Monoclonal antibodies to ThB detect close linkage ofLy-6 and a gene regulating ThB expression

We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976).Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene,Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying theThb ^h allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry theThb ^l allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.This work was supported in part by grants from the National Institutes of Health (AI-08917, CA-04681, GM-17367).     

Isolation and characterization of cDNA clones for the mouse thymocyte B cell antigen (ThB).

The Thb locus is responsible for the expression of 15-kDa phosphatidyl inositol anchored molecules (ThB) on murine thymocytes and B cells. Thb expression as detected with mAb is polymorphic on B cells with two alleles, Thbh and Thb1 responsible for the high and low expression of ThB on B cells. The regulatory locus for Thb expression had been mapped with the Ly-6 cluster of genes to Chr 15. In our study we used expression cloning in COS cells to isolate cDNA clones that code for ThB after transfection; the cDNA products react with anti- ThB antibodies, but not with Ly-6A.2, -6B.2, -6C.2, or -6D.2 antibodies. One of these clones, pThB-A contains insert of 702 bases which was sequenced. The translated amino acid sequence has 11 cysteine residues, and together with the absence of potential N-linked glycosylation sites is similar to the structure of the Ly-6 molecules. The nucleotide and amino acid sequences of ThB cDNA were compared to those of Ly-6 genes and the Ly-6 related human CD59 and show clear homology. Finally using interspecies crosses, the structural Thb gene has been mapped to Chr 15; thus both structural and regulatory genes map to a similar site. The genetic map location near Ly-6 and the sequence similarity suggest that Thb and Ly-6 may have been derived from the same progenitor by gene duplication.     

The relationship between airway immunoglobulin activity and eosinophils in COPD

In chronic obstructive pulmonary disease (COPD), the effects of inhaled corticosteroids are predicted by blood eosinophil counts. We previously briefly reported increased immunoglobulin (Ig)A and IgM levels in bronchoalveolar lavage (BAL) of COPD patients with higher (eosinophil^high) compared to lower (eosinophil^low) blood eosinophils (>250/μL versus < 150/μL), suggesting differences in adaptive immune function. An inverse relationship exists between eosinophil counts and airway pathogenic bacteria levels. The mechanistic reasons for these associations between eosinophils, corticosteroids and pathogenic bacteria are unclear. IgA, IgM and IgG levels were assessed in BAL, bronchial biopsies and epithelium collected from eosinophil^high (n = 20) and eosinophil^low (n = 21) patients. Bronchial B-cell numbers were measured by immunohistochemistry. B-cell activity was assessed in bronchial samples and following exposure to BAL from eosinophil^high and eosinophil^low patients. BAL levels of non-typeable Haemophilus influenza (NTHi)-specific immunoglobulins were quantified. Results showed airway expression of IgA, IgG1 and IgM were lower in eosinophil^low compared to eosinophil^high patients, with lower levels of NTHi-specific IgA and IgM. Bronchial B-cell numbers were similar in both groups, but B-cell activity was lower in eosinophil^low patients. In conclusion, COPD eosinophil^low patients show differences in adaptive immune function compared to COPD eosinophil^high patients. These differences may cause different microbiomes in these COPD phenotypes.     

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44^high/EGFR^low, CD44^high/EGFR^high and CD44^low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44^high/EGFR^low population showed a spindle-shaped EMT-like morphology, while the CD44^low population was dominated by cobblestone-shaped cells. The CD44^high/EGFR^low population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44^high/EGFR^low cells in comparison to CD44^low cells. Moreover, CD44^high/EGFR^low cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44^high/EGFR^high and CD44^low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.     

Selective Loss of Early Differentiated,Highly Functional PD1high CD4 T Cells with HIV Progression

The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127^high CD4 T cells within the early/intermediate differentiated (EI) (CD27^highCD45RA^low) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1^highCTLA-4^low CD4 T cells was found specifically in the CD127^highCD27^highCD45RA^low compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1^high compared to PD-1^low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1^low EI CD4 T cells. In line with our previous findings, PD-1^high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.     

Interrelationships of the “Ly-6 complex” antigens

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes—Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10–20% or >90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B,2 antigens are distinct from Ly-6A.2 and Ly-6B.2, however. ThB is a 16–18 kd antigen which is not associated on the cell surface with any other Ly-6 antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.     

