Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human,cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach

The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.     

An extensive monoclonal antibody panel for the phenotyping of leukocyte subsets in the common marmoset and the cotton-top tamarin.

New World monkeys are valuable animal models to study human diseases. To determine the phenotype of cells involved in immune responses, we used flow cytometry to screen a large panel of anti-human monoclonal antibodies (mAb) for cross-reactivity with cells of the common marmoset and the cotton-top tamarin. Certain antigens (e.g., CD2, CD8, CD20) are well conserved. However, CD10, CD23, and CD33 showed a clear discrepancy in their reaction patterns in both species, indicating that significant differences on the epitope level occurred during evolution. Epstein-Barr virus-transformed B-cell lines were shown to be a valuable tool for screening B-cell-specific reagents. In some cases, fluorescein isothiocyanate (FITC) and phycoerythrin (PE) modification of mAbs had a negative effect on the binding capacity, which stressed the importance of choosing the right label. Despite the fact that some CD antigens were not detected, adequate numbers of cross-reactive mAbs were identified to perform extensive studies on immunological functions in both the common marmoset and the cotton-top tamarin.     

Comparative Plasma Lipidome between Human and Cynomolgus Monkey: Are Plasma Polar Lipids Good Biomarkers for Diabetic Monkeys?

Background

Non-human primates (NHP) are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis.

Methodologies/Principal Findings

We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS). We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3). Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005) and plasmalogen PC species (p<0.0005), while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA) in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005) and GM₃ (p<0.0005), but higher level of Cer (p<0.0005) in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls.

Conclusions

For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys exhibited decreased levels of sphingolipids, which are microdomain-associated lipids and are thought to be associated with insulin sensitivity. Significant increases in PG species, which are precursors for cardiolipin biosynthesis in mitochondria, were found in fasted diabetic monkeys (n = 8).     

E. coli expression and purification of human and cynomolgus IL-15

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)₆-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)₆-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3⁻ CD8⁺ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

 Interleukin 16 (IL-16) is synthesized as a 67 000 M _r precursor (pro-IL-16), but only a carboxy terminal part of 12 000–14 000 M _r is secreted by CD8(⁺) lymphocytes. This lymphokine binds to CD4 and has been shown to induce migration, affect the activation state of T cells, and inhibit immunodeficiency virus replication. It has been suggested that CD8(⁺) cell-derived soluble factors play a pivotal role in protecting natural-host nonhuman primates from developing immunodeficiency following SIV infection. In a first attempt to address this question, we cloned and sequenced the IL-16 cDNA from different primates. Here we report the pro-IL-16 sequence from chimpanzees, African green monkeys (AGM), rhesus macaques, and cynomolgus macaques. In order to compare and analyze structural motifs possibly involved in processing, intracellular targeting, or secretion, we extended our study to the New World monkeys saimiri and aotus and to the mouse. Alignments of deduced amino acids reveal that the human protein shares 99% similarity to that of chimpanzees, approximately 95% to rhesus, cynomolgus and AGM, about 90% to aotus and saimiri, and 77.5% to the mouse. Phylogenetic analyses revealed the expected evolutionary groupings. Received: 27 August 1997 / Revised: 7 October 1997     

北京地区实验恒河猴外周血T淋巴细胞亚群分析

目的测定正常实验恒河猴外周血T淋巴细胞亚群,获取基础数值。方法使用BD FACS Calibur流式细胞仪,Cell QuestTM3.3分析软件,应用CD3-Percp、CD4-FITC、CD8-PE荧光标记的抗体,对北京地区84只实验恒河猴的外周血T淋巴细胞亚群进行统计分析。结果北京地区实验恒河猴外周血CD3＋,CD4＋T淋巴细胞占淋巴细胞（LYM）的百分比为32.82%±5.93%;CD3^＋CD8^＋T淋巴细胞占淋巴细胞百分比为（23.99±6.34）%;CD4^＋/CD8^＋的比值为1.45±0.41;LYM百分比为白细胞的（49.70±14.00）%;LYMF为（4.634±2.181）×106/mL。结论实验用恒河猴T淋巴细胞亚群的基础数值测定为相关比较医学研究奠定基础。     

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.     

