B-cell subsets in blood and lymphoid organs in Macaca fascicularis.

BACKGROUND: Cynomolgus monkeys (Macaca fascicularis) are widely used animal models in biomedical research. However, the phenotypic characteristics of cynomolgus monkey (CM) B cells in peripheral blood (PB) and lymphoid organs are poorly understood. METHODS: FACS analyses of PB-, spleen-, lymph node (LN)-, and bone marrow (BM)-derived B cells were performed. RESULTS: CM peripheral blood B cells have a smaller fraction of CD27(-) (naive) cells ( approximately 40%), as compared to human blood samples ( approximately 70%). Similar to humans, an early activation marker, CD23, is expressed more on CD27(-) CM naive B cells, as compared to CD27(+) B cells. The mean fraction of B cells exhibiting a memory phenotype is similar to that seen in human blood. Unlike humans, CM blood contains a subset of CD20(++)CD80(+)CD21(-)IgM(+/-)CD27(+)CD19(+)FSC(++)BAFF-R(low) B cells that are likely of germinal center origin. Thus, CM blood contains (i) a higher percentage of B cells that express the co-stimulatory molecule CD80, and (ii) a lower fraction of B cells that are CD21(+), as compared to human blood. Due to the relative paucity of information on B-cell subsets in organs of healthy humans, a direct comparison between human and CM lymphoid organ data is limited. The fraction of CD27(+) and CD23(+) B cells appears to be similar, while the fraction of CD80(+) B cells appears to be higher than that seen in human lymphoid organs. CM spleens and to some extent lymph nodes have a distinct subset of CD21(++) cells that are also CD80(+/-)CD23(low)IgM(++)CD27(+/-)FSC(++). This subset is phenotypically similar to the marginal zone B cells present in human spleen and LN samples. We also provide detailed analyses on the fraction of lymphoid organ B cells that express CD21, CD23, CD32, and/or CD80 B-cell markers. CONCLUSIONS: In general, cynomolgus monkey B-cell subsets are similar to those seen in humans, as well as to those seen in other nonhuman primates. However, there are some clear differences between human and cynomolgus monkey B-cell subsets. These findings have direct implications for a variety of in vivo studies in cynomolgus monkeys, ranging from basic research on primate B-cell differentiation to models of infectious diseases and trials of new B-cell targeting therapeutic agents.     

Protective immune responses induced by a non-pathogenic simian/human immunodeficiency virus (SHIV) against a challenge of a pathogenic SHIV in monkeys

A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.     

A nonfucosylated human antibody to CD19 with potent B-cell depletive activity for therapy of B-cell malignancies

A human anti-CD19 antibody was expressed in fucosyltransferase-deficient CHO cells to generate nonfucosylated MDX-1342. Binding of MDX-1342 to human CD19-expressing cells was similar to its fucosylated parental antibody. However, MDX-1342 exhibited increased affinity for FcγRIIIa-Phe158 and FcγRIIIa-Val158 receptors as well as enhanced effector cell function, as demonstrated by increased potency and efficacy in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. MDX-1342 showed dose-dependent improvement in survival using a murine B-cell lymphoma model in which Ramos cells were administered systemically. In addition, low nanomolar binding to cynomolgus monkey CD19 and increased affinity for cynomolgus monkey FcγRIIIa was observed. In vivo administration of MDX-1342 in cynomolgus monkeys revealed potent B-cell depletion, suggesting its potential utility as a B-lymphocyte depletive therapy for malignancies and autoimmune indications.     

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.     

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques

The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC-LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC-LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC-LAMP and S100b expression.     

