Jun 2, 1997 - ation methods were used, the magnetic cell sorting system (MACS) and ... canine hematopoietic progenitor cells are comparable to those of ...
Copyright 0 Munksgaard 1997
Tissue Antigens 1997: 50: 466474 Printed in Denmark . All rights reserved
TISSUE ANTIGENS ISSN 0001-2815
Immune phenotype of canine hematopoietic progenitor cells E. Neuner, M. Schumm, E.-M. Schneider, W. Guenther, J. Ellwart, Kolb. Immune E. Kremmer, C. Vogl, M. Buettner, S. Thierfelder, H.-J. phenotype of canine hematopoietic progenitor cells. Tissue Antigens 1997: 50: 466474. Q Munksgaard, 1997 The immune phenotype of canine hematopoietic progenitor cells w a s studied by immunoseparation and culturing of separated cells. Two separation methods were used, the magnetic cell sorting system (MACS) and the fluorescence activated cell sorter (FACS). For separation rat anti dog antibodies Dog 13 and Dog 14 directed against Thy-1, and Dog 26 as well as cross-reactive mouse anti human antibodies IOT2a and 7.2 directed against MHC class I1 were used. Separated cell populations were cultured in semisolid agar before and after long-term culture on a pre-established irradiated stromal cell layer. After 28 days, adherent and nonadherent cells were harvested from long-term culture. The MACS system allowed separation of cells into positive and negative fractions. Long-term culture-initiating cells (LTC-IC) were found in both the Thy-l+ and the Thy-1- fraction, but the content of LTC-IC was higher in the Thy-l+ fraction. The MACS system did not allow separation of progenitor cells according to the expression of MHC class I1 antigen detected by Dog 26 and the crossreactive antibodies IOT2a and 7.2. In contrast to the MACS system the FACS allowed separation of negative, low-positive and high-positive cell populations. Low-positive fractions were well defined for Thy-1 and less well defmed for MHC class 11. CFU before and after long-term culture were exclusively observed in the ,low positive fraction (Thy-l'O+). Using MHC class I1 antibody Dog 26 LTC-IC were found mainly in the negative and low positive fraction, and CFU were observed mainly in the low and high positive fraction. In conclusion pluripotent canine hematopoietic precursor cells are low positive for Thy-I and for MHC class 11. In this respect canine hematopoietic progenitor cells are comparable to those of mouse
Experimental studies in dogs have made important contributions to the progress of transplantation of solid organs and hematopoietic tissues. Unfortunately, the components of the canine hematopoietic system are less well defined than those of mice and humans. Definition of the immune phenotype of canine hematopoietic precursor cells is a prerequisite for further studies of stem cell expansion, of stem cell transplantation as well as of immune reactions against stem cells. The long-term bone marrow culture (LTC) system described by Dexter mimics the in vivo hematopoietic environment and supports proliferation of more primitive cells (1). Immature progenitor cells are believed to lodge and to proliferate 466
E. Neunerl, M. Schumd, E . 4 . Schneldef, W. Guenthef, J. Ellwaff, E. Kreminerl, C. Vogll , M. Buettnd, S. Thlerfelder' and H . 4 Kolb1#2 'Instiiut fur Immunologie. 21nstltut fiir Klinische Haematolopie and, %stiiut fiir Experimentelle Haematolopie, GSF-Forschunpszentrumfur Urnwelt und Gesundheit, Munich, 4Eundesforschunpsanstalt fur Wruskrankheiten der Tiere, Tirbingen, Germany
Key words: canine - LTC-IC - Thy-1 - MHC class II Received 19 Aupust 1996, revised, accepted for publication 2 June 1997
in the adherent stromal layer and to be released into the overlying supernatant as differentiation proceeds (2). This method has been adjusted for the culture of canine hematopoietic progenitor cells (3). Recently, a number of monoclonal antibodies (mAb) directed against a wide range of antigens on canine leukocytes have been developed by us and others (4, 5). We studied separation of canine long-term culture initiating cells (LTC-IC) using antibodies against canine Thy-1 and MHC class 11. Two rat antibodies against canine Thy-1 (Dog 13, Dog 14), one rat antibody against canine MHC class I1 (Dog 26),and two cross-reactive murine antibodies against human MHC class I1 (IOT2a, 7.2) were selected for these studies. In most spe-