Tumor genetic heterogeneity analysis of chronic sun‐damaged melanoma

Chronic sun‐damaged (CSD) melanoma represents 10%–20% of cutaneous melanomas and is characterized by infrequent BRAF V600E mutations and high mutational load. However, the order of genetic events or the extent of intra‐tumor heterogeneity (ITH) in CSD^high melanoma is still unknown. Ultra‐deep targeted sequencing of 40 cancer‐associated genes was performed in 72 in situ or invasive CMM, including 23 CSD^high cases. In addition, we performed whole exome and RNA sequencing on multiple regions of primary tumor and multiple in‐transit metastases from one CSD^high melanoma patient. We found no significant difference in mutation frequency in melanoma‐related genes or in mutational load between in situ and invasive CSD^high lesions, while this difference was observed in CSD^low lesions. In addition, increased frequency of BRAF V600K, NF1, and TP53 mutations (p < .01, Fisher's exact test) was found in CSD^high melanomas. Sequencing of multiple specimens from one CSD^high patient revealed strikingly limited ITH with >95% shared mutations. Our results provide evidence that CSD^high and CSD^low melanomas are distinct molecular entities that progress via different genetic routes.     

Functional and Phenotypical Analysis of Subsets of Rat CD4+ T Cells

Rat CD4⁺ T cells were divided into two distinct subsets by a monoclonal antibody RTH-1 recognizing a unique epitope on rat CD45R. Cellular distribution of OX-22- and RTH-1-defined antigens was the same. However, OX-22 and RTH-1 recognized different epitopes that exist on rat CD45R. The expression of IL-4 gene was detected only in RTH-1^low CD4+ T cell subset upon various stimulations. In contrast, the expression of IL-2 and IFN-γ gene varied depending upon the nature of stimuli. The increased cell surface expression of CD44 was detected in RTH-1^high CD4+ T cell subset. Conversely the increased expression of CD2 was detected in RTH-1^low CD4+ T cell subset. The expression of CD3 and LFA-1 was not significantly different between RTH-1^high and RTH-1^low subsets.     

CLEC1B Expression and PD-L1 Expression Predict Clinical Outcome in Hepatocellular Carcinoma with Tumor Hemorrhage

Spontaneous tumor hemorrhage (TH) is frequently observed in solid tumors including human hepatocellular carcinoma (HCC). TH implies fast-growing and worse tumor immunological microenvironment; however, the underlying mechanism remains largely unknown. CLEC1B is a signature gene highly associated with tumor progression. PD-L1 expression is a key biomarker predictive of immune checkpoint therapies, which showed astonishing effect on various types of tumor. We assume that, in HCC, TH may closely associate with the expression of these two molecules. In this study, 136 patients with HCC were enrolled. qRT-PCR showed that CLEC1B expression is significantly lower in HCC tumor tissue. Immunohistochemistry of HCC tissue microarrays demonstrated that PD-L1_high and CLEC1B_low expressions were significantly correlated with TH and clinicopathological features indicating worse HCC progression. According to univariate/multivariate analysis, a combination of PD-L1_high and CLEC1B_low expression was an independent prognostic factor indicating the poor outcome. The prognostic value of PD-L1_high and CLEC1B_low was validated by Cox proportional-hazard analyses. Collectively, tumor with TH is closely associated with CLEC1B_low & PD-L1_high expression, which may imply high response of PD-L1/PD-1 immune checkpoint therapies. CLEC1B may be a potential therapeutic target for PD-L1/PD-1 immunotherapy. PD-L1_high and CLEC1B_low can be a valuable prognosis factor implying worse clinical outcomes.     

Imaging Axl expression in pancreatic and prostate cancer xenografts

The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl–Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with ¹²⁵I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC (Axl^high) and Panc1 (Axl^low) pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [¹²⁵I]Axl mAb was also validated in mice harboring orthotopic Panc1 or CFPAC tumors and in mice harboring subcutaneous 22Rv1 (Axl^low) or DU145 (Axl^high) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [¹²⁵I]Axl mAb in Axl^high (CFPAC and DU145) expression tumors compared to the Axl^low (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [¹²⁵I]IgG1 antibody in the Axl^high and Axl^low expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.     