Integrated pharmacokinetic,pharmacodynamic and immunogenicity profiling of an anti-CCL21 monoclonal antibody in cynomolgus monkeys

QBP359 is an IgG1 human monoclonal antibody that binds with high affinity to human CCL21, a chemokine hypothesized to play a role in inflammatory disease conditions through activation of resident CCR7-expressing fibroblasts/myofibroblasts. The pharmacokinetics (PK) and pharmacodynamics (PD) of QBP359 in non-human primates were characterized through an integrated approach, combining PK, PD, immunogenicity, immunohistochemistry (IHC) and tissue profiling data from single- and multiple-dose experiments in cynomolgus monkeys. When compared with regular immunoglobulin typical kinetics, faster drug clearance was observed in serum following intravenous administration of 10 mg/kg and 50 mg/kg of QBP359. We have shown by means of PK/PD modeling that clearance of mAb-ligand complex is the most likely explanation for the rapid clearance of QBP359 in cynomolgus monkey. IHC and liquid chromatography mass spectrometry data suggested a high turnover and synthesis rate of CCL21 in tissues. Although lymphoid tissue was expected to accumulate drug due to the high levels of CCL21 present, bioavailability following subcutaneous administration in monkeys was 52%. In human disease states, where CCL21 expression is believed to be expressed at 10-fold higher concentrations compared with cynomolgus monkeys, the PK/PD model of QBP359 and its binding to CCL21 suggested that very large doses requiring frequent administration of mAb would be required to maintain suppression of CCL21 in the clinical setting. This highlights the difficulty in targeting soluble proteins with high synthesis rates.     

Establishment of a B-lymphoblastoid cell line infected with Epstein-Barr-related virus from a cynomolgus monkey (Macaca fascicularis)

A B-lymphoblastoid cell line (Ts-B) was established from lymph nodes of an apparently healthy cynomolgus monkey. The cells were demonstrated to contain Epstein-Barr virus-related antigens. Moreover, herpesvirus particles were found in the cells by electron microscopy. The cell-free culture medium transformed lymphocytes of cynomolgus rhesus monkeys, and of Japanese monkeys.     

2型糖尿病相关基因在人和食蟹猴外周血白细胞中的表达模式

2型糖尿病(T2DM)是一种多因素相关的复杂的代谢性疾病。采用实时PCR技术了解51个T2DM相关基因mRNA在健康和糖尿病人/食蟹猴外周血白细胞中的表达模式。结果表明,与健康组相比,糖尿病猴中有34个基因表达量差异显著(P<0.05),约为66.7%,其中24个基因表达上调(P<0.05);而糖尿病人中有26个基因表达量差异显著(P<0.05),约为50.1%,其中16个基因表达上调(P<0.05),这些基因表达受到糖代谢、脂肪代谢、炎症等方面的影响。糖尿病人与食蟹猴样本中有18个基因都存在差异性表达(P<0.05),表达模式基本一致。因此,食蟹猴可以作为研究糖尿病的较理想的模式动物,外周血白细胞基因的表达模式可以为糖尿病早期诊断和预后评价提供重要指标。     

Macaca fascicularis, a nonpermissive host for the human filarial parasite Loa loa

The ability of the filarial nematode Loa loa to infect 2 species of primates was studied. The primate species selected were closely related to species known to be susceptible. A mandrill (Mandrillus sphinx) and 6 cynomolgus monkeys (Macaca fascularis) were infected by subcutaneous injection of third-stage larvae of human L. loa from Gabon. The mandrill developed microfilaremia with an estimated prepatent period of 147 days, but microfilariae were not detected in any of the cynomolgus monkeys. Thus, mandrills appear permissive to human L. loa, whereas cynomolgus monkeys are not. Serum antibody responses were examined on western blots of adult L. loa antigens. Preinfection sera from all animals gave no reactions, but, after infection, sera from cynomolgus monkeys reacted more intensely and with more antigens than mandrill sera. Antibodies were still detectable in cynomolgus monkeys 15 mo postinfection. These reactions were compared with those found using human infection sera. Reactions with the cynomolgus monkey sera resembled those found with resistant endemic and amicrofilaremic human sera.     