Phenotype of canine LTC-IC cies Thy-1 is expressed on prothymocytes , thymocytes and mature T-cells (6), but also in the brain and a variety of other non-lymphoid tissues (7). In rat and mice Thy-1 has been described on immature myeloid and erythroid progenitors (CFUGM, BFU-E), while more mature progenitors were found to be Thy-l- (8). Pluripotent stem cells however were found in the low positive fraction of Thy-1 (9). The present study investigated whether Thy-1 and MHC class I1 as differentiation antigens are expressed on the surface of pluripotent hematopoietic progenitor cells of dogs. Material and methods Monoclonal antibodies
Monoclonal antibodies against canine cells have been produced by immunizing LodC rats as described elsewhere (5). All antibodies are of IgG2a subclass. Dog 15 recognizes about 80 YOof canine peripheral blood and bone marrow granulocytes and 20 YOof bone marrow lymphocytes, but was found almost negative on peripheral blood and spleen lymphocytes. Dog 17 recognizes canine CD5. Dog 22 recognizes canine IgG. Dog 26 recognizes canine MHC 11. Dog 13 and Dog 14 recognize different epitopes on canine Thy-l(5). Monoclonal antibodies were used as cell culture supernatants in a dilution of 1:lO which has been found a saturating concentration for all antibodies. The specificities of the mAbs were defined in the First International Canine Leukocyte Antigen Workshop (CLAW) (4). IOT 2a (Dianova, Hamburg), an IgG2b mouse antibody directed against HLADR cross-reacts with approximately 80% of canine lymphocytes (10). Mouse mAb 7.2 (IgG2b) (generously provided by Paul Martin, Seattle, USA) reacts with a framework determinant of human Ialike antigen and cross-reacts with the canine Ia antigen (1 1). High gradient magnetic cell sorting (MACS)
The magnetic cell separation system used in this investigation has been described in detail by Miltenyi et al. (12). Mononuclear cells were separated on a Ficoll density gradient. They were adjusted to 2X107 MNC/ml and incubated with 10 percent of cell culture supernatant of monoclonal antibodies, washed twice and 10 p1 of microbeads coated with goat-anti-mouse or -rat antibodies per 1X107 MNC were added. The cell suspension was then transferred to the MACS BS column installed in the magnetic field, where magnetically labeled cells were trapped in the column while unlabeled cells were flushed through at 3.5 ml/ min using a 21 G needle. The purity of the separ-
ated populations was determined by FACS analysis after incubation of separated cells with FITClabeled goat-anti-rat antibody as proposed by the manufacturer. Fluorescence-activated cell sorting (FACS)
Ficoll-separated bone marrow cells were incubated with Dog 13, Dog 14, Dog 26, or mAb 7.2 for 30 min at 4°C. After two washes, they were incubated with F(ab’) 2 fragments of goat-anti-rat or goatanti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) (Jackson, West Grove, PA, USA) for 30 min at 4°C and washed twice. For cell labeling with mouse anti-HLA-DRY we incubated cells with FITC-conjugated anti-HLA-DR antibody 30 min at 4°C and washed twice. Cells were sorted on a FACS-Star flow cytometry system (Becton Dickinson, San Jose, CAYUSA). The sorting gates were set to isolate cells expressing lowdensity, and high-density FITC-conjugated antigen, and cells lacking the antigen. The purity of the sorted cells was later determined with a FACScan. Short-term agar assay
