Non-HLDA8 animal homologue section anti-leukocyte mAbs tested for reactivity with equine leukocytes

Non-HLDA8 animal homologue section anti-leukocyte

mAbs tested for reactivity with equine leukocytes

Sherif Ibrahim a, Falko Steinbach a,b,*a Institute for Zoo and Wildlife Research, Alfred Kowalke Str. 17, 10315 Berlin, Germany

b Virology Department, Veterinary Laboratories Agency (VLA), Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK

Abstract

In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs

were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose,

one colour flow-cytometric analysis was performed as screening test.

This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with

weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern

from that expected from human immunology, 2 mAbs with questionable variable staining, 13 mAbs with weak-positive

expression and alternate pattern, and 78 negative mAbs. In 23 cases, more appropriate target cells, such as thymocytes or stem

cells, were not available for screening. The results support and add to the value of the ‘‘cross-reactivity’’ approach for equine

immunology.

Crown Copyright # 2007 Published by Elsevier B.V. All rights reserved.

Keywords: Horse; Immunology; Leukocyte differentiation

www.elsevier.com/locate/vetimm

Veterinary Immunology and Immunopathology 119 (2007) 81–91

1. Introduction

Well characterized, commercially available, mono-

clonal antibodies directed against surface antigens of

human cells are an important tool for veterinary

immunology (e.g. Steinbach et al., 2002; Saalmuller

et al., 2005). Based on this knowledge, anti-human

monoclonal antibodies were tested for their reactivity

against equine immune cells to broaden the toolbox for

equine immunology.

In the present study, we used flow-cytometry to

screen mAbs obtained in addition to the mAb derived

* Corresponding author at: Virology Department, Veterinary

Laboratories Agency (VLA), Woodham Lane, New Haw, Addlestone,

Surrey KT15 3NB, UK. Tel.: +44 1932 357 566;

fax: +44 1932 357 239.

E-mail address: [email protected] (F. Steinbach).

0165-2427/$ – see front matter. Crown Copyright # 2007 Published by E

doi:10.1016/j.vetimm.2007.06.033

from the animal homologues section of the 8th

international workshop on Human Leukocyte Differ-

entiation Antigens (HLDA8). These mAbs again were

tested for reactivity on equine leukocytes and platelets.

Taking into account that some of the previous

descriptions of cross-reactive mAbs were not confirmed

later, we put strong emphasis on the comparability of

equine and human staining patterns and on data and

experiences using human cells. Some of the mAbs

analysed have been described before and were used as

controls – but also for re-analysis. Only mAbs that

stained comparable to human leukocytes in several

consecutive tests with independent animals were finally

assigned ‘‘positive’’ in this study. The study comple-

ments the HLDA8 equine part (Ibrahim et al., 2007) and

the positive mAbs were later added to further analysis

such as double labelling or immunohistochemistry

(Flaminio et al., 2007).

lsevier B.V. All rights reserved.

mailto:[email protected]

http://dx.doi.org/10.1016/j.vetimm.2007.06.033

S. Ibrahim, F. Steinbach / Veterinary Immunology and Immunopathology 119 (2007) 81–9182

Table 1

mAbs detected positive during the flow cytometric screening

Donator CD no. Clone Isotype Result

NatuTec/VMRD CD2 HB88A mIgG1 ++

NatuTec/VMRD CD5 HT23A mIgG1 ++

MACS CD11b M1/70.15.11.5 mIgG2b ++

Biometec CD14 7H3 (big 10) mIgG1 ++

Biometec CD14 big 11 mIgG1 ++

Biometec CD14 big 12 mIgG1 ++

Biometec CD14 7D6 (big 13) mIgG1 ++

BD CD21 B-Ly4 mIgG1 ++

Seroteca CD68 Ki-M6 mIgG1 ++

Coulter CD83 HB15a mIgG2b (++)

Coulter CD206 (MMR) 3.29B1.10 mIgG1 ++

Serotec CD283 (TLR3) TLR3-7 mIgG2a (++)

Coulter HLA-ABC (MHC-I) B9.12.1 mIgG2a ++

NatuTec/VMRD MHCII EqT2 mIgG1 ++

Seroteca HLA-DR (MHC-II) D-F1 mIgG2a ++

Serotec B cells (Canine) CA2.1D6 IgG1 (++)

‘‘++’’ indicates a clear positive result, consitent with human staining pattern described. ‘‘(++)’’ refers to a clear positive result where minor doubts

remain due to a lack of expierence (e.g. with mAb or CD molecule). All antibodies were directed against human antigens, if not specified

otherwise.a These mAbs are not distributed by Serotec any more.

