British Journal of Haematology, 1994, 88, 24-30

CD4 5 isoform expression on human haemopoietic cells at different stages of development

WILLIAM CRAIG, SIBRAND POPPEMA,* MARIE-TERESE LITTLE, WIESLAWA DRAGOWSKA AND PETER M. LANSDORP Terry Fox Laboratory, British Columbia Cancer Agency, and Department of Medicine, University of British Columbia, and *Department of Laboratory Medicine, Cross Cancer Institute, University of Alberta, Edmonton, Canada

Received 9 March 1994; accepted for publication 9 May 1994

Summary. Alternate splicing and glycosylation produce multiple CD45 isoforms which are selectively expressed on the surface of cells of the haemopoietic system. The expression of CD45RA, CD45RB and CD45RO on CD34' and CD34- haemopoietic cells from umbilical cord blood, bone marrow and fetal liver were studied by flow cytometry. CD34' subpopulations defined by CD45 isoform expression were sorted from bone marrow and tested in long-term culture assays. By combining results of functional studies with phenotypic data and previously published information, the following pattern of CD45 isoform expression on early haemopoietic cells was established. The most primitive CD34' cells are CD45RO' CD45RB' and express low or undetectable levels of CD45RA. Upon erythroid differentia- tion, CD34' cells remain CD45RO' CD45RB'. whereas

commitment into the myeloid and lymphoid lineages coincides with down-regulation of CD45RO and up-regula- tion of CD45RA. As a result, the majority of CD34' cells can be divided into two mutually exclusive populations of cells which express either CD45RO or CD45RA. This notion was c o d e d in this study by three-colour immunofluores- cence. The alternative expression of various CD45 isoforms on functionally distinct haemopoietic cells suggests an important role for these molecules in the proliferation and differentiation of haemopoietic cells.

Keywords: haemopoietic stem cells, cell surface markers, CD45 isoforms, alternative splicing, haemopoietic differentiation.

The leucocyte common antigen (CD45) is a highly glycosylated cell surface protein expressed only on cells of the haemopoietic system (Trowbridge, 1991; Thomas, 1989; Lai et al, 1991). All haemopoietic cells, except platelets and mature erythrocytes, are CD45' (Thomas, 1989; Shaw et al. 1988). Alternate splicing of three exons (4, 5 and 6, or A, B and C) can produce up to eight possible isoforms (Trowbridge, 1991: Thomas, 1989: Streuli et al, 1988), all of which have been detected at the mRNA level in different cell types (Trowbridge, 199 1). Additional hetero- geneity exists due to differential glycosylation of multiple potential 0 and N linked glycosylation sites (Thomas, 1989; Thomas & Lefrancois, 1988: Poppema et al, 1991). Monoclonal antibodies (mABs) specific for CD45 are classified as either anti-CD45 (reacting with determinants present on all isoforms) or anti-CD45R (specific for restricted determinants) (Thomas & Lefrancois, 1988). Anti-CD45R mABs are further defined by the specific isoform that is

Correspondence: Dr Peter M. Lansdorp, Terry Fox Laboratory, B.C. Cancer Research Centre, 601 West 10th Avenue, Vancouver, B.C., Canada V5Z 1L3.

recognized: RA, RB, RC or RO, specific for isoforms containing sequences encoded by exons A, B, C, or none respectively (Lai et al, 1991; Poppema et al, 1991). Anti-RO antibodies recognize the 180 kD low molecular weight isoform, anti-RB the 190, 205 and 220kD isoforms, and anti-RA the 205 and 220kD isoforms (Poppema et al, 1991). Cells may express more than one CD45 isoform (Thomas, 1989; Lai et al, 1991: Pilarski & Deans, 1989; Thomas & Lefrancois. 1988; Jensen et al, 1989).