Marrow cells were separated by density gradient centrifugation over Ficoll (d= 1.077) and 1X lo5 MNC cells per ml were cultured in 0.3% agar (Difco laboratories, Detroit, USA) with Mc Coy’s 5A medium supplemented with 20% heat-inactivated fetal calf serum (Pansystems, Aidenbach, Germany), 0.75% sodium bicarbonate (Gibco, Eggenstein, Germany), 1.25% sodium pyruvate (Gibco, Eggenstein, Germany), 0.5% Eagle’s MEM vitamins (Gibco, Eggenstein, Germany), 1% Eagle’s MEM amino acids (Gibco, Eggenstein, Germany), 0.5% non-amino acids (Gibco, Eggenstein, Germany), 0.5% L-glutamine (Gibco, Eggenstein, Germany), 0.5% L-serine (21 mg/ml) (Merck, Darmstadt, Germany), 0.2% Lasparagine (10 mg/ml) (Merck, Darmstadt, Germany), 1.5% penicillin-streptomycin (Gibco, Eggenstein, Germany) and 20 U/mlrhu-erythropoietin (Boehringer, Mannheim, Germany), 200 ng/ml rhu-GM-CSF (Essex, Munich, Germany), 1% hemin (Sigma, Deisenhofen, Germany) and 20 ngl ml recombinant canine stem cell factor (rcanSCF) (Amgen, Thousand Oaks, CA, USA). Cultures were incubated in a humidified atmosphere at 38°C and 5% COz for 14 days. The elevated incubation temperature was chosen taking into account the higher body temperature of dogs. High potential proliferative-colony-forming cells (HPPCFC), which are described as early progenitor cells responding to factor combinations as SCF 467
Neuner et al.
cells was defined as the number of colonies grown in these cultures.
and GM-CSF by producing a huge amount of cells within 3 weeks (13), were defined as cells forming colonies with a diameter of more than 0.5 mm at day 21.
Results Long-term marrow culture
Canine lineage-specific antigens
Normal allogeneic stromal layers were cultured in 25-cm2 flasks (Nunc) for 1 4 2 8 days, as previously described (3) and irradiated at. 15 Gy. Separated cell populations were then plated on allogeneic irradiated stroma in LTBMC media (Iscoves medium with 20% horse serum (Sigma, Deisenhofen, Germany), 1% L-glutamine (200 mM, lOOX), 1% penicillin (5000 U/ml) and 1% streptomycin (5000 pg/ml; Gibco) and 10'' moVl hydrocortisone-21-phosphate (Sigma)). All LTBMC were maintained in a humidified atmosphere at 37°C with 5% COz. At weekly intervals, the cultures were fed by removing half of the supernatant and replacing it with fresh media. Non-adherent and adherent cells were recovered after 4 5 weeks and assayed in a short-term agar culture system for the presence of colony-forming cells. The number of long-term culture-initiating
Monoclonal antibody Dog 15 directed against antigens on canine granulocytes and monocytes recognizes 440/0+-31.6% of canine mononuclear bone marrow cells enriched over Ficoll-Isopaque (n=4). 7.8%?1.7% of these cells are labeled with Dog 17 which is directed against canine T-cells, and 25.3%?10,4% are labeled with Dog 22 a monoclonal antibody against canine IgG (n=4). Combination of these three antibodies results in 57.2Ohk26.8% antibody-positive cells (n=6). Purity of the negative fractions was between 93% and 95%, in the positive fractions it was between 36% and 69%. CFU are enriched in the negative fraction of each of the three antibodies as well as in the negative fraction of the antibody combination (Table 1). Comparison of lineage-negative and unseparated .bone marrow shows an enrichment of CFU and long-term culture-initiating cells
Table 1. Cell recovery, CFU growth and CFU recovery of canine bone marrow cells after immuno-magnetic separation with monoclonal antibodies Recovery of cell numbers %=SD
CFU-GM per 2x105 MNCzSD
Recovery of FCU numbers percent2SD
antibody
n
negative fraction
positive fraction
before MACS
negative fraction
posftive fraction
negative fraction
positive fraction
Dog 15
3 3 3 2 2
43514.8 58.852.6 56.2212.6 61 472
30.8217 12.828.5 11.826.1 22.7 23.5
91 2 2 0 91 2 2 0 91 220 91 2 2 8 91228
212243 156232 175226 171 445
12214 13216 321 14 1
98225.1 101.8+13 112238.2 115.9 232.4
5.428.1 2.323.5 0.520.4 3.5 0.26
Dog 17 Dog 22 Dog 15+Dog 17 Dog 15+DOg 17+Dog 22
+
CFU growth and CFU recovery of bone marrow cells before and after separation with MACS-technique. Cells were labeled with Dog 15, Dog 17 and/or Dog 22 and plated (before MACS) or plated after immuno-magnetic separation (neglpos. fraction). Zx105 MNC were plated into agar with the growth factors rcanSCF, rhGM-CSF and erythropoietin (n- numbers of experiments, SD = standard deviation)
Table 2. CFU and LTC-IC am negative for antigens detected by monoclonal antibodies Dog 15, Dog 17 and Dog 22 CFU (day 0) unseparated marrow exp. no.
