CROSS REACTIVITY OF ANTI-HUMAN MONOCLONAL ANTIBODIES WITH MONKEY PERIPHERAL BLOOD CELLS BY FLOW CYTOMETRY T. Ramananarivo, M. Herbert, F. Montero, C. Fornelli Beckman Coulter, Immunotech, R&D Cellular Analysis, Marseille, France. Monoclonal Antibodies (mAbs) The following mouse mAbs were used to develop the three color combinations: • IOTest® Anti-NHP CD3-FITC / CD4-PC7 / CD8-APC (PN A44224): anti-Rhesus monkey CD3, clone FN18, IgG1; anti-human CD4, clone M-T477, IgG2ak; anti-Human CD8, clone B9.11, IgG1. • IOTest® Anti-NHP CD3-FITC /CD20-PC7 /CD16-APC (PN A44225): anti-Rhesus monkey CD3, clone FN18, IgG1; anti-Human CD20, clone B9.E9, IgG2ak; anti-Human CD16, clone 3G8, IgG1. FITC and PE conjugated antibodies used in this study are listed in figure 2. Sample Preparation & Staining • Analysis of Whole Blood: 25µL of EDTA whole blood sample (≤ 24 hrs) was incubated with 20µl of PE conjugated antibody, in presence or absence of 25 µL of anti-NHP combination, and incubated for 20 minutes at room temperature (RT), protect from light. The red blood cells were then lysed with 1 ml of a “Fix-and-Lyse” mixture, based on the combination of VersaLyse™ Lysing Solution (PN IM3648) and IOTest 3 Fixative Solution (PN IM3515). Samples were incubated for additional 10 minutes at RT, protect from light and kept at 2-8°C until use. • Activation markers: heparinized blood samples were subjected to Ficoll-Histopaque gradient centrifugation (Sigma, Réf.10831), in order to obtain Peripheral Blood Mononuclear Cells. PBMC were subsequently cultured alone, with PHA (Sigma, Réf. L4144 5µg/ml) or with PMA (Calbiochem, Réf. 524400 20ng/ml) plus Calcimycin (Sigma, Réf. C7522 125ng/ml). Samples were further processed as described for whole blood. Illustration on figure 3. Flow Cytometry For simultaneous analysis of lymphocyte sub-populations and expression of a lineage specific marker, the flow cytometer must be equipped to detect Forward and Side Scatter and four fluorescence channels allowing the analysis of FITC-, PE-, PC7-, and APC-conjugated antibodies (respectively 525 15 / 575 15 / 755 15 / 675 15 nm maximal peak emission). APC conjugates require an exciting source of 633 nm (He-Ne laser) or 635 nm (Red diode laser). Prepared samples were analyzed on FC 500 / Multi Carousel Loader (MCL) using Beckman Coulter Cytomics™ CXP™ analysis software. In all experiments, a mixture of Flow-SetTM Fluorospheres (PN 6607007) and Flow-Set 675 Fluorospheres (PN 6607120) were used to set up the PMT values while compensations were set by using Cyto-Comp Cells (PN 6607023) stained separately with either CD45-FITC (PN IM0782), CD45-PE (PN IM2078), CD45-PC7 (PN IM3548) or CD45-APC (PN IM2473). Each (T, B, NK)-lymphocyte subset was analyzed as described in Figure.1 . The results of phenotype is summarized in figure 2. Absolute count 25µl of Flow Count (Réf 7547053) were added to stained & lysed samples before analysis on FC500. The linearity of