CD45 has been demonstrated to have a role in signal transduction (Ostergaard et aI, 1989; Peyron et al, 1991). The large cytoplasmic domain contains multiple potential serine phosphorylation sites (Trowbridge, 1991) and has sequence homology with placental phosphatase activity (Charbonneau et al, 1988). The large external amino domain contairis multiple potential 0-linked glycosylation sites within exons 3-8 (Thomas, 1989; Thomas & Lefrancois, 1988), and multiple N-linked sites between the sites of 0-linked glycosylation and transmembrane regions (Thomas, 1989) producing additional heterogeneity. CD45 may interact with other cell surface molecules through its various carbohydrate determinants (Thomas, 1989), and

24

CD45 Zsoform Expression on Haemopoietic Cells 2 5 5E10 at 4 pg/ml. Biotinylated goat anti-mouse IgG (biotin- anti-mIg, 11 5-065-062, Jackson, Westgrove, Pa.) was diluted 200x in HFN containing 5% normal sheep serum (NSS) and 5% NHS. 6B6 x anti-R-phycoerythrin (RPE) bispecfic tetrameric antibody complexes (Wognum et al, 1987) were used for double-staining experiments with 6B6 and UCHL1. Anti-RPE (1D3) and equimolar amounts of 6B6 (or an irrelevant control Ab) were linked by WLP9 rat anti- mouse immunoglobulin F(ab)2 fragments. and mixed with RPE as described previously (Wognum et al. 1987). Tetramers were used at a final concentration of 5pg 1D3/ml.

Flow cytometry. All staining procedures were performed at a cell concentration of 2-10 x 106/ml. FL. CB and BM samples were stained in parallel under identical conditions, and multiple experiments were performed. Cells were incubated with unlabelled anti-CD45 mAbs or irrelevant isotype matched control Abs specific for trinitrophenol (TNP) or peroxidase (PO) for 30 min at 4% followed by two washes with HFN. Samples were resuspended in biotin-anti-mIg diluted 200x in HFN + 5% NSS + 5% NHS, incubated for 30 min at 4°C. washed twice in HFN, and resuspended in an irrelevant control mAb (100 pg/ml of anti-TNP or anti-PO in HFN) to block any residual free binding sites of cell bound biotin-anti-mIg. After 10 min at 4°C. directly conjugated mAbs, tetramer/RPE complexes and/or streptavidin-R- phycoerythrin (SA-RPE Molecular Probes Eugene, Or.) at 1 : 500 or avidin-FITC (Becton Dickinson, San Jose, Cali.) at 3pg/ml were added and cells were incubated for another 30 min at 4°C. followed by two washes with HFN. Cells were then resuspended in HFN + propidium iodide (PI, cat. no. p- 5264. Sigma) at I pg/ml for analysis on a FACStarf (Becton Dickinson. San Jose, Calif.) equipped with a 5 W argon and a 30 mW helium neon laser.

Long-term bone marrow cultures and progenitor assays. Long- term bone marrow cultures and progenitor assays were performed as previously described (Sutherland et al, 1989). except for the addition to methylcellulose cultures of 20 ng/ ml of the GM-CSFIIL-3 fusion protein (Curtis et al, 1991) and 20 ng/ml of human mast cell growth factor (Williams et al, 1990) (MGF. a c-kit ligand) which were both kindly provided by Dr D. E. Williams (Immunex, Seattle, Wash.). Sorted cells were plated in long-term cultures and methylcellulose progenitor assays. Methylcellulose colonies were scored on day 14 to determine the number of progenitor cells (colony forming units, CFU, including myeloid. erythroid and mixed myeloid-erythroid colony forming cells) present in the original sample. Long-term cultures were harvested after 5 and 8 weeks and colony assays performed to determine the number of long-term culture-initiating cells (LTC-IC) present in the original sample. Individual LTC-IC were assumed to produce an average of four CFU after 5-8 weeks in culture (Sutherland et al, 1990).

differences in these carbohydrate structures may alter the proteins and/or cell types that can interact with CD45 (Poppema et al, 1991).