Table 2

mAbs analysed by flow cytometry without homologous staining to

humans

CD no. Clone Isotype Result

Donator

Biometec

CD14 RoMo1 mIgG2a –

CD14 4A7 mIgG1 –

CD14 7F11 mIgG2a A

CD14 big 16 mIgG1 A

Chemicon

CD1b WM-25 mIgG1 NA/–

CD18 68-5A5 mIgG2a W

CD23 Tu1 mIgG3 –

CD64 10.1. mIgG1 –

Coulter

CD1a BL-6 mIgG1 NA/–

CD28 CD28.2 mIgG1 –

CD33 D3Hl60.251 mIgG1 –

2. Material and methods

Most materials (except for the mAbs used) and

methods were described in depth in Ibrahim et al.

(2007). In addition, monocytes were differentiated

towards macrophages (MF) and dendritic cells

(DC), respectively. For this purpose, monocytes were

isolated by adherence such as described (Steinbach

and Thiele, 1994) and differentiated for DC thereafter

with EqGM-CSF (250 U/ml) and EqIL-4 (100 U/ml)

or EqGM-CSF (500 U/ml) and LPS (0.5 mg/ml) to

induce MF, such as described and deduced from the

human system (Vandergrifft et al., 1994; Steinbach

et al., 1998, 2005; Dohmann et al., 2000; Mauel et al.,

2006).

Additionally, intracellular staining for some anti-

gens, such as CD68, was performed. For this purpose,

two methods were applied. First, the cells were

pelleted for 5 min 2000 rpm in an Eppendorf tube,

washed once with PBS and fixed thereafter with

ice-cold (�18 8C) 96% EtOH for 5 min. Thereafter

the cells were washed again in PBS and used for

staining such as described including the use of the

respective isotype controls (Ibrahim et al., 2007).

Alternatively, cells were treated with permeabilizing

solution 2 (BD Biosciences, Heidelberg, Germany)

according to the manufacturers protocol before

staining. Acquisition and analysis by flow cytometry

was performed as described and discussed (Ibrahim

et al., 2007).

3. Results and discussion

Flow-cytometric screening of this panel of antibodies

indicated for 16 mAbs a positive reaction which is in

accordance to human staining pattern. The data of these

mAbs are summarized in Table 1 and will be presented

and discussed in more detail. Further 26 mAbs showed a

weak, alternate, questionable or positive staining that was

not in accordancewith staining patterns in humans. These

results will be discussed briefly and are summarized in

Table 2. Twenty-three mAbs could not be tested on most

S. Ibrahim, F. Steinbach / Veterinary Immunology and Immunopathology 119 (2007) 81–91 83

Table 2 (Continued )


CD34a 581 mIgG1 NA

CD40 MAB89 mIgG1 –

CD203 97A6 mIgG1 –

Immunotools

CD1a HI149 IgG1 NA/–

CD1a WM35 IgG2b NA/?