H5N1接种恒河猴和食蟹猴后外周血细胞变化的分析

目的测定H5N1型禽流感病毒感染恒河猴、食蟹猴后,其外周血细胞的变化,为H5N1模型猴提供基础数据及研究参考。方法健康合格食蟹猴、恒河猴各4只,经滴鼻方式接种H5N1病毒107TCID50,确认发病后,在不同时间点进行血细胞及T淋巴细亚群的分析。结果与接种H5N1病毒前比较,接种后白细胞总数（WBC）在第6天时有所降低,至第9天时回升;红细胞总数（RBC）在第3天有所降低,之后回升;淋巴细胞比例及数量分别在第6天、第9天升高并达到最高值。至第9天时,CD4^＋T细胞数明显高于接种前,CD8^＋T细胞数上升显著,导致CD4^＋/CD8^＋T细胞比例下降,甚至在2只食蟹猴出现了比例倒置。结论实验用猴感染H5N1后,可导致WBC,CD4^＋,CD8^＋T等血液细胞的变化,应作为H5N1模型动物的检测指标。     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.

Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose.

At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.

⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

Characterizing the induction of diabetes in juvenile cynomolgus monkeys with different doses of streptozotocin

Juvenile (2–23 years old) cynomolgus monkeys are frequently used as recipients in non-human primate islet transplantation studies. The aim of this study was to examine the effects of different doses of streptozotocin (STZ), and find the optimal dose for inducing diabetes in these monkeys. Fifteen juvenile (2–3 years old) cynomolgus monkeys were separated into three groups and administered with different doses of STZ (100, 68 or 60 mg kg⁻¹). Basal and glucose-stimulated blood glucose, insulin, and C-peptide levels, as well as body weights were monitored. Hepatic and renal function tests and pancreatic immunohistochemistry were performed before and after STZ treatment. Monkeys treated with both 100 and 68 mg kg⁻¹ of STZ exhibited continuous hyperglycemia, which coincided with a nearly complete loss of islet β-cells. Two monkeys received 60 mg kg⁻¹ of STZ, but only one became completely diabetic. During the first week following STZ treatment, hepatic and renal function slightly increased in these three groups. However, 24 hours post-STZ, serum total bile acid levels were significantly increased in monkeys treated with 100 mg kg⁻¹ than those treated with 68 mg kg⁻¹ of STZ (P<0.05). These data suggest that 100 mg kg⁻¹ and 68 mg kg⁻¹ of STZ can safely induce diabetes in cynomolgus monkeys aged 2–3 years, but 68 mg kg⁻¹ of STZ, rather than 100 mg kg⁻¹ of STZ, may be more appropriate for inducing diabetes in these monkeys. Furthermore, body surface area, rather than body weight, was a more reliable determinant of dosage, where 700 mg m⁻² of STZ should be the lower limit for inducing diabetes in juvenile monkeys.     

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r² = 0.83, r = 0.91, p < 0.01) better than NHP (r² = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to Phase 1 clinical studies

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake. Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

Possible role of genetic factor(s) on age-related increase of peripheral CD4+CD8+ double positive T cells in cynomolgus monkeys.

Mature TCR alpha beta T cells in peripheral blood are generally classified into either CD4 single positive (sp) T cells or CD8sp T cells. Several studies demonstrated that considerable amounts of CD4+CD8+ double positive (DP) T cells exist in peripheral blood of human and several animals. In particular, we previously reported that peripheral DP T cells increase in an age-related manner in cynomolgus monkeys (Macaca fascicularis), but the finding that DP T cells in some aged monkeys were maintained at a low proportion (under 5%), suggests that the increase in peripheral DP T cells might be genetically controlled in cynomolgus monkeys. To test this hypothesis, 24 families were randomly selected and used in a formal genetic analysis of the proportion of DP T cells. Parents and offspring in selected families were classified into DP-High and DP-Low groups based on a 5% cutoff level of DP T cells. The cutoff value was set by analysis of the distribution of the proportion of DP T cells. Nine out of 13 offspring (69.2%) with DP-High x DP-High parents belonged to the DP-High group, whereas three out of nine offspring (33.3%) belonged to DP-High group in the case of DP-High x DP-Low mating pairs. No offspring (0%) of two offspring with DP-Low x DP-Low parents belonged to the DP-High group. In addition, heritability (h2: narrow sense) obtained from the regression coefficient of offspring on mid-parent values was 0.54 +/- 0.19. Both findings suggest that increases in DP T cells in cynomolgus monkeys may be genetically controlled.     

