanized NSG mice. Tumors from mice treated with two anti-B7-H4 scFv or with a control scFv were sectioned and labeled by immunofluorescence to characterize tumor cells and immune cells. Tumor cells were co-stained with EpCAM and Ki-67, and leukocytes with CD45 and Ki-67. Preliminary studies suggested that in vivo delivery of anti-B7-H4 scFv decreased tumor cell proliferation as evidenced by a reduction of cells co-stained by EpCAM and Ki-67 compared with the staining of tumor cells from mice treated with the control antibody. Concurrently, tumors from mice receiving anti-B7-H4 scFv presented an increase in cells that stained positive for both CD45 and Ki-67 compared with tumors from mice receiving a control scFv. These results suggest that anti-B7-H4 scFv enhances leukocyte proliferation. Additional work will be performed to statistically analyze these results over all experimental groups, to functionally phenotype the tumor-infiltrating leukocytes, and to assess the impact of B7-H4 scFv administration on tumor hypoxia, apoptosis and angiogenesis. We tentatively conclude that B7-H4 is another immune checkpoint and its inhibition by antibody targeting may open novel immunotherapeutic avenues for ovarian cancer. Citation Format: Bhairavi (Vivi) Tolani, Denarda Dangaj, Daniel J. Powell, Jr., Nathalie Scholler. Characterization of the tumor microenvironment of a humanized mouse model of ovarian cancer after treatment with anti-B7-H4 recombinant antibodies. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A56. doi:10.1158/1538-7445.CHTME14-A56

IFITM3 (CD225) Regulates CD19 Surface Expression and CD19-Mediated Activation of PI3K Signaling in Pre-B Acute Lymphoblastic Leukemia Cells

Background & Hypothesis:IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21 on mouse and human B cells. However, besides its interaction with CD19, specific functions of Ifitm3 in normal B cells development and, potentially leukemogenesis, are not known. Recent approaches based on CD19-specific chimeric antigen receptors (CAR) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (e.g. Grupp et al., N Engl J Med 2013). However, in some cases, CD19 CAR treatment was followed by ALL relapse developing from a clone that lacked CD19 surface expression. Therefore, we studied factors that can potentially regulate CD19 surface on normal and leukemic pre-B cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in near-complete loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. Reconstitution of IFITM3 in patient-derived pre-B ALL cells rescued both CD19 expression and increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT, pointing to a central role of IFITM3 in regulating CD19/PI3K-AKT signaling in pre-B cells and pre-B ALL.Deletion of Ifitm3 also resulted in significant cell cycle arrest in the G0/G1 phase (P<0.001) and increased protein levels of p53 and p21 compared to wild-type pre-B ALL cells. Moreover, treatment with low dose adriamycin (25 ng ml-1) for induction of modest DNA damage significantly induced cellular senescence in Ifitm3-/- but not wild-type pre-B ALL cells. Consistent with Ifitm3-mediated proliferative defects of Ph+ ALL cells, Ifitm3-/- Ph+ ALL cells exhibited reduced self-renewal capacity (P=0.0004) decreased colony in colony forming assay compared to wild-type cells. Furthermore, Ifitm3 deficient B cell progenitors also showed significant inhibition of proliferation with inhibition of phosphorylation of Stat5Y694and c-Myc expression.4-hydroxytamoxifen (4-OHT)-inducible activation of CD19 rescued proliferative defects in Ifitm3 deficient B cell progenitors with release from G0/G1 cell cycle arrest associated with upregulation of phosphorylation of Stat5 Y694 and c-Myc expression. In addition, we found that CD19 positively regulates the surface expression of IL7R in B cell progenitors. Interestingly, CD19low population in Ifitm3 deficient B cell progenitors showed lower surface expression of IL7R compared to CD19high population. In contrast to B cell progenitors, forced expression of CD19 did not increase the proliferation of wild-type Ph+ ALL cells. Surprisingly, upregulation of CD19 induced apoptosis in Ifitm3-deficient Ph+ ALL cells with significant inhibition of BCR-ABL1 activity, phosphorylation of Stat5Y694and Bcl2 expression.Conclusion: These findings identify novel role of IFITM3 in regulating CD19 surface expression and CD19-Y513 mediated PI3K-AKT signaling in pre-B cells and pre-B ALL. Preliminary experiment showed that agonistic antibodies against IFITM3/CD225 increased CD19/PI3K-AKT signaling and proliferation in pre-B ALL cells and future studies will elucidate whether blocking antibodies (e.g. decoy Fc-fusion molecules) will have useful effects in targeting CD19/PI3K-AKT signaling in human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.