Identification of an amino acid responsible for the CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis)

The FN18 monoclonal antibody (mAb), directed to CD3 molecules, did not react with the lymphocytes of some cynomolgus monkeys (Macaca fascicularis), because of the polymorphism of the CD3epsilon chain. The epitope recognized by the FN18 mAb was successfully expressed on COS7 cells upon transfection of plasmid DNA coding for the CD3epsilon derived from T cells of a FN18 positive cynomolgus monkey. By construction and expression of plasmid DNA encoding the mutant CD3epsilon, the amino acid residue at position 67 was demonstrated to be involved in the formation of an epitope recognizable by the FN18 mAb.     

Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys

A non-opportunistic, generalized giant cell disease (GCD) was found in 12 out of 25 (48%) cynomolgus monkeys infected with SIVsm. Most organs were affected notably the lymph nodes (LN), spleen, gut, liver, lungs and CNS. The multinucleated GC varied considerably in cell size and in the number and cytoplasmic distribution of the nuclei. Immunohistochemically most GC expressed SIV antigens and markers of mononuclear phagocytes (CD68), CD4 and also occasionally the T-cell markers CD45RO, CD43 and CD2. Monkeys with GCD had more pronounced immunosuppression with lower CD4-cell counts, more often demonstrable SIV antigen in the blood and LN and had been infected for a longer time oeriod, as compared to monkeys without GCD.These findings show that SIV infection in cynomolgus monkeys is frequently associated with extensive formation of multinucleated GC of macrophage origin, which appears to be related to the pathogenesis of the infection and the degree of immunosuppression.     

A Monoclonal Antibody Selection for Immunohistochemical Examination of Lymphoid Tissues From Non-human Primates

Non-human primates (NHPs) offer valuable animal models for basic research into human diseases and for the preclinical validation of new therapeutics. Detailed in situ examination of the involved cell types using immunohistochemistry is often hampered by the lack of cross-reactive antibodies (Abs). In the current study, we have tested a large panel of monoclonal antibodies raised against human leukocyte differentiation and activation markers for cross-reactivity on cryosections of lymphoid tissue from six NHP species. In total, we have tested 130 Abs against 69 antigens expressed in tissues from one great ape species (chimpanzee/Pan troglodytes), two Old World species (rhesus macaque/Macaca mulatta and cynomolgus macaque/Macaca fascicularis), and three New World species (common marmoset/Callithrix jacchus, cotton-top tamarin/Saguinus oedipus, and owl monkey/Aotus triviogatus). We have found a large panel of cross-reactive Abs: 93 of 102 (91%) in chimpanzee, 97 of 125 (78%) in rhesus macaque, 70 of 109 (64%) in cynomolgus macaque, 69 of 116 (60%) in common marmoset, 40 of 81 (49%) in cotton-top tamarin, and 35 of 80 (44%) in owl monkey. The availability of a reliable panel of cross-reactive markers is important to gaining further insight into immunological processes in disease-affected tissues from NHP species. (J Histochem Cytochem 57:1159–1167, 2009)     

Wild-type measles virus with the hemagglutinin protein of the edmonston vaccine strain retains wild-type tropism in macaques

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM(+) as well as CD46(+) cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM(+) cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM(+) lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo.     

Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis

Abstract: Cross-reactivity between several commercially available mouse antihuman monoclonal antibodies (mAbs), conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC) fluorochromes, and peripheral blood leukocyte surface antigens, has been established in infant cynomolgus (Macaca fascicularis) monkeys using whole blood lysis, and two-color, PE and FITC flow cytometric analysis. With the exception of the CD8 marker, the bivariate dot-plot patterns for all other markers were similar in infant monkeys and in humans. For the CD8 marker, however, a CD8⁺CD2^? population of cells was observed in the majority of monkeys tested (10 out of 12). The number of CD8⁺CD2^? cells was higher (13%) in infant monkeys compared to the 3% reported for adult human blood. The mean percentage and absolute numbers for the cell surface markers identified with the human mAbs CD2 (FITC, Ortho, Paritan, NJ), CD4 (PE, B-D, Mountain View, CA), and CD8 (PE, B-D) when these were combined with a series of PE- or FITC-labelled human mAbs were similar across all combinations tested. Statistically significant differences were observed between male and female monkeys for the mean percentage levels of CD4 (females > males) and for the CD4/CD8 ratio (females > males). Such gender differences need to be taken into consideration when infant cynomolgus monkeys are used as models for chemical-induced immunotoxicity studies. Measurement of the interleukin-2 (IL-2) and transferrin proved to be useful in monitoring in vitro cellular activation in infant cynomolgus and possibly in rhesus (M. mulatta) monkeys.     