CD2 4G4 IgG1 –

CD3 MEM-57 IgG2a W/A

CD3 16A9 IgG2a W/A

CD4 MEM-241 IgG1 –

CD4 6D10 IgG1 –

CD5 1C12 IgG1 –

CD6 MEM-98 IgG1 –


CD7 7F3 IgG2a W/A

CD8 MEM-31 IgG2a W/A

CD8 4H8 IgG2b –

CD9 MEM-61 IgG1 –

CD9 4E1 IgG2a –

CD10 4F9 IgG2a +

CD11a MEM-25 IgG1 A

CD11a TB-133 IgG2a –

CD11b B2 IgM –

CD13 Q20 IgG2a –


CD14 8G3 IgG2a –

CD15 B4 IgM –

CD16 MEM-154 IgG1 +

CD16 5D2 IgG2a –

CD18 MEM-48 IgG1 W/A

CD18 LFA-1/1,54 IgG1 –

CD19 LT19 IgG1 –

CD19 11G1 IgG1 –

CD20 MEM-97 IgG1 W/A

CD20 NKI-IH4 IgG1 –

CD21 5D1 IgG1 –

CD22 6B11 IgG1 –

CD23 tu1 IgG3 –

CD24 IB5 IgG1 –


CD25 TB30 IgG2b +

CD27 9F4 IgG2a –

CD28 Kolt-2 IgG1 –

CD29 2A4 IgG1 –

CD31 HEC-75 IgG1 –

CD33 MD33.6 IgG1 NA/–

CD34a 4H11 IgG1 NA

CD34a QBEnd10 IgG1 NA

CD35 E11 IgG1 W/A

CD36 IVC7 IgG1 –

CD37 NMN46 IgG2a –

CD41a 6C9 IgG1 –

CD42b MB45 IgG1 –

CD43 DFT1 IgG1 A

CD44 NKI-P2 IgG1 –


CD45 15D9 IgG1 –

CD45RA MEM-56 IgG2b +

CD45RA FB-11-13 IgG1 W/A



CD45RB MEM-55 IgG1 –

CD45RO UCHL1 IgG2a W/A

CD49b 10G11 IgG1 –

CD49f NKI-GoH3 IgG2a W/A

CD54 MEM-111 IgG2a –

CD54 15.2 IgG1 –

CD57 NC1 IgM –

CD59 MEM-43 IgG2a –

CD61 C17 IgG1 –


CD65 88H7 IgM –

CD66acde IH4Fc IgG1 –

CD66b B13.9 IgG1 –

CD71 RVS10 IgG1 –


CD95 FAS19 IgG1 –

CD103 2G5 IgG2a +

CD122 MIK-beta1 IgG2a A

CD235a AME-1 IgG1 –

HLA-DR (MHC-II) 1E5 IgG1 +

MPO 7.17 IgG1 +

Miltenyi Biotec

BDCA-1 (CD1c) AD5-8E7 mIgG2a NA/–

BDCA-2 AC144 mIgG1 NA/–

BDCA-3 AD5-14H12 mIgG1 NA/–

BDCA-3 AD5-5E8.12.3 mIgG1 NA/–

BDCA-4 AD5-17F6 mIgG1 NA/–

CD2 LT2 mIgG2b A

CD3 OKT3 mIgG2a +

CD3 BW264/56 mIgG2a –

CD4 M-T466 mIgG1 +

CD6 M-T411 mIgG1 –

CD7 6B7 mIgG2a W/A

CD8 BW135/80 mIgG2a –

CD14 TuK4 mIgG2a –

CD15 VIMC6 mIgM –

CD16 VEP13 mIgM –

CD19 LT19 mIgG1 –

CD19 SJ25-C1 Rat IgG2a A

CD20 LT20 mIgG1 A

CD25 4E3 mIgG2b –

CD27 LT27 mIgG1 –

CD30 Ki-2 mIgG1 A

CD34a AC136 mIgG2a NA

CD36 AC106.3 mIgG2a W/A

CD43 6F5 Rat IgG2a –

CD45 5B1 mIgG2a –

CD45R RA3-6B2 rIgG2a +

CD56 AF12-7H3 mIgG1 –

CD56 MZ3-26G9 mIgG1 –

CD61 Y2/51 mIgG1 –

CD90.2 30-H12 Rat IgG2b W/A

CD123 AC145 mIgG2a A

CD 133/1 AC133 mIgG1 NA

CD 133/2 AC141 mIgG2a NA

CD133/2 293C3 mIgG1 NA

CD138 B-B4 mIgG1 A

CD205 NLDC-145 mIgG2a NA/–

CD235a AC107 mIgG1 NA




TCR alpha/beta BW242/412 mIgG2b A

MHCII M5/114.15.2 Rat IgG2b –

Serotec

CD1a NA1/34 mIgG1 NA/?

CD1a O10 mIgG1 NA/–

CD1c L161 mIgG1 NA/?

CD86 BU63 mIgG1 NA/–

CD91 a2MRa-2 mIgG1 –

TLR2 TLR2-3 mIgG1 ?

TLR4 HTA125 mIgG2a –

TLR9 5G5 mIgG2a –

VMRD

CD5 B29A mIgG2a ?

CD90 (Thy-1) DH24A IgM +

‘‘W’’ a weak likely unusable but positive reactivity. ‘‘+’’ indicates a

positive staining pattern that was not consitent with human staining

pattern. These results might still be due to homologous staining.