LTCIC (day 35) llneage negative
unseparated marrow
lineage negative
HPP-CFC
CFU-GM
HPP-CFC
CFU-GM
HPP-CFC
CFU-GM
HPP-CFC
CFU-GM
0 0 0 0
96 119 308 392
0 0 0 0
146 564 481 772
2 11
1278 3786 539 92
46 30
2737 5734 1221 6000
0 0
0 8
Comparison of CFU-GM growth on day 0 per 2x105 MNC and content of LTC-IC after 5 weeks of long-term culture per 2 x 1 0 MNC which were seeded on a pre-established feeder-layer - unseparated and lineage-negative bone marrow. Lineage depletion was performed using monoclonal antibodies Dog 15, Dog 17 and Dog 22 and MACS. HPP-CFC: High potential proliferative-colony-forming cells
468
Phenotype of canine LTC-IC Table 3. CFU and LTC-IC after immuno-magnetic separation for Thy-1 CFU exp. no.
1 2 3 4
LTC-IC
before separation
negative fraction
positive fraction
before separation
66 96
0 1
104 180
1500
119 74
17
1500
0
54
negative fraction
positive fraction
192 231 0
1500
56 1500 16
0
P=0.067
88 1500 414
f=0.141 ~~
~
Content of directly CFU on day 0 and LTC-IC (CFU after week 5 from adherent and nonadherent cells from long-term culture) per 2x105 MNC, plated in agar short-term culture. Bone marrow cells were separated with monoclonal antibodies Dog 13 and Dog 14 directed against canine Thy-1 using MACS technique. Wilcoxon-test for matched pairs.
(LTCIC) in the lineage-negative bone marrow fraction (Table 2).
purity of separated fractions was greater in the negative fraction (90-93%) than in the positive fraction (69-96%). Colony-forming units (CFU) and the majority of LTC-IC were found in the Thy-1 positive fraction (Table 3). FAC-Sorting of marrow cells allowed separation according to fluorescence intensity into negative,
Canine Thy-1
Between 62% and 95% of cells were recovered after magnetic separation with Dog 13 and Dog 14. The before FAC-Sorting
neg
low
high
.
Thy- 1 negative
Sorted as outlined
Figure 1. Fluorescence pattern of Thy-1 labeled cells. Fluorescence intensity of canine bone marrow cells before and after sorting with monoclonal antibody against Thy1. Sorted cells were analyzed on a FACScan after separation on a FACS Star Plus. Therefore. the level of fluorescence appears to be higher after sorting.
Thy-I low positive
Thy-1 high positive
469
Neuner et al. Table 4. CFU and LTC-IC after FAC-Sorting for Thy-1 CFU Exp. no.
LTC-IC
negative
low pos.
high pos.
negative
0 0 0 0 0
325 402 725 195 800
28 0 17 0 0
0 0 0
1 2 3 4 5
low pos. 1500 1500 53 11 52
0 0
high pos. 45 0
0 0 1
Content of directly CFU and LTC-IC after FAC-Sorting. Canine marrow cells were stained with monoclonal antlbodies against Thy-1 (Dog 13 + Dog 14) and separated by fluorescence intensity as indicated In Figure 1. The number of CFU was determined in agar culture. LTC-IC is given as CFU growth after 5 weeks of long-term culture started with 3x105 MNC.