Comparison of pharmacokinetic behaviors of two human interferons (Lb-IFN-alpha and Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

Two human interferons (IFNs), natural lymphoblastoid IFN-alpha (Lb-IFN-alpha) and recombinant IFN-alpha (Re-IFN-alpha A) were compared for their induction of 2'-5' oligoadenylate (2-5A) synthetase and pharmacokinetic behaviors in cynomolgus monkeys. In in vitro experiments, the enzyme activity induced in an epithelial cell strain from human amniotic membrane (FL) cells was much higher than that in primary culture of cynomolgus monkey kidney (PMK) cells, and Lb-IFN-alpha induced higher levels of the enzyme than did Re-IFN-alpha A in both cells. In in vivo experiments, no difference was found in clearance from the blood between the two IFNs, but the half life of the enzyme induced in peripheral leukocytes by Lb-IFN-alpha was about twice longer than that by Re-IFN-alpha A, and the enzyme levels detected in various tissues were IFN-dose dependent. These results indicate that natural Lb-IFN-alpha is more effective in inducing 2-5A synthetase than Re-IFN-alpha A, and that the enzyme activity of various tissues as well as peripheral blood lymphocytes (PBL) increased in the cynomolgus monkeys administered with human IFNs.     

Expressed sequence tags from cynomolgus monkey (Macaca fascicularis) liver: a systematic identification of drug-metabolizing enzymes

The liver, a major organ for drug metabolism, is physiologically similar between monkeys and humans. However, the paucity of identified genes has hampered a deep understanding of drug metabolism in monkeys. To provide such a genetic resource, 28655 expressed sequence tags (ESTs) were generated from a cynomolgus monkey liver full-length enriched cDNA library, which contained 23 unique ESTs homologous to human drug-metabolizing enzymes. Our comparative genomics approach identified nine lineage-specific candidate ESTs, including three drug-metabolizing enzymes, which could be important for understanding the physiological differences between monkeys and humans.     

A species comparison of low density lipoprotein heterogeneity in nonhuman primates fed atherogenic diets

Six male cynomolgus monkeys and five male African green monkeys were fed dietary cholesterol to induce hypercholesterolemia. The two groups studied had equivalent total plasma cholesterol concentrations. Low density lipoproteins (LDL) were isolated from whole plasma by ultracentrifugation and separated from other lipoprotein classes by agarose column chromatography. LDL were further subfractionated by density gradient ultracentrifugation in a VTi-50 vertical rotor. The material within five density regions was pooled from each sample and molecular weight, electrophoretic mobility, apoprotein heterogeneity, and percentage composition were determined for each subfraction. In general, cynomolgus monkey LDL were larger and more polydisperse than African green monkey LDL, and the LDL subfractions of cynomolgus monkeys were generally of lower densities although molecular weights at any density were in the same range for both species. ApoB-100 was the major apoprotein in each subfraction. ApoE was frequently present in the less dense subfractions while apoA-I was often seen in the more dense subfractions. Cynomolgus monkey LDL appeared to contain more apoE than African green monkey LDL. Over the entire spectrum of LDL, the percentage composition of the particles at any given density was indistinguishable between the species. In general, the average cynomolgus monkey LDL was larger, more polydisperse, less dense, and appeared to contain more apoE than the average African green monkey LDL. One or all of these differences might help explain the increased susceptibility to diet-induced atherosclerosis seen in cynomolgus monkeys.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.