‘‘A‘‘ indicates a clear alternate expression pattern from that

expected from humans. More unlikely to reflect homologous stain-

ing. ‘‘?’’ is a questionable result in staining, especially due to

staining variablility between animals. ‘‘NA’’ was not tested on

the appropriate target cells. Designations may be combined, such

as ‘‘W/A’’. All antibodies were directed against human antigens, if

not specified otherwise.a Anti-human CD34 mAbs were also analysed on the available

equine leukocyte cell lines (Steinbach et al., 2007).

appropriate targets and the remaining 91 mAbs showed a

weak alternate (W/A) or negative pattern. These results

are summarized in Table 2 only.

Two mAbs directed against equine leukocyte

markers were included in this study as positive controls

especially as a basis for the further studies (in particular

the double labelling). Both mAbs were distributed by

VMRD, Pullman, USA. The mAb HB88A is directed

Fig. 1. Detection of equine T lymphocytes by use of equine specific mAbs.

the vast majority of lymphocytes. Both mAbs served also as controls for the

Both mAbs were analysed by indirect staining using a PE-labelled second

against EqCD2. This mAb stained, as expected, the vast

majority of lymphocytes but no other cells (Fig. 1A).

Likewise the mAb HT23A stained such a lymphocyte

population and served as positive control for equine

CD5 (Fig. 1B).

The clone M1/70.15.11.5 from Miltenyi Biotec

(Bergisch Gladbach, Germany) is a rat IgG2b mAb

directed against mouse and human CD11b. This mAb

stained equine monocytes and granulocytes and a small

population of lymphocytes weakly (Fig. 2). Such

staining is in accordance with staining of human cells,

where, next to myeloid cells, NK cells are also CD11b+.

A significant number of antibodies in this study were

directed against human CD14 and four of them (clones

big 11, big 12, 7D6/big 13 all from Biometec

(Greifswald, Germany) reacted strongly with equine

monocytes only (Fig. 3). CD14 has been described in

low amounts on populations of human B cells and

granulocytes also, but our previous attempts to detect

significant amounts of CD14 there in humans failed

(Steinbach and Thiele, 1994). Therefore, the present

results are in accordance with human staining and we

assume the detection of equine CD14 by these

antibodies. It should be mentioned that one additional

CD14 mAb (7H3, big 10) was described recently

(Steinbach et al., 2005). All hybridomas produce IgG1

and recognize a conformational epitope of aa9–13 and

39–44 (Schutt et al., 1995; http://www.biometec.de).

This result is slightly surprising as aa9–13 (human

system) are conserved but aa40 is changed (E! G). In

contrast, another clone (RoMo1) that recognises the

fully conserved aa147–152 at least as part of the epitope

does not react with equine monocytes. Thereby, it

becomes evident that the homology in a certain area is

not sufficient to deduce the reactivity of mAbs

The mAbs HB88A (EqCD2 (A)) and HT23A (EqCD5 (B)) both detect

consecutive two-colour flow-cytometry study (Flaminio et al., 2007).

ary antibody (FL-2).

http://www.biometec.de/


Fig. 2. Analysis of equine leukocytes using the anti-mouse CD11b mAb M1/70.15.11.5. All granulocytes (C) all monocytes (B) but only a

population of lymphocytes (A) were stained by this mAb. M1/70.15.11.5 was directly FITC conjugated (FL-1).

Fig. 3. Analysis of equine monocytes using anti-human CD14 mAbs. The mAbs big 11 (A), big 12 (B), and big 13 (C) all detect equine monocytes

but no other leukocytes (data not shown). All mAbs were analysed by indirect staining using a PE-labelled secondary antibody.

Fig. 4. Analysis of equine lymphocytes using the anti-human CD21

mAb B-Ly4. A population of 9–24.5% of the lymphocytes were

stained by this mAb, presumably representing B lymphocytes.

B-Ly4 was directly PE-conjugated (FL-2).

associated. Even more, the result underlines the

necessity for a controlled study, as clone 7F11 (big

14) was initially suggested to cross-react with horses

(but did not in our hands). In the case of CD14, further

experiments may be performed to assess the functional

activity of the antibodies as some might or should

inhibit LPS binding also (Schutt et al., 1995).