Table 5. CFU and LTC-IC after immunomaanetic smaration for MHC class II
CFU
LTC-1C
exp. no.
unsep.
neg.
pos.
unsep.
neg.
pos.
Dog 26
1 2 3 4
401 233 96 74
239 185 0 45
10 1 181 0
1500 996 1500 16
1000 636 1500 232
1000 76 1500 301
IOT 2a
1 2 3 4 5
401 233 96 74 17
155 34 81 240 474
319 38 136 149 307
1500 996 1500 16 1500
1500 508 250 1500 1500
326 56 1500 1500 1500
7.2
1 2 3
233 96 17
144 138 175
0 88 22
996 1500 1500
192 1500 1500
16 348 1500
Content of directly CFU and LTC-IC per 2x105 MNC plated in agar at day 0 and 6 weeks after long-term culture. Marrow cells were separated using monoclonal antibodies against canine class II and immunomagnetic beads (MACS) into positive and negative fractions. Unseparated marrow cells were used as control.
low positive and high positive fractions. Fluorescence pattern of Thy-1 labeled canine marrow cells showed 76.152.8% negative cells, 8.2%t5.7% Thy-1 low positive and 15.9+2.9% high positive cells (n=5). The cells were investigated after sorting by a second analysis with a flow cytometer. The purity of the fractions was 95.8% for the negative cells, 76.1% for the low positive cells and 92.6% for the high positive cells (Figure 1). CFU and LTCIC were almost exclusively found in the Thy-1 low positive fraction (Table 4). Canine MHC class II
Three monoclonal antibodies were used to investigate CFU and LTC-IC after immuno-magnetic separation. The recovery of cells was comparable to that of the Thy-1 separation (59-94%). Again the purity of the negative fraction was excellent (93-99.80/;)). The purity of the positive fractions was good using Dog 26 (80-910/;), but it was poor 470
using the cross-reactive antibodies IOT 2a (1890%) and 7.2 (15-62N). CFU growth was variable after separation. Using mAb Dog 26 CFU were found almost exclusively in the negative fraction in 3 of 4 cases. In experiments with mAb IOT 2a CFU growth varied between positive and negative fractions, and using mAb 7.2 the majority of CFU was also found in the negative fraction (Table 5). However, colonies with high proliferative potency (HPP-CFC) were only found in the positive fractions. LTC-IC were found in both fractions. FAC-Sorting improved separation results and made it possible to distinguish cells according to their antigen density. Fluorescence pattern of Dog 26 labeled marrow cells showed 67.1+7.5% negative cells, 9.4+4.3% low positive and 23.5+9.0% high positive cells. Separation into negative. low and high positive fractions revealed a purity of 98.6%)of the negative cells, 44.9% of the low positive and 92.8% of the high positive cells (Figure 2).
Phenotype of canine LTC-IC Figure 2. Fluorescence pattern of Dog 261abeled cells. Fluorescence intensity of canine bone marrow cells before and after sorting with monoclonal antibody against MHC class 11. An irrelevant antibody (anti-EBNA 2b) was used to define the population of fluorescence negative cells (overlay). Sorted cells were analyzed on a FACScan after separation. The higher level of fluorescence after sorting is due to change in calibration.