Our goals in this study were to examine CD45 isoform expression in relation to the developmental stage and functional properties of distinct haemopoietic cells that express CD34 (Lansdorp et aZ, 1993). CD34 is a highly glycosylated cell surface protein expressed on 1-4% of low- density bone marrow mononuclear cells (Civin et al, 1989), including all cells capable of forming colonies in methyl- cellulose (CFU) and maintaining haemopoiesis in long-term culture (long-term culture initiating cells, LTC-IC) (Suther- land et al, 1989). CD34' cells have been shown capable of reconstituting recipients who have undergone marrow ablative therapy (Berenson et al, 1991). Expression of CD34 is highest on the most primitive cells and is lost as cells mature (Sutherland et al, 1989; Terstappen et al, 1991). CD45 expression is lower on CD34' cells than on the majority of the nucleated CD34- cells of the haemopoietic system (Shaw et al, 1988), and LTC-IC as well as BFU-E are highly enriched in the CD45RO' (Lansdorp et al, 1990) and CD45RA- cell fractions (Lansdorp & Dragowska, 1992). Previous studies also documented that the majority of myeloid colony-fonning cells (CFU-G, CFU-M and CFU-G/M) are CD34' CD45RA' and CD45RO-, suggesting that ex- pression of CD45RA and CD45RO isoforms on CD34' cells could be mutually exclusive.

MATERIALS AND METHODS

Cells. Heparinized bone marrow (BM) samples were obtained from informed and consenting individuals donat- ing for allogeneic transplantation. Cord blood (CB) samples were obtained from clamped umbilical cords at the time of birth from full-term normal pregnancies (kindly provided by Dr J. O'Toole and colleagues at the Royal Columbian Hospital, Coquitlam, B.C.). Fetal liver (FL) cells were obtained from selective. therapeutic first and second trimester abortions. Low-density mononuclear cells ( 4 . 0 7 7 g/cm3) of the various tissues were isolated by density separation using Ficoll-Paque (Pharmacia LKB, Uppsala, Sweden). Multiple samples (three to eight) from each tissue were examined. Interphase cells were removed and washed twice in Hanks Hepes Buffered Salt Solution containing 2% fetal calf serum and 0.1% sodium a ide (HFN) before being resuspended at lo7 cells/ml in HFN containing 5% normal human serum (NHS) to block F, receptors. The use of human material for this study was approved by the Ethical Review Board of the University of British Columbia.

Antibodies. Monoclonal IgGl antibodies (mABs) specilk for CD34 (8G12), CD45RA (8d2), CD45RB (6B6), Thy-1 (5E10; Craig et al, 1993) and CD45RO (UCHL1, IgG2,) were purified from tissue culture supernatants. UCHLl was a kind gift from Dr P. Beverley (ICRF, London, U.K.). 8G12 was labelled with Cy5 (Biological Detection Systems Inc., Pittsburgh, Pa.) as described (Southwick et al, 1990) and used at 10pg/ml. 8d2 was labelled with fluorescein isothiocyanate (FITC, product F-7250, Sigma, St Louis, Mo.) and used at 2 pg/ml. Unlabelled 6B6 and UCHLl were both used at 5 pg/ml and

RESULTS

CD45 isoform expression on bone marrow cells Low-density BM mononuclear cells were analysed for CD45 isoform expression after staining with Abs specific for CD34,

26 William Craig et a1 CD45RA and either CD45RO or CD45RB (Fig 1). The light- scatter gates and the gate set to exclude dead (PI+) cells as well as the threshold used to separate CD34+ from CD34- cells were set up as previously described (Craig et al, 1993). A sizeable proportion of BM cells expressed none of the CD45 isoforms and presumably these cells represent cells at late stages of erythroid differentiation (Figs 1C and lE), as mature erythrocytes and their immediate precursors do not express CD45 (Thomas, 1989; Shaw et al, 1988). In contrast, CD34+ cells from BM could be subdivided into

C 034- cel Is CD34' cells A I B

.. . . - I .: . . ....