The mAb B-Ly4 directed against human CD21 was

designated to cross-react with equine cells before and

indeed reacts with a population of lymphocytes (Lin

et al., 2002). In contrast to the mAb BL13 with its very

weak staining (Ibrahim et al., 2007), B-Ly4 stains a

population of lymphocytes brightly and additionally

fewer cells less intense (Fig. 4). The positive cells

comprised of 9–24.5% (n = 5 horses) of the lympho-

cytes. This labelling itself, the previous description of

reactivity and the knowledge on the amount of B-cells

expected lead to the result that B-Ly4 most likely

stains equine CD21 (Tumas et al., 1994). In addition,

we tested an antibody from Serotec (CA2.1D6)

that was indicated to cross-react from canine to


Fig. 5. Analysis of equine monocytic cells using the anti-human

CD68 mAb Ki-M6. Monocytes were differentiated towards macro-

phages and stained intracellular. All monocytic cells reacted clearly

positive, whereas controls and extracellular staining remained nega-

tive (not shown). Ki-M6 was analysed by indirect staining using a PE-

labelled secondary antibody (FL-2).

equine B cells with the same result (Table 1). In this

case, however, the mAb was classified conditionally

positive (++), as we do not have further information

about the epitope that is recognised and for which

homology is assumed.

The mAb Ki-M6 (Serotec) is directed against CD68

and has been suggested in an immunohistochemistry

(IHC) study to detect equine CD68 alike (Siedek et al.,

2000). In the context of another study, we were, not able

to confirm the staining pattern described with in situ

hybridization, performed with a cDNA of equine CD68

cloned recently (Steinbach et al., 2005). Indeed, Ki-M6

did not stain PBM nor LPS activated monocytes in

standard flow cytometry. However, when intracellular

Fig. 6. Analysis of equine leukocytes using the anti-human CD83 mAb HB-

positive, possibly representing B lymphocytes (A). Additionally, in some hor

never detected (C). HB-15a was directly PE-conjugated (FL-2).

staining was applied Ki-M6 did stain cultured equine

monocytes clearly (Fig. 5). This is in principle

accordance with human cells, where CD68 is mainly

expressed intracellularily (Stockinger, 1989). Using Ki-

M6 as positive control, we returned to all anti-CD68

mAbs of the animal homologues section of the HLDA8

(Ibrahim et al., 2007) and other anti-CD68 mAbs and

analysed them by intracellular staining, but Ki-M6

remained the only mAb with positive staining pattern.

The mAb HB15a (Beckman Coulter) directed

against CD83 was also analysed as part of this study.

This mAb is not identical to the clone HB15e of the

HLDA8 study (Ibrahim et al., 2007). The mAb HB15a

repetitively reacted with a population of 14–25% of

lymphocytes, which were presumed to be B-lympho-

cytes (Fig. 6). Additionally, the mAb did show reactivity

in some but not all horses with monocytes, but never

with granulocytes (Fig. 6C). The data on CD83

expression in humans is slightly complex. In blood,

only a population of DC stains positive. The marker has

therefore been suggested also as the unique marker for

mature DC. However, CD83 was originally cloned from

activated B cells and was also detected in LCs, which

are certainly the hallmark cells for immature myeloid

DC (Zhou et al., 1992; Kozlow et al., 1993).

Additionally, CD83 may at least on MoDC be induced

during maturation and accordingly may appear on

myeloid cells (Zhou and Tedder, 1995). Overall, the

staining of CD83 was considered (++), awaiting further

characterization by double labelling and other methods.

The mAb 3.29B1.10 (Beckman Coulter) is directed

against the human macrophage mannose receptor

(MMR) and has been described elsewhere in more

detail (Steinbach et al., 2005). CD206 is not expressed

by most PBM but up-regulated within 48 h during in

vitro culture (Stahl and Gordon, 1982). The staining of

15a. In all horses analysed, a population of lymphocytes was detected

ses, most or all monocytes were also positive (B) but granulocytes were


Fig. 7. Analysis of equine monocytic cells using the anti-human

CD283 (TLR3) mAb TLR3-7. Monocytes were differentiated towards

macrophages and stained intracellular. In contrast to CD68, only a

population of monocytes reacted with this mAb. We assume that this

was due to regulated expression and suboptimal conditions in stimu-

lation. TLR3-7 was analysed by indirect staining using a PE-labelled

secondary antibody (FL-2).

this mAb was fully in accordance with our expectation

from the human system and was therefore classified ++

(Table 1).

The mAb TLR3-7 (Serotec) is directed against the

toll like receptor 3 (TLR 3), recently classified CD283

(Zola et al., 2005). This receptor which detects dsRNA

is located mainly intracellularily (Sen and Sarkar,

2005), where we detected the reaction of TLR3-7 using

differentiated and activated monocyte-derived macro-

phages (Fig. 7). The result remains conditionally

positive only (++) as many TLRs (including TLR-3)

are expressed by some cells only and the situation in

horses has not been investigated thus far to our

knowledge.