before FAC-Sorting
1 Dog 26 negative cells
Dog 26 low positive
The majority (about 90%)) of CFU and LTC-IC was found in the low positive fraction (Table 6). Further differentiation into eight fractions of fluorescence showed the majority of CFU in fraction. I11 to V and the majority of LTC-IC in fraction IV to VI (Figure 3). Dlscussion
Hematopoietic progenitor cells occur with a low frequency in the bone marrow, one of several hundred mononuclear cells is capable of forming colonies in semisolid agar and the frequency of pluripotent progenitor cells as LTC-IC is even lower (14). The immune phenotype of these hematopoietic progenitor cells can be defined by culture of immunologically separated cells. The results depend on the avidity of the antibodies, the separation technique and the culture conditions. We found contradictory and inconsistent results after immunomagnetic separation with anti Thy- 1 antibodies and no separational effect of the anti MHC
Dog 26 high positive
class I1 antibodies. In the case of the class I1 antibodies this may reflect the low avidity of the antibodies which can be seen in the poor purity of the separated fractions. Especially the cross-reactive human antibody 7.2 showed much lower purity of separated cells as the antibody raised against canine class 11. In the case of Thy-1 the purity of the separated cells was much better, but we also observed contradictory culture results. This indicates that the avidity of the antibodies used simultaneously was much higher and therefore improved the immunomagnetic separation. However, the contradictory results could only be explained by an low or intermediate expression of both antigens, which could only be investigated using fluorescence-based sorting of cells. In mice, primitive hematopoietic progenitor cells are found: Thy-l'O (9). Previous investigations in our laboratory with the rnAb F3.20.7 - one of the first canine T-cell antibodies (7) - did not show that hematopoietic precursor cells of dogs express Thy1 (10). However, imniuno-rosetting with antibody471
Neuner et al. Table 6. CFU growth and LTC-IC after separation (FACS) for MHC class II CFU exp. no.
1 2 3 4 5 6 7
LTC-IC
negative
low positive
high positive
0 39 0 204 3a 1
0
384 524 749 256 a14 292 1214
0 0 0 0 12 1 22
1
524
0
median
negative
low positive
high positive
0
9a
n. e.
n. e.
n. e.
69
0
375 331 1770 2512 2916
11 7 175 431 94
43
1072
52
ia
70 03
0
CFU growth after FAC-Sorting of Dog 26 labeled marrow cells before (CFU12xlP MNC) and after long-term culture (LTC-IC/3x1@ MNC). Marrow cells were stained with monoclonal antibody Dog 26 and separated according to their fluorescence. n.e.=not evaluable. sorting gate LTC-IC CFU
I
II
111
IV
v
VI
VII
Vlll
0
4 5
99 27
15 364
212 317
1 2
0 0
8 94
0 0
0 0
0
ExP 1 ExP 2
3
2 11
Exp 1 Exp 2
o o
o
1
3
3 3 9 45 a3
2
coated erythrocytes and centrifugation on a percoll density gradient separated only the high positive cells and left the low positive cells in the interphase (10). Here we used the novel mAbs Dog 13
L v
VI
VII
sortinggate
I
I1
111
IV
V
VI
VII
Vlll
LTC-IC Expl
0
2
4
99
15
212
1
0
Exp2
3
11
5
27
364
317
2
0
Expl
0
1
33
28
0
0
0
up2
o
o
45
39 a3
94
o
o
o
CFU
,
Figure 3. CFU growth and LTC-IC after sorting for MHC class 11. Fluorescence profile of MHC class 11-labeled bone marrow cells and content of directly CFU and LTC-IC after FAC-Sorting. Canine marrow cells were stained with monoclonal antibody (Dog 26) against canine MHC class 11 and separated into eight fractions by fluorescence intensity (I-VIII). Given is the number of CFU/50,000 cells (CFU) directly after sorting and the total number of CFU after 5 weeks of long-term culture (LTC-IC).