C l I D

I cl I

Lu a,

oc in CD45RA-F

mlgG,-F Fig 1. CD45 isoform expression on BM cells. Dot plots of green fluorescence (FITC) versus red fluorescence (PE) of samples stained 'with 8G12-Cy5 (anti-CD34). and mAbs specific for one (A, B, G and H) or two of the indicated CD45 isoforms (C, D, E and F). An irrelevant control Ab, isotype matched with the used anti-CD45 mAbs, was included in samples stained for a single isoform. Analysis was restricted to viable (PI-) cells in the selected light-scatter gate (excluding very small or large events as well as cells with a high perpendicular light scatter). Gated cells were divided into CD34- cells (left panels) and CD34+ cells (right panels). Fluorescence is plotted on a log scale.

two populations of about equal size that appeared largely non-overlapping and mutually exclusive: CD45RO- CD45RA+ and CD45RO+CD45RA- (Fig 1D). Expression of CD45 isoforms on CD34' cells was low relative to some CD34- cells (i.e. compare Fig 1D with Fig 1C and Fig 1F with Fig 1E). CD34+CD45RA- cells expressed low to intermediate levels of CD45RB (Fig 1F) which, together with the profile shown in Fig lD, indicates that most of these cells co-express CD45RB and CD45RO. All CD34+CD45RAf cells were also CD45RB' (Fig 1F). Whereas CD45RA and CD45RO expression appeared to be mutually exclusive on CD34+ cells, CD45RA and CD45RB expression were correlated, suggesting that CD45RA' cells co-express CD45RB.

Primitive haemopoietic cells from bone marrow are CD34' CD45RO+CD45RB+ CD45RB' was expressed on >90% of BM CD34+ cells (Fig 1F). In order to examine CD45RB expression on functionally distinct haemopoietic cells, BM cells were labelled and sorted on the basis of CD34, CD45RO and CD4RB expression and plated in long-term cultures (Fig 2). The majority of LTC-IC were recovered in the fraction B that contained CD34' CD45RO' cells expressing CD45RB+ (Table I). Additional three-colour staining experiments indicated that CD34' CD38- cells (Terstappen et al. 1991) and CD34+CD71- cells (Lansdorp et al, 1992) were also clearly CD45RB+, indicating that the most primitive haemopoietic cells express CD45RB as well as CD45RO (results not shown).

CD45 isoform expression on cord blood cells CD45 expression on CB cells (Fig 3) was quite different from that on BM cells (Fig 1). The most noticeable exceptions were the populations of CD34+CD45RB+++CD45RAff cells found in CB which was not observed in BM samples (compare Fig 3F with Fig 1F) and the absence of CD45- erythroid cells in the density-separated cord blood cells (compare Figs 1C and 1E with Figs 3C and 3E). For these experiments, a lower

I . , I . ; . , . . I

I I I

CD45RB-PE Fig 2. Selection of sort windows of CD34+ bone marrow cells based on co-expression of CD45RO and/or CD45RB. Viable CD34+ cells in the selected light-scatter gate were sorted into three fractions: (A) CD45RO+CD45RB-. (B) CD45RO+CD45RB+ and (C) CD45RO- CD45RB'.

CD45 lsoform Expression on Haernopoietic Cells 2 7 Table I. LTC-IC are CD34+CD45RO+CD45RB+. Bone marrow cells were sorted on the basis of CD34. CD45RO and CD45RB expression as shown in Fig 2 and placed in long-term cultures. Long-term cultures were harvested at week 5 or 8, plated in methylcellulose, and myeloid and erythroid colony-forming cells were scored on day 14. LTC-IC were assumed to produce an average of four CFU after 5-8 weeks in culture (Sutherland et al, 1990). Enrichments were calculated by dividing the frequency of LTC-IC in puritied CD34+ fractions by the frequency for sorted total viable cells in the selected light-scatter gate ( 100% sorted). Recoveries were calculated by multiplying the enrichment factor by the fraction of sorted cells.

Fraction Phenotype of cells sorted in PI- and LS gate

Week 5 LTC-IC Week 8 LTC-IC

1 Per Enrichment Recovery (YO) 2 x lo6 Enrichment Recovery (%)

Sorted ('m) 100 1 100 540 1 100

A CD34' CD45RO' CD45RB-

B CD34' CD45RO' CD45RB'

C CD34' CD45RO- CD45RE3+

1.16: 24 28 6000 11 13

1.20 52 62 19500 36 43

1.14 4.5 5.1 1500 2.8 3.2

*The relative high proportion of CD34+CD45RO'CD45RB- cells in this experiment probably resulted from the relatively weak (direct) staining for CD45RB required to detect expression of CD45RO (Fig 2) . Sorted cells with this phenotype probably included a significant proportion of CD45RB'OW cells.