the flow count measurement in our system is shown in figure 4. Hist 1 Hist 2 on LY-MO Hist 3 on CD3+ Hist 4 on CD3- FS vs. SS SS vs. FL1 CD8 vs. CD4 CD16 vs. CD20 T cells Figure.4 : Absolute count using IOTest Anti-NHP combos Flow count Fluorospheres were collected on FS/FL5 channel as illustrated on the histogram (gate CAL983) Histo 3: combo CD3/CD4/CD8 FL4 (CD8) vs. FL5 (CD4) conditioned on CD3+ T cells to analyse CD8+ CD4- and CD4+ CD8- T cells subsets. Rhesus BABOUIN CYNO RHESUS 8 Baboon R2 = 0,9996 R2 = 0,982 R2 = 0,998 6 CD3+ 4 2 0 0 0,25 0,5 0,75 6 1 Dilution du sang 2 R = 0,9996 4 CD4+ 2 R = 0,9969 R2 = 0,9708 2 0 0 0,25 0,5 0,75 Dilution du sang CD2 39C1C5 CD5 BL1A CD7 8H8.1 CD8b 2ST8.5H7 CD11a (LFA1-a) 25.3 CD11b (Mac-1) BEAR.1 CD11c BU15 CD14 RMO52 CD15 80H5 CD18 7E4 CD19 J4.119 CD21 BL13 CD21 B120 CD23 HD50 CD23 9P25 CD24 ALB9 CD27 1A4CD27 CD28 CD28.2 CD29 K20 CD31 5.6E CD31 1F11 CD32 2E1 CD33 D3HL60 CD35 J3D3 CD38 LS198-4-3 CD38 T16 CD40 MAB89 CD43 DFT1 CD44 J.173 CD45 KC56 CD45 ALB12 CD45 IMMU19.2 CD45 J33 CD45 B3821F4A CD45RA ALB11 CD45RO UCHL1 CD50 HP2/19 CD54 84H10 CD56 N901 CD57 NC1 CD81 JS64 CD83 HB15a CD85a (ILT5) 7H5 CD85D (ILT4) 42D1 CD85J (ILT2) HP-F1 CD85K (ILT3) ZM3.8 CD86 HA5.2B7 CD89 A3 CD90 F15-42-1-5 CD94 HP-3B1 CD95 (Fas) 7C11 CD95 (Fas) UB2 CD105 1G2 CD116 (GM-CSFR)SCO6 CD120A H398 CD120B 80M2 MONOCYTES FORM PN CYNO RHE BAB PE PE PE PE PE PE PE PE PE PE PE PE RD1 RD1 PE PE PE PE FITC FITC PE PE PE FITC PE PE PE PE PE PE FITC PE PE PE PE PE PE PE PE FITC PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE IM0443 IM0469 IM1429 IM2217 IM1433 IM2581 IM1760 IM0650 IM1954 IM1570 IM1285 A32536 6603196 6604426 A33099 IM1428 IM2578 IM2071 IM0791 IM1431 IM2409 IM1935 IM1179 IM1836 IM2371 IM1832 IM1936 R&D A32560 6603839 IM0647 IM1833 IM2078 4660704 IM1834 IM1307 IM1601 IM1239 IM2073 IM0466 IM2579 IM2218 A44226 A22334 A07408 IM3579 IM2729 IM1614 IM1840 IM2276 IM2446 IM1739 A07414 IM1977 A22359 A22360 T, NK T, NK T, NK T, B, NK T, B, NK NK T, B, NK T, B, NK NK, B T, NK T, B, NK T, B, NK NK T, B, NK B B T, B, NK B B T, B, NK B B B B B B B B T, B, NK T, B, NK T, B, NK T (B) NK T, B, NK T (B) NK T, B, NK T, B, NK B, NK T, B, NK T, B, NK B, NK T, B, NK T, B, NK B, NK B T, B, NK T, B, NK B T, B, NK T, B, NK B T, B, NK T, B, NK T, B, NK T T, B, NK T, B, NK T, B, NK T, B, NK T, B, NK T, B, NK T, B, NK T, NK GRANUS CYNO RHE BAB CYNO RHE BAB LYMPHOCYTES SPECIFICITE CLONE CD122 (IL2Rb) CF1 CD123 (IL3Ra) 107D2 CD126 (IL6Ra) M91 CD130 (IL6Rb) TR5.7.5 CD134 (OX40) Ber-act35 CD135 (FLT3) SF1.340 CD138 BB4 CD144 TEA 1/31 CD136 ID1 CD158a EB6B CD158b GL183 CD158e Z27.3.7 CD158i FES172 CD159a (NKG2A) Z199 CD160 BY55 CD161 (NKRP1A) 191B8 CD164 67.D2 CD166 3A6 CD184 (CXCR4) 12G5 CD203C 97A6 CD226 KRA236 CD244 (2B4) C1.7 CD300a (IRp60) E59.126 CD314 (NKG2d) ON72 CDw328 (p75AIRM) Z176 CD335 (NKP46) BAB281 CD337 (NKP30) Z25 CRTH2 BM16 NKp80 HLA-ABC B9.12.1 HLA-DR, DP, DQ 9-43 HLADR B8.12.2 HLADR IMMU357 