T-Cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx)

Background Although a wide variety of non‐human primates are susceptible to simian T‐cell leukaemia virus type 1 (STLV‐1), little is known about the virological or molecular determinants of natural STLV‐1 infection. Methods We determined STLV‐1 virus tropism in vivo and its relation to the immune response by evaluating cytokine production and T‐cell subsets in naturally infected and uninfected mandrills. Results With real‐time PCR methods, we found that STLV‐1 in mandrills infects both CD4⁺ and CD8⁺ T cells; however, proviral loads were significantly higher (P = 0.01) in CD4⁺ than in CD8⁺ cells (mean STLV‐1 copies number per 100 cells (± SD) was 7.8 ± 8 in CD4⁺ T cells and 3.9 ± 4.5 in CD8⁺ T cells). After culture, STLV‐1 provirus was detected in enriched CD4⁺ but not in enriched CD8⁺ T cells. After 6 months of culture, STLV‐1‐transformed cell lines expressing CD3⁺, CD4⁺ and HLADR⁺ were established, and STLV‐1 proteins and tax/rex mRNA were detected. In STLV‐1 infected monkeys, there was a correlation between high proviral load and elevated levels of interleukin (IL)‐2, IL‐6, IL‐10, interferon‐γ and tumour necrosis factor‐α. The two monkeys with the highest STLV‐1 proviral load had activated CD4⁺HLADR⁺ and CD8⁺HLADR⁺ T‐cell subsets and a high percentage of CD25⁺ in CD4⁺ and CD8⁺ T cells. Conclusions Our study provides the first cellular, immunological and virological characterization of natural STLV‐1 infection in mandrills and shows that they are an appropriate animal model for further physiopathological studies of the natural history of human T‐cell leukaemia viruses.     

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>)

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2–10), and average heterozygosity was 0.63 (range 0.10–0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.     

Decreased expression of MMP-9 in CD8+ cells in placenta with severe preeclampsia

We compared the number of CD4-positive (CD4⁺) and CD8-positive (CD8⁺) cells in severe and non-severe preeclampsia (PE), and in normal pregnancy. We also evaluated the expression of matrix metalloproteinase 9 (MMP-9) in CD4⁺ and CD8⁺ cells. Immunohistochemistry for CD4⁺ and CD8⁺ was performed on the decidua basalis of 15 severe and 13 non-severe PE women and compared to decidual tissue of 19 normal pregnancies (control group). Co-expression of MMP-9 with CD8⁺ and CD4⁺ cells was determined by double immunofluorescence staining. The median number of CD8⁺ cells/mm² was significantly lower for the severe PE group than for the normal pregnancy group, as was the number of CD4⁺ cells and MMP-9⁺CD8⁺ cells. No statistical difference was found between the non-severe PE group and the normal pregnancy group. The significant decrease of CD4⁺, CD8⁺ and MMP-9⁺CD8⁺ cells at the fetal-maternal interface only in the severe PE group suggests that immunological disorders play a role in the pathophysiology of severe PE.     

Heat Shock Cognate Protein 71-Associated Peptides Function as an Epitope for Toxoplasma gondii-Specific CD4+ CTL

HLA-DR-restricted CD4⁺ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4⁺ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4⁺ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4⁺ CTL.     

Triple immunofluorescence confocal laser scanning microscopy: spatial correlation of novel cellular differentiation markers in human muscle biopsies.