Fig. 8. Analysis of equine leukocytes using the anti-human MHC class I

granulocytes) of all horses tested were brightly stained. B9.12.1 was direc

The mAb B9.12.1 (Beckman Coulter) directed

against the human MHC class I HLA-ABC locus

reacted with all equine leukocytes (Fig. 8). According to

the fact that cross-reactivity of anti-MHC mAbs

between various species including horses has been

described many times before, we do not see reason to

assume non-specific staining here and classified the

mAb ++.

For the more restrictedly expressed MHC class II, we

had the positive control mAb EqT2 (VMRD) and

detected the mAb D-F1 (Serotec). While EqT2 is

designated to react with pan-MHC II, mAb D-F1 is

directed against the human HLA-DR only. Accordingly,

it was not surprising that this mAb stained slightly

weaker than EqT2. Notably: while all monocytes

(Fig. 9B) and no granulocytes (Fig. 9C) were stained, a

significant population of both resting and activated PBL

(not shown) were stained also (Fig. 9). It has been

described earlier that equine lymphocytes (all B and

some T cells) are, MHC class II positive (Crepaldi et al.,

1986; Lunn et al., 1993). The phenomenon of cross-

reactivity of anti-MHC mAbs is not new and many more

mAbs cross-react alike (e.g. Crepaldi et al., 1986;

Monos et al., 1989). Equine specific mAbs and cross-

reacting anti-human mAbs open the gate returning to

the analysis of MHC II subclasses and expression of

MHC II by subpopulations of lymphocytes.

Of the remaining 139 mAbs that were tested in this

study (Table 2), the mAb 68-5A5 from Cymbus

Biotechnology (distributed via Chemicon, UK)

most likely stains the equine homologue of CD18

weakly (Table 2). As there were already some anti-

CD18 mAbs with better staining found in the HLDA8

study (Ibrahim et al., 2007) we did not analyse this mAb

further.

In 11 cases we observed a clear staining we could,

however not interpret as homologous or alternate.

mAb B9.12.1. All leukocytes (A, lymphocytes; B, monocytes; C,

tly FITC-conjugated (FL-1).


Fig. 9. Analysis of equine leukocytes using the anti-human MHC class II mAbs EqT2 (A–C) and D-F1 (E–F). While all peripheral blood monocytes

(B and E) reacted positively with both mAbs, the number of positive lymphocytes was variable (A and D), but in some instances (such as represented

by (A)) many T-lymphocytes were also MHC II+. Granulocytes (C and F) were never MHC II+. Both mAbs were analysed by indirect staining using a

PE-labelled secondary antibody (FL-2).

Among these were antibodies against CD3 (OKT3),

CD4 (M-T466) and CD10 (4F9) that were later

also included into double labelling, but did not perform

as to be expected in case of homologous staining

(Flaminio et al., 2007). For the anti-CD16 clone MEM-

154 we did not observe the required population of

positive monocytes and for the anti-CD25 clone TB30

failed to observe any up regulation after activation.

In case of the mAb MEM-56, directed against

CD45RA we observed the staining of a population of

lymphocytes that we could not correlate with CD45RA

pattern.

The rat mAb RA3-6B2 is designated to react against

mouse CD45R (Miltenyi Biotec) and is described to be

specific for the mouse B cell B220-antigen that is the

220kD large ABC-isoform of CD45 (Marvel and

Mayer, 1988; Hathcock et al., 1992; Rodig et al.,

2005). CD45 is a marker expressed in various isoforms

and B220 is an especially enigmatic variant. On the

one hand used as a valuable B cell marker for mice, it is

also expressed on some mouse T cells (Marvel and

Mayer, 1988). In humans, B220 is expressed by only a

subset of B cells that do not express the memory B-cell

marker CD27 (Rodig et al., 2005). It must be

mentioned in this context that isoforms of CD45

may occur in parallel. For example, the CD45R/B220/

ABC isoform is specific for mouse B cells, but those

cells also express other isoforms, such as CD45RA or

RC, which are not exclusive for mouse B cells. It was

not possible to obtain much additional information

on this mAb but it seems to be specific for the ABC

form. The reaction with horse leukocytes was

clearly positive, but in contrast to the mAb DH16A

(Ibrahim et al., 2007), RA3-6B2 recognises all equine

leukocytes (Fig. 10). Therefore, the mAb had to be

designated + until further studies are performed to

resolve the nature of equine CD45.

