HEMATOPATHOLOGY Original Article

Surface and Cytoplasmic Expression of CD45 Antigen Isoforms in Normal and Malignant Myeloid Cell Differentiation CHARLES W. CALDWELL, M.D., PH.D., WILLIAM P. PATTERSON, M.D., BRIAN D. TOALSON, B.A., AND YOHANNES W. YESUS, M.D.

The CD45 antigen, sometimes referred to as "leukocyte common" or "T200" antigen in humans, is a family of high molecular weight transmembrane glycoproteins expressed in various relative molecular mass (Mr) forms on different types of lymphohematopoietic cells but is absent on platelets and mature erythrocytes. 12 This antigen family consists of at least four (and conceivably as many as eight) M r isoforms ranging from 180 to 220 kD. Subsets of mature T cells express combinations of the 180-, 190, 200-, and 220-kD isoforms, whereas immature thymo-

the cell surface at all stages. Only a small percentage (3-15%) of PMNs expressed surface CD45RA. However, there was a cytoplasmic pool of each isoform associated with membranebound granules found throughout differentiation, with remarkable increases in expression at the terminal stages. In the case of acute myeloid leukemias (AMLs), most cases expressed surface CD45RA with, or without, CD45RO, regardless of their FrenchAmerican-British (FAB) classification. This appeared to be a stable process at diagnosis and relapse in individual patients and may therefore serve as a diagnostic aid. The biologic significance of this aberrant expression of CD45RA by malignant cells is unknown but raises important questions regarding the cellular processes of phosphorylation/dephosphorylation in normal and malignant cells. (Key words: CD45; CD45RA; CD45RO; Cell differentiation; Protein tyrosine phosphatase) Am J Clin Pathol 1991;95:180-187

cytes express only the 180-kD protein. "Virgin," or unstimulated, T cells express the 220-kD isoform, but, on antigenic stimulation, the "memory" T cells express the 180- but not the 220-kD isoform.3 B cells express predominantly the higher M r forms of 200 and 220 kD. Human granulocytes express one to two forms of CD45 and bind the monoclonal antibody (MoAb) UCHL 1, which reacts with the low M r isoform.4"6 Quantitatively, the expression of CD45 on the surface of granulocytes is less than that of lymphocytes.7"9 However, in addition to surface antigen, it appears that granulocytes contain an intracellular pool of CD45 that may be responsible for in10 From the Departments ofPathology and Medicine. University ofcreases Mis- in surface expression on stimulation. souri School of Medicine, Columbia. Missouri. Recent studies have shown that, although all isoforms arise from a single gene, the molecular basis for CD45 Received February 22, 1990; received revised manuscript and accepted isoform heterogeneity is based on alternative splicing of for publication April 26, 1990. Supported in part by grants from the Fraternal Order of Eagles of three variable exons in the mRNA.1-2 Eight possible Missouri, and the Cancer Research Center, Columbia, Missouri. mRNAs might be generated by this mechanism, of which Address reprint requests to Dr. Caldwell: Department of Pathology, six have been isolated as cDNAs."" 15 There is very high University of Missouri School of Medicine, 1 Hospital Drive, Columbia, Missouri 65212. sequence homology over approximately 705 