concentration of 6B6 (anti-CD45-RB) was used (I pg/mI) because of the extremely bright CD45RB staining that interfered with accurate detection of the FL4 signal (8G12- CY5). Samples stained with higher concentrations of 6B6 produced identical profiles except that there were fewer or no CD45RB- cells. The light-scatter profiles of the CD34+CD45 RB+++CD45RAff cells was similar to that of lymphocytes (data not shown) and possibly these cells are early lymphoid precursors. As with BM, CD45RO expression appeared inversely related to CD45RA expression on CD34' cells, although a larger population of cells with low expression levels of both CD45RO and CD45RA than in BM was observed (Fig 3D).

CD45 isoform expression on fetal liver cells A significant proportion of fetal liver cells stained with control antibodies (Figs 4A, 4B, 4G and 4H) complicating analysis of CD45 isoform expression. However, compared to CB and BM. the mutually exclusive expression of CD45RA and CD45RO on CD34' and the correlated expression of CD45RA and CD45RB cells were largely conserved (Figs 4D and 4F respectively).

In contrast to CB and similar to BM, most FL CD34- cells lacked CD45 expression, particularly CD45RA and CD45R0, and probably these cells represent various stages of late erythroid differentiation. Up to 30% of CD34- cells were CD45RB' (Fig 4E). Almost all CD34' cells were CD45RB+ (Fig 4F) and these cells co-expressed either CD45RO or CD45RA (Fig 4D).

DISCUSSION

Previous studies have shown that the most primitive haemopoietic cells in humans that can be measured in long-term cultures (LTC-IC) have a CD34+CD45RO+CD45 RA- phenotype (Lansdorp et al, 1990; Lansdorp & Dragowska, 1992). This study indicates that CD45RO and CD45RA expression on CD34+ cells is largely mutually exclusive on all tissues studied (Figs lD, 3D and 4D). Together with previous studies (Lansdorp et al, 1990; Lansdorp & Dragowska, 1992), these observations indicate that CD34' cells, like CD4+ lymphocytes (Rudd et al, 1987; Thomas, 1989), can be divided into functionally distinct subpopulations on the basis of the CD45 isoforms which are expressed.

When CD34+CD45RO+ cells were sorted on the basis of CD45RB phenotype expression, the majority of LTC-IC were recovered in the CD4 5RB' fraction (Table I), indicating that LTC-IC are CD34+CD4 5ROfCD4 5RB+CD4 5RA-. Expres- sion of CD45RB on the most primitive human haemopoietic cells is also suggested by three-colour staining experiments indicating that CD34+CD71dU" cells and CD34+CD38- cells from BM and FL were also CD45RB' (data not shown). These findings suggest the following pattern of CD45 isoform expression in early haemopoiesis. The majority of primitive CD34' cells. including fetal liver cells, co-express both CD45RB and CD45RO. Upon further maturation along the erythroid lineage, the total level of CD45 expression decreases and is lost altogether on erythrocytes and their immediate precursors. The exact stage at which CD45

28 William Craig et a1

CD34- cel Is CD34+ cel Is B

. .

CD34- cel Is CD34' cel Is

-

L L - a

CD45RA-F U CD45RA-F K Ln

I I I I I I- . .

mlgG,-F Fig 3. CD45 isoform expression on CB cells. CB cells were stained in parallel with BM and FL samples, and analysed with the same gates and FACS settings described in Fig 1. CD45 isoform expression on CD34- and CD34' cells is shown in the left and right panels respectively.