The mAb 2G5 that is directed against CD103 stained

more lymphocytes (2–10%) than recommended from

the literature (0.5–5%) but this could be a species-

specific difference we cannot exclude. For TLR-2 we

observed a staining of a sub-population of differen-

tiated myeloid cells only, which we considered

questionable (?) on the assumption that expression in


Fig. 10. Analysis of equine leukocytes using the anti-mouse CD45R mAb RA3-6B2. All lymphocytes (A), all monocytes (B), and all granulocytes

(C) were stained by this mAb. RA3-6B2 was directly PE-conjugated (FL-2).

humans and horses need not necessarily have to be

identical.

The myeloperoxidase (MPO) is a heme-enzyme

present in the granules of granulocytes and has been

demonstrated to participate in the oxygen dependent

microbicidal activity of these cells (Nauseef et al., 1983).

When internal staining was applied, mAb 7.17 stained all

neutrophils but also a subpopulation of lymphocytes.

Therefore, the data are not in accordance with human.

An analogous problem occurs with the mAb DH24A,

which detects CD90 in dogs (Cobbold and Metcalfe,

1994) but reacts clearly (+) with equine granulocytes

only (data not shown). It may thus be a valuable marker

for equine granulocytes (which we did not put emphasis

to investigate further) but most likely does not stain the

equine CD90 molecule, which would be expected on T

cells and their precursors (Table 2).

The mAb B29A distributed from VMRD was

generated using PBL from multiple species for

immunization and especially on the detection of

IgM-pos cells was presumed to detect equine B-cells

(Tumas et al., 1994). The staining, however, was weak

and it remained unclear if B29A detects all equine B

cells (Kydd et al., 1994; Tumas et al., 1994). We have

also seen a weak staining of a population of equine PBL,

thereby confirming the earlier results (data not shown).

The principle problem occurs in how to interpret this

data. This mAb is bovine CD5-specific (Davis et al.,

1990) and it is not rational to assume that this mAb

stains a B-cell specific variant of CD5, even though

CD5 has been described on human and mouse B cells,

especially at pathological conditions (Berland and

Wortis, 2002) Almost certainly, B29A does not stain an

equine homologue of CD5 – as it was intended as part of

this workshop. The staining itself with high variability

remains questionable (?).

In 12 cases we observed a clear staining that was

contradictory to our expectations from the human

situation and these mAbs were therefore termed ‘‘A’’

(Table 2). Good examples are an anti-human CD14

mAb staining populations of lymphocytes or an anti-

human CD19 mAb, staining granulocytes most

brightly.

Twenty-three mAbs could not be tested by appro-

priate cells or tissue and were listed NA, including the

stem cell markers CD34 or CD133. For some cells,

additional experiments were performed such as in the

main study and designation thereafter was performed

accordingly NA/nn (Ibrahim et al., 2007). Briefly, CD33

should be expressed by PBM also and the anti-CD33

mAb was accordingly termed NA/–. Additionally, we

differentiated MoDC and tested markers specific for DC

(such as CD1 or B-7). Two of the anti-CD1 mAbs from

Serotec (NA1/34 against CD1a and L161 against CD1c)

did show some reactivity with a subset of DC and

were therefore termed NA/? (Table 2) underlining the

need for further studies. It should be mentioned also

that all anti-human CD34 mAbs were analysed on an

available equine leukocyte cell line also (Steinbach

et al., 2007).

Ninety-one mAbs showed a weak alternate (W/A) or

no staining (–).

In summary, 155 mAbs were analysed in this study

for their reactivity against equine leukocytes, using flow

cytometry as screening technology. Next to anti-human

mAbs, this study also included some mAbs that were

originally directed against mouse or other animal

antigens. Additionally, a few mAbs were included that

were generated against equine leukocytes. Like in the

HLDA8 study, only a few mAbs directed against other

species reacted with horse leukocytes but add further

antibodies to the toolbox of equine immunology.


Acknowledgments

The authors thank the companies that provided

additional mAbs for this part of the study: Beckman

Coulter, Krefeld, Germany; Biometec, Greifswald,

Germany; Miltenyi Biotec, Bergisch Gladbach, Ger-

many; Immunotools, Friesoythe, Germany; Serotec,

Dusseldorf, Germany; VMRD, Pullman, USA.

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