The phenotype and function of naturally existing regulatory dendritic cells in nematode-infected mice

Immunosuppression associated with chronic helminth infections has been documented in many studies and regulatory T (Treg) cells have been shown to mediate the nematode-induced immunosuppression, but the role of dendritic cells (DCs) in the induction of Treg cell response and immunosuppression has not yet been fully determined. We analysed the response and function of DCs in mesenteric lymph node (MLNs) of mice infected with a gastrointestinal nematode, Heligmosomoides polygyrus, and observed a substantial expansion of DCs in MLNs following the infection. The CD11c⁺ DCs in MLNs of infected mice showed reduced expression of co-stimulatory molecules CD40, CD86 and MHC-II, and production of inflammatory cytokines IL-12 and IL-6. Analysis of MLN DC subsets defined by CD11c and CD45RB expression showed that the CD11c^lowCD45RB^mid subset increased rapidly following H. polygyrus infection and the CD11c^midCD45RB^high subset expanded from the third week after infection. In the co-culture of sorted DC subsets with ovalbumin-(OVA-)specific T cell receptor (TCR) transgenic CD4⁺ T cells, CD11c^lowCD45RB^mid DCs induced a low proliferation response and a high level of IL-10 production in CD4⁺ T cells, whereas CD11c^midCD45RB^high DCs induced more IFN-γ and IL-4 producing CD4⁺ T cells. Intracellular staining revealed that CD11c^lowCD45RB^mid DCs promoted CD4⁺ Foxp3⁺ differentiations. These results indicate that nematode infections selectively induce expansion of the CD11c^lowCD45RB^mid regulatory DC subset that promotes development of Foxp3⁺ and IL-10 producing Treg cells. The Treg cell responses and immunoregulatory cytokines induced by this regulatory DC subset in turn play an important role in mediation of the nematode-induced immunosuppression.     

Myeloperoxidase expression in human colonic mucosa is related to systemic oxidative balance in healthy subjects

Objectives: To improve understanding of the preclinical stage of colonic inflammation by exploring the existence of a link between early inflammatory changes in the colonic mucosa and the systemic redox balance.

Methods: Clinical characteristics, a fasting blood draw, and mucosal biopsies from the right, left, and sigmoid-rectum colonic tracts collected from 28 healthy individuals (14/14 males/females) who underwent colonoscopy. Myeloperoxidase (MPO) positive cells infiltrating colonic mucosa specimens were assessed by immunohistochemistry, and patients divided into high or low MPO expressing cells/optical field groups (MPO^high or MPO^low, respectively).The systemic oxidative balance has been studied through derived-Reactive Oxygen Metabolites (d-ROMs), Biological Antioxidant Potential (BAP), and Lipoperoxide-cholesterol Oxidizing (LP-CHOLOX) tests on serum.

Results: MPO^high patients demonstrated an increased systemic oxidative stress compared to MPO^low individuals (P?=?0.035), especially when MPO is referred to the left-sided colonic mucosa (P?=?0.007). MPO^low subjects in the sigmoid-rectum showed a significant higher antioxidant capacity in the serum (P?P?=?0.044), and a decreasing gradient in MPO expression moving from the cecum to the rectum (ascendant, descendant, and sigmoid-rectum: 3.7?±?2.8, 3.1?±?1.7, and 1.4?±?0.5, respectively, P?=?0.012) were also found and discussed.

Discussion: The study is the first demonstrating a connection between systemic redox balance and MPO expression in the colonic mucosa, according to the colonic tract and patient gender. Further research evaluating the MPO expression in the human colon and its relationship with pathological conditions could benefit from these results.     

Chromosomal location of the gene encoding the murine acute-phase protein serum amyloid P-component (SAP)

A full-length cDNA clone, pmSAP3, encoding the serum P component (SAP), has been used to search for DNA fragment length variation among mouse strains previously analyzed for differences in endogenous SAP levels. Three alleles were found usingEcoRI-digested DNA. The finding of a single 5.4-kb fragment, alleled, in DNA from DBA/2J mice suggests the presence of a singleSap locus. Segregation of DNA fragment associated withSap ^b andSap ^d alleles was analyzed in three sets of recombinant inbred (RI) strains. The strain distribution pattern found for theSap alleles was identical to that of alleles ofLy-9 in 43 individual RI strains, suggesting tight linkage withLy-9 on mouse chromosome 1. In the BXD RI strains, the SDP of theSap locus, defined by the difference in the endogenous SAP level, is also identical to the SDP of the DNA fragments. We propose to redesignate theSap locus to include both the structural element defined by the DNA polymorphism and the regulatory element involved in the regulation of SAP synthesis. TheSap locus is the major genetic element contributing to the regulation of SAP production. Other genetic factors are also involved, as shown by the presence of nonparental phenotypes in the individual BXH RI strains. This study was performed through special Coordination Funds of the Science and Technology Agency of the Japanese Government and PHS Grant GM24464 to R.W.E.     