I I I I 1-

mlgG,-F Fig 4. CD45 expression on FL cells. Cells were labelled and analysed as BM and CB samples (Figs 1 and 3) . Profiles of CD45 isoform expression on CD34- and CD34' cells are shown in the left and right panels respectively.

disappears on erythroid cells has not been established in this study. Commitment of CD34' cells into the granulocytic/ monocytic as well as lymphoid lineage coincides with up- regulation of CD45RA and loss of CD45RO. Outside the CD34 compartment the total level of CD45 increases significantly in the lymphocyte and (to a lesser extent) in the monocyte and granulocytic cell lineage (Shaw et al, 1988). At some point along the monocyte and granulocyte differentiation pathway, CD45RA expression of CD34- myeloid cells must decrease because most mature mono- cytes and granulocytes express CD45RO and CD45RB but not CD45RA (Lai et al, 1991: Shaw et al, 1988).

Our finding that the most primitive haemopoietic cells appear to express CD45RB is surprising in view of previous reports demonstrating that rat stem cells are CD45RB- (McCarthy et al, 1987) and the observed conservation of

CD45 isoform expression between species (Trowbridge. 1991). This contradiction may reflect differences in experi- mental conditions or antibodies used and also real biological differences. One possibility is that the Ab used in the rat studies (0x22) may recognize a more restricted (i.e. carbohydrate dependent) epitope than the Ab (6B6) used in our study.

Our experimental approach does not allow detection of all possible CD45 isoforms encoded by one or more of the alternatively spliced exons. As a result, we cannot distinguish between the possibility that cells are switching from the co-expression of CD45RO and CD45RB to expression of isoforms containing a single A or B exon encoded sequences, or, alternatively, that CD45RE3 expression remains constant as cells switch from expression of CD45RO to an isoform containing both exon A and B encoded sequences. Immunoprecipitation and Western blotting could

CD45 lsoform Expression on Huemopoietic Cells 29 provide a clearer answer, but such studies are complicated by the difficulties in obtaining sufficient numbers of CD34+ cells from normal tissue.

Little is known about the mechanisms involved in alternate splicing of CD45 isoforms. It has been proposed that the basic splicing machinery of CD45 gene products is unable to recognize all exons and splices either all alternate exons 4, 5 and 6 (giving rise to CD45RO) or exons 4 and 6 (giving rise to CD45RB) (Streuli & Saito, 1989). Co- expression of CD45RO and CD45RB on candidate human stem cells could indicate that such cells express the basic splicing machinery. Expression of specific factors which interfere with the basic splicing mechanism or down- regulation of essential components of this machinery could coincide or initiate commitment into myeloid and/or lymphoid differentiation pathways and allow expression of exons 4, 5 and 6 and expression of CD45RA (Streuli & Saito, 1989). Whatever the exact mechanism involved in the regulation of alternative splicing, the selective expression of CD45 isoforms on functionally distinct subpopulations of CD34' cells implies an important role for CD45 molecules in early haemopoietic differentiation. In view of the known phosphatase activity of CD45 (Trowbridge, 1991), it is tempting to speculate that this role could involve modulation of the signals transducted via various haemopoietic cytokine receptor complexes. The finding that anti-CD45 Abs or antisense RNA can inhibit the actions of early-acting growth factors on haemopoietic progenitors (Broxmeyer et ul, 199 1) is in agreement with this theory. In one of the resulting models, primitive uncommitted haemopoietic cells could express multiple cytokine receptor complexes which, by association with particular CD45 isoforms, would require different levels of cytokines for effective signalling. By changing the responsiveness to various cytokines without altering the levels of cytokine receptors, CD45 isoforms could modulate activation, proliferation and differentiation of haemopoietic cells. Alternatively, expression of different CD45 isoforms could allow alternate interactions between cells in the haemopoietic system. Further studies of the ligands or molecules to which the various CD45 isoforms bind or with which they associate could help to clarify these issues.