472
o
and Dog 14 directed against canine Thy-1 and the MACS cell sorting system ( 5 ) . Nearly all CFU and most of the LTC-IC were found in the Thy-1 positive fraction. These results indicate that Dog 13 and Dog 14 are of high avidity and the MACS system retains even low positive cells. The fluorescence pattern with two positive populations, a population with low and varying fluorescence and a population with more defined high fluorescence of Dog 13 and Dog 14 in the bone marrow supports this hypothesis. Using FACS technique we received almost pure cell populations (90-99%), and we were able to sort three fractions according to their fluorescence intensity. By this technique almost all directly clonogenic cells and complete CFU growth after long-term culture was found in the low positive fraction. This corresponds to findings of Prendergast et al. (15 ) who found complete inhibition of CFU growth and graft failure after complement lysis with a monoclonal antibody against Thy-1. Monoclonal antibodies to class I1 MHC antigens frequently cross-react between species; and the antibodies IOT2a and 7.2 have been used in the dog before (10, 11, 16). With the MACS technique the purity of the positive fractions was so poor that we could not use it for further studies. However, separation with the canine MHC class I1 antibody was much better, but we could not find a clear difference in neither CFU nor LTC-IC growth of positive and negative fractions. Although the numbers were not different the size of CFU was greater in the positive fractions of MHC
Phenotype of canine LTC-IC class 11. These colonies may be an equivalent of HPP-CFC (= high proliferative potential colonyforming cells) described in mice (17). In the immunofluorescence analysis a low positive population for MHC class I1 could not be as distinctly defined as with the Thy-I antibody. However, a low positive fraction was enriched for CFU and LTC-IC (Table 6). The expression of MHC class I1 on canine stem cells is not clear. Prendergast et al. suggested from transplantation experiments that class I1 is present on more mature progenitor cells, while stem cells are class I1 negative (15). In contrast, Berenson et al. were able to get complete engraftment after transplantation of class I1 positively selected marrow cells after separation with immunoabsorption (18) or FACS sorting (19). The authors proposed that at least part of the stem cells are positive for class 11. This corresponds to our findings. Later, Greinix et al. showed late graft failure after infusion of anti-class I1 monoclonal antibody (20), suggesting the depletion of stem cells by the antibody. Recent results of that group, however, support the opinion that infused class I1 antibodies inhibit the binding of progenitor cells to class I1 positive cells of the microenvironment and that at least some of the progenitor cells promoting engraftment are class I1 negative (21). It may very well be that the expression of MHC class I1 antigens is closely associated with the recruitment of early hematopoietic precursor cells, and therefore MHC class I1 expression varies from negative to highly positive according to the recruitment status. Sutherland et al. were able to show this for human marrow cells (14). In the dog however, we could not confirm that; both CFU and LTCIC were found with equal density of class 11. The first step towards the purification of progenitor cells can be done by depletion of mature cells with lineage specific markers as described for murine and human cells (22, 23). We were able to show that for this purpose three novel monoclonal antibodies from our laboratory may be useful. None of the antibodies neither impaired CFU growth nor depleted more primitive long-term culture-initiating cells. Depletion of marrow cells with these antibodies has the advantage that the negative fraction of cells can be used for further investigations, and purification can easily be done with large cell numbers with immunomagnetic systems. In contrast, purification with antibodies against class I1 or Thy-1 is only feasible with a cell sorter due to their low density. Separated cells bear some of the antibody on their surface which can influence further experiments. In conclusion, canine progenitor cells can easily be enriched by immutiomagnetic depletion of lin-
eage marker positive cells. Stronger purification can be achieved with a cell sorter, where cells can be separated by their antigen density. Here, canine clonogenic cells as well as LTC-IC express low levels of Thy-1 antigen. The expression of class I1 antigen is more variable with higher density on the more primitive progenitor cell. Acknowledgments
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anti-class I1 monoclonal antibody. Blood 1991: 78: 2131-8. 21. Greinix HT, Storb R, Bartelmez SH. Specific growth inhibition of primitive hematopoietic progenitor cells mediated through monoclonal antibody binding to major histocompatibility class I1 molecules. Blood 1992: 80: 1950-6. 22. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells [published erratum appears in Science 1989: 244: 10301. Science 1988: 241: 58-62. 23. Verfaillie C, Blakolmer K, McGlave I! Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med 1990: 172: 509-2. Address: Hans-Jochem Kolb GSF-Institut fur Immunologie Marchioninistr. 25 D-81377 MIlnchen Germany Fax +49 89-7099-300
