Transcript Ramananarivo, M. Herbert, F.Montero, C. Fornelli

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