ACKNOWLEDGMENTS

These studies were supported by grants from the NIH (A129524 to P.L.) and the Medical Research Council of Canada. Dr M. Strong and colleagues (North West Tissue Centre, Seattle) and colleagues from the Bone Marrow Transplantation programme of British Columbia and the Terry Fox Laboratory are thanked for their help in making tissue samples, growth factors and components of tissue culture medium available for this study. Steel factor (mast cell growth factor) was kindly provided by Dr D. Williams (Immunex, Seattle). Dr P. Beverley (ICRF, London) kindly provided monoclonal antibody UCHL1. Dr J. O'Toole and his colleagues at the Royal Columbian Hospital, Coquitlam. B.C., are thanked for umbilical cord specimens. Excellent technical assistance was provided by G. Thornbury,

C. Smith and C. McAloney. The manuscript was typed by Colleen MacKinnon.

REFERENCES

Berenson, R.J.. Bensinger, W.I., Hdl. R.S., Andrews. R.G., Garcia- Lopez, J., Kalamasz, D.F.. Still, B.J.. Spitzer. G.. Buckner, C.D., Bernstein, I.D. & Thomas. E.D. (1991) Engraftment after infusion of CD34' marrow cells in patients with breast cancer or neuroblastoma. Blood, 77, 1717-1722.

Broxmeyer, H.E., Lu, L., Hangoc. G.. Cooper, S.. Hendrie, P.C., Ledbetter, J.A., Xiao, M., Williams, D.E. & Shen. F.W. (1991) CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 ( P 3 ) , granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein. and mast cell growth factor (a c-kit ligand). Journal of Experimental Medicine, 174, 447-458.

Charbonneau. H., Tonks, N.K., Walsh. K.A. & Fischer. E.H. (1988) The leukocyte common antigen (CD45): a putative receptor- linked protein tyrosine phosphatase. Proceedings of the National Academy of Sciences of the United States of America. 8 5 , 7182- 7186.

Civin. C.I., Trischmann. T.M.. Fackler, MJ.. Bernstein. I.D.. Buhring. H-J.. Campos, L.. Greaves. M.F.. Kamoun. M.. Katz. D.R.. Lansdorp, P.M.. Look, A.T.. Seed, B.. Sutherland, D.R.. Tindle, R.W. & Uchanska-Ziegler, B. (1989) Report on the CD34 cluster workshop. Leucocyte Typing. IV. White Cell Diferentiation Antigens (ed. by W. Knapp et al), pp. 818-825. Oxford University Press.

Craig. W., Kay. R., Cutler, R.L. & Lansdorp. P.M. (1993) Expression of Thy-1 on human hematopoietic progenitor cells. Journal of Experimental Medicine, 177, 133 1-1 342.

Curtis, B.M., WiIliams, D.E., Broxmeyer, H.E., Dunn. J., Farrah, T., JefTrey, E.. Clevenger. W., deRoos, P., Martin, U., Friend, D., Craig, V., Gayle. R.. Price. V., Cosrnan. D.. March, C.J. & Park, L.S. (1991) Enhanced hematopoietic activity of a human granulocyte/ macrophage colony-stimulating factor-interleukin 3 fusion protein. Proceedings of the National Academy of Sciences of the United States of America, 88, 5809-5813.

Jensen, G.S., Poppema. S., Mant, M.J. & Pilarski, L.M. (1989) Transition in CD45 isoform expression during differentiation of normal and abnormal B cells. International Immunology, 1,229-236.

Lai. R.. Visser, L. & Poppema, S. (1991) Tissue distribution of restricted leukocyte common antigens: a comprehensive study with protein- and carbohydrate-specific CD4 5R antibodies. Laboratory Investigation, 64, 844-854.

Lansdorp, P.M. & Dragowska. W. (1992) Long-term erythropoiesis from constant numbers of CD34' cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. Journal of Experimental Medicine. 175, 1501-1509.

Lansdorp, P.M.. Dragowska. W. & Mayani, H. (1993) Ontogeny- related changes in proliferative potential of human hematopoietic cells. Journal of Experimental Medicine. 178, 787-791.

Lansdorp, P.M., Schmitt, C.. Sutherland. H.J., Craig, W.H.. Dragowska. W.. Thomas, T.E. & Eaves, C.J. (1992) Hemopoietic stem cell characterization. Advances in Bone Marrow Purging and Processing. 377 (ed. by D. A. Worthington-White et al), pp. 475- 486. Wiley-Liss, New York.

Lansdorp, P.M.. Sutherland, H.J. & Eaves, CJ. (1990) Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow. Journal of Experimental Medicine, 172, 363-366.