Two different methods for triple immunofluorescence imaging with a confocal laser scanning microscope (CLSM) are described. The methods enable spatial "mapping" of 3 different epitope distribution patterns simultaneously in one tissue section. The key to triple imaging includes: (a) specific immunolabeling with 3 different mouse monoclonal antibodies (mAbs), (b) localization of the antibody binding sites by 3 different dyes, (c) spectral isolation of each dye by using selective band pass or long pass emission filters, (d) computerized imaging of the fluorescences as colored overlays or as selective signals in each optical section through the tissue in the z-direction. Method 1 consists of the combination of fluorescein isothiocyanate (FITC), phycoerythrin R (PE), and Texas Red (TR) as fluorescent markers. These dyes can be imaged by using 488 nm and 543 nm excitation laser lines. In method 2 aminomethyl coumarin acetic acid (AMCA) was combined with FITC and PE. For this application the CLSM was adapted to ultraviolet microscopy that enabled the use of 3 laser lines (364 nm, 488 nm, 543 nm) for excitations. Cryostat sections of diagnostic human muscle biopsies (n = 9) were studied which were normal by ordinary light microscopic examination. Sections were incubated with mAbs specific for: (1) the fast myosin heavy chain (My32); (2) the major histocompatibility class II antigen HLA-DR; (3) the lymph node homing receptor Leu8, and (4) the cell adhesion receptor OKM5 (CD36). By combining these mAbs in triple staining procedures, 3 capillary types and 4 different phenotypes expressed by muscle fibers were identified simultaneously. The mAbs Leu8 and OKM5, widely used as leukocyte typing antibodies in the blood, exhibit hitherto unrecognized specificities for antigens displayed by muscle fibers. At the level of these markers, specific spatial correlations between OKM5 reactive capillaries and both OKM5 reactive and nonreactive muscle fiber types become visible. The presented results provide direct evidence for cellular complexity and novel insight into the immunoanatomical architecture of skeletal muscle. The methods may be of general significance for the construction and quantification of three-dimensional multiparameter "maps" of cells and tissues.     

One-round determination of seven leukocyte subsets in rhesus macaque blood by flow cytometry

BACKGROUND: Rhesus macaques are frequently used in biomedical research as experimental models for studying infectious diseases and for preclinical vaccination trials. The infection of these monkeys with simian immunodeficiency viruses (SIV) or simian-human immunodeficiency viruses (SHIV) reproduces the clinical and immunological characteristics of human infection by human immunodeficiency virus (HIV). Evolution of the immune response in the infected animals is generally analyzed by determining the lymphocyte subsets on blood samples using flow cytometry but requiring multiple, blood consuming, determinations. METHODS: Cell subsets present in whole-blood samples were labeled with a combination of anti-human monoclonal antibodies to CD2, CD20, CD4, CD8, and CD14 coupled to FITC or PE and analyzed by flow cytometry. RESULTS: In one round, we obtained the precise determination of macaque blood cell composition by flow cytometry. Monocytes, granulocytes, eosinophils, B lymphocytes, helper, and cytotoxic T lymphocytes were distinguished. Results obtained correlated strongly with those obtained with conventional blood cell differential systems and with separate staining of lymphocytes. The analysis of blood from healthy rhesus macaques and SHIV-infected animals demonstrated the accuracy of the determination even in very pathological situations such as macaques with simian AIDS. CONCLUSIONS: Our method allows fast determination of the blood cell composition and will be particularly useful to evaluate the cell subset evolution of macaques involved in large-scale experimental trials.     

The detection of a breast cancer antigen on MCF-7 cells reactive with the TCR(α) of a specific killer T cell line

We established several immortalized human T cell lines by cotransfecting primary lymphocytes derived from breast cancer patients with human c-myc and human c-Ha-ras oncogenes. A CD8 positive (CD8⁺) killer T cell line, FT-8, exhibitedin vitro specific cytotoxicity to a human breast cancer cell line, MCF-7. The FT-8 cells suppressed the growth of MCF-7 cells transplanted to athymic mice. The cytotoxic reaction was caused via T cell antigen receptor (TCR) on MCF-7 cells, because monoclonal antibodies against the TCR inhibited the cytotoxicity of FT-8 cells. The TCR() cDNA of FT-8 was cloned by using a PCR amplification technique and expressed by a cell-freein vitro translation system. The TCR() protein recognized a target antigen of 32 KDa on MCF-7 cells.Abbreviations FCS fetal calf serum - FITC fluorescein isothiocynate - MAb monoclonal antibody - PE phycoerythrin - TCR T cell recepter - TCR() chain of TCR - TPBS PBS containing Tween 20