IL5R H17N NTB-A MA.127 OSCAR 11.1CN5 TCRgd IMMU510 TCRab BMA031 SPECIFICITE CD25 CD25 CD45RO CD69 CD71 CD86 CD134 (OX40) CD154 (CD40L) CLONE B1.49.9 1HT44H3 UCHL1 TP1.55.3 YDJ1 HA5.2B7 BER-ACT35 TRAP-1 FORM PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE PE FITC PE PE PE PE PE PE PE PE PN CYNO IM1978 NK A32535 IM1979 A22362 A24986 IM2234 IM2759 A07481 A22363 A09778 IM2278 IM3292 IM3337 IM3291 NK IM3657 IM3450 A32347 T, B, NK A22361 A07409 T B (NK) IM3575 A44227 IM1608 A22328 A08934 T, NK A22330 IM3711 NK IM3709 NK A07413 R&D T, NK IM1838 T, B, NK 6604366 B, NK IM0464 T, B, NK IM1639 T, B, NK A31894 A40926 A24987 IM1418 IM1467 FORM PN PE IM0479 RD1 6604422 PE IM1307 PE IM1943 PE IM2001 PE IM2729 PE A24986 PE IM2216 CYNO T, B, NK T++ T, B, NK B MONOCYTES RHE BAB NK NK GRANUS CYNO RHE BAB CYNO RHE BAB T, NK T, NK T, NK T, B, NK T, B, NK T (B) NK T T T, NK T, NK T, NK T, B, NK B, NK T, B, NK T, B, NK NK NK NK T, NK T, B, NK B, NK T, B, NK T, B, NK R2 = 0,9996 R2 = 0,9995 R2 = 0,9845 CD8+ 2 0 0 0,25 0,5 0,75 Dilution du sang 1 2 R2 = 0,9996 R2 = 0,9953 R2 = 0,9896 CD20+ 1 0 0 0,25 0,5 0,75 Dilution du sang 1 1 R2 = 0,9996 R2 = 0,9838 R2 = 0,9747 CD16+ 1 0 0 0,25 0,5 0,75 Dilution du sang Our 4-color analysis combined to a no wash lysis procedure is a powerfull tool to analyse Leukocytes, activated PBMC and more deeply, to delineate lymphocyte subsets. This methodology is perfectly compatible with the Flow count fluorospheres, which can be included in the test to determine absolute count. From the 102 antihuman mAbs analysed, an average of 40% showed positive cross-reactivity with Rhesus, Cynomolgus and Baboon. The positive results obtained in this study were largely correlated to published data3-4 and extended to Baboon species. Among the few differences found between these species: CD7, CD126 & CD226 were only found on Baboon in contrary to CD14 marker exclusively expressed on macaques. The only significant differencies observed with human, were the staining of Cynomolgus myeloid cells with CD203c and the expression of CD90 on the overall leukocyte populations. REFERENCES PBMC RHE BAB T, B, NK T, B, NK T, B, NK T, B, NK T, B B T T POSITIVE 1 DISCUSSION Figure.2 : Crossreactivity of anti-human mAb with cynomolgus (CYNO), Rhesus (RHE) and Baboon (BAB) antigens LYMPHOCYTES 1 3 4 Cells/µl (*103) Histo 4: combo CD3/CD20/CD16 FL4 (CD16) vs. FL5 (CD20) conditioned on CD3- T cells to analyse CD16+CD20- NK cells and CD20+ CD16-B cells. CLONE NK cells Cyno Histo 2: S. Scatter vs FL1 A gate is drawn around CD3+ and another one around CD3lymphocytes. SPECIFICITE B cells Cells/µl (*103) Animals Cynomolgus (Maccaca Fascicularis) and Rhesus (Maccaca mulatta) monkeys were obtained from the Primatology Center- Louis Pasteur University (Strasbourg, France) and Baboon monkeys from the Primatologie Center- CNRS (Rousset, France). All animals were serologically free of pathogenic enterobacteries, tuberculosis and were