McCarthy. K.F., Hale, M.L. & Fehnel, P.L. (1987) Purification and analysis of rat hematopoietic stem cells by flow cytometry. Cytometry. 8, 296-305.

30 William Craig et a1

Ostergaard, H.L., Shackelford, D.A., Hurley, T.R.. Johnson, P., Hyrnan. R., Sefton, B.M. & Trowbridge, I.S. (1989) Expression of CD45 alters phosphorylation of the Ick-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proceedings of the National Academy of Sciences of the United States of America, 86, 8959- 8963.

Peyron. J-F.. Verma. S., de Waal Malefyt, R., Sancho, J., Terhorst. C. & Spits, H. (1991) The CD45 protein tyrosine phosphatase is required for the completion of the activation program leading to lymphokine production in the Jurkat human T cell line. International Immunology, 3, 13 5 7-1 366.

Pilarski, L.M. & Deans, J.P. (1989) Selective expression of CD45 isoforms and of maturation antigens during human thymocyte differentiation: observations and hypothesis. (Letter). Immunology, 21,187-398.

Poppema. S., Lai. R. & Visser. L. (1991) Antibody MT3 is reactive with a novel exon B-associated 190-kDa sialic acid-dependent epitope of the leukocyte common antigen complex. Journal of Immunology. 147, 2 18-22 3.

Rudd, C.E., Morimoto, C., Wong, L.L. &Schlossman, S.F. (1987) The subdivision of the T4 (CD4) subset on the basis of the differential expression of L-C/T200 antigens. Journal of Experimental Medicine, 166, 1758.

Shaw, O.V., Civin, (2.1. & Loken, M.R. (1988) Flow cytometric anaiysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis. JournuZ of Immunology. 140, 1861.

Southwick, P.L., Ernst, LA. , TauielIo, E.W., Parker, S.R., Mujumdar. R.B., Mujumdar, S.R., CIever. H.A. & Waggoner, A S . (1990) Cyanine dye labeling reagents: carboxymethylindo- cyanine succinimidyl esters. Cytometry, 11, 418-430.

Streuli. M., Morimoto, C., Schrieber, M.. Schlossman, S.F. & Saito, H. (1988) Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express

individual human leukocyte common antigens. Journal of Immunology, 141, 3910-3914.

Streuli, M. & Saito. H. (1989) Regulation of tissue-specific alternative splicing: exon-specific ciselements govern the splicing of leukocyte common antigen pre-mRNA. EMBO Journal, 8, 787-796.

Sutherland, H.J., Eaves, CJ.. Eaves, A.C., Dragowska, W. & Lansdorp, P.M. (1989) Characterization and partial purification of human marrow cells capable of initiating long-term hemato- poiesis in vitro. Blood, 74, 1563-1570.

Sutherland. H.J., Lansdorp, P.M., Henkelman, D.H.. Eaves, A.C. & Eaves, CJ. (1990) Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proceedings of the National Academy of Sciences of the United States of America. 87, 3584- 3588.

Terstappen. L.W.M., Huang, S., Sdord, M.. Lansdorp. P.M. &Loken, M.R. (1991) Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- pro- genitor cells. Blood, 77, 1218-1227.

Thomas, M.L. (1989) The leukocyte common antigen family. Annual Review of Immunology, 7, 339-369.

Thomas, M.L. & Lefrancois. L. (1988) Differential expression of the leukocyte-common antigen family. Immunology Today. 9, 320- 326.

Trowbridge, I.S. (1991) A prototype for transmembrane protein tyrosine phosphatases. Journal of Biological Chemistry. 266,

Williams, D.E., Eisenman. J., Baird. A.. Rauch. C.. Van Ness. K.. March, C.J.. Park, L.S., Martin, U.. Mochizuki. D.Y.. Boswell. H.S.. Burgess, G.S., Cosman, D. & Lyman, S.D. (1 990) Identification of a ligand for the c-kit proto-oncogene. Cell, 63, 167-174.

Wognum, A.W., Thomas, T.E. & Lansdorp, P.M. (1987) Use of tetrameric antibody complexes to stain cells for flow cytometry. Cytometry. 8, 366-371.

2351 7-23520.