Further definition of the Ly-5 system

Ly-5 is expressed by cells of the hematopoietic branch of development. Further serological analysis of the Ly-5 system, aided by Ly-5 monoclonal antibodies and by two Ly-5 congenic mouse strains, reveals two new Ly-5 alloantigens, Ly-5. 3 and Ly-5.4. The data define three thymocyte phenotypes, Ly-5.1,3, Ly-5.2,4, and Ly-5.2,3, and three corresponding genotypes, Ly-5 ^a, Ly-5 ^b, and Ly-5 ^c, respectively. Ly-5 ^ais by far the most common allele. The Ly-5 ^callele is found only in the ST/bJ strain, a finding that accords with the presently unique pattern of restriction fragments previously observed in Southern blotting of ST/bJ DNA with an Ly-5 cDNA probe. Present serological and biochemical data favor the interpretation that the compound Ly-5 phenotype of thymocytes is attributable to two separate Ly-5 molecular isoforms that exhibit a discrete difference in protein composition, bear different Ly-5 antigens, and are produced jointly by thymocytes, unlike other Ly-5 isoforms previously shown to distinguish different hematopoietic cell lineages.     

Ly6Clow and Not Ly6Chigh Macrophages Accumulate First in the Heart in a Model of Murine Pressure-Overload

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6C^low and Ly6C^high macrophages. Ly6C^low macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6C^low but not on Ly6C^high macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6C^low macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6C^low macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.     

Phospholipase A2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells

Phospholipase A₂ (PLA₂)-activating protein (PLAA) is a novel signaling molecule that regulates eicosanoid production and participates in inflammatory responses. In our current study, we revealed that PLAA production was induced by the chemotherapeutic drug cisplatin in HeLa cervical carcinoma cells. To determine the potential pro-apoptotic effects of PLAA induction by cisplatin, we utilized HeLa (Tet-off) cells overexpressing the plaa gene (plaa ^high) and compared them with control (plaa ^low) cells, which produce endogenous plaa from the chromosome. Cisplatin-stimulated plaa ^high cells contained significantly higher levels of DNA fragmentation, caspase 3, 8 and 9 activities, PLA₂ enzyme activity, and cytochrome c leakage from mitochondria than did the cisplatin-stimulated plaa ^low cells. Importantly, siRNA against PLAA (siRNA–PLAA) reduced the levels of cisplatin-induced PLAA, DNA fragmentation, and PLA₂ activation, while promoting cell viability in both plaa ^high and plaa ^low cells. Cisplatin-induced-cytochrome c leakage in plaa ^high cells was reduced by siRNA–PLAA and restored by the addition of exogenous arachidonic acid (AA), suggesting to us that PLAA induction by cisplatin promoted cytochrome c leakage/mitochondrial damage partially by accumulating AA. In addition, cisplatin-stimulated plaa ^high cells produced less cytoprotective clusterin than did the cisplatin-stimulated plaa ^low cells, and siRNA–PLAA promoted clusterin production from both plaa ^high and plaa ^low cells. We showed that clusterin reduced DNA fragmentation in cisplatin-stimulated plaa ^high and plaa ^low cells, which is consistent with the notion that clusterin promotes cancer chemoresistance. Furthermore, cisplatin-stimulated plaa ^high cells produced more IL-32 (a pro-apoptotic protein) than did cisplatin-stimulated plaa ^low cells, and siRNA–PLAA reduced IL-32 production from both plaa ^high and plaa ^low cells. Finally, our proteomic analysis revealed that cisplatin-stimulated plaa ^high cells contained higher levels of phosphorylated JNK/c-Jun and FasL than did plaa ^low cells treated the same way. In summary, our data indicated that PLAA induction enhanced cisplatin-induced-apoptosis through four pathways, namely by: 1) accumulation of AA and mitochondrial damage, 2) downregulation of the cytoprotective clusterin, 3) upregulation of the pro-apoptotic IL-32, and 4) induction of JNK/c-Jun signaling and FasL expression.     

Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.