negative for Herpes B and simian retroviruses. Blood sampling were performed under anesthesia. Blood samples were collected by venipuncture with EDTA (for lysed whole blood screening) or heparin (for PBMC activation study). Histo 1: F.Scatter vs S. Scatter A large gate is drawn around lymphocytes and monocytes, to include nearly all lymphocytes, that vary greatly in light scatter characteristics . Cynomolgus PBMC stimulated 1day with PHA and stained with CD3/CD20/CD16 + CD86. Lymphocytes B and NK are gated on CD3- cells. Cells/µl (*103) MATERIALS AND METHODS Figure.1 : Representative FACS analyses of Monkey Whole blood samples- Gating strategy Cells/µl (*103) Non Human Primate (NHP) animal models represent an important tool1 for the development of vaccines or retroviral agents, the evaluation of immune responses to human pathogens and are particularly attractive for efficacy testing of human-specific pharmaceutical compounds that are not reactive in less closely related species as rodents. Among Different Immunotoxicology tests recommended by the International Conference on Harmonisation (ICH) Harmonized Tripartite Guideline2, Immunophenotyping by Flow Cytometry is a useful assay. The ever-growing list of anti-human mAb, together with the identification of novel structures on human cells that play an important role in immune functions, prompted us to examine the cross-reactivity of a number of usual as well as seldom anti-human antibodies, with three monkey species commonly used: cynomolgus & rhesus (Asian Macaques) and Baboon (African). To address this, and in order to determine the distribution of lymphocyte subsets in peripheral blood , we developed a standardized Four Color Cytometry tool to screen the panel of anti-human antibodies. By using a cocktail of pre-calibrated labeled antibodies that give minimum emission spectral overlap (FITC/PC7/APC), we enable the addition of a wide variety of PE-labeled antibodies. This flexible 4-color Lymphocyte cytometric assay was optimized in a No Wash Red Blood Cell lysing protocol for enhanced accuracy and auto-setup panels were developped for analysis on FC500 cytometer. Moreover, our 4-color no wash methodology can be used in combination with Flow-Count™ Fluorospheres to enable direct determination of absolute counts in each lymphocyte subset. Figure.3 : Illustration of a 4-color analysis. Cells/µl (*103) INTRODUCTION 1- Parker J « Toward better health. The role of primates in biomedical research at the regional primate research centers » Primate News, (1990), 24: 3-31 2- Immunotoxicity for Human pharmaceuticals. ICH Harmonised Tripartite Guideline S8. (2005) 3- NIH Non Human Primate reagent Resource (www. nhpreagents. bidmc. harvard. Edu) NEGATIVE WEAK UNCLEAR 4- R. Biassoni « Molecular and Functionnal Characterisation of NKG2D, NKP80, and NKG2C Triggering NK cell Receptors in Rhesus and Cynomolgus Macaques.. » The Journal of Immunology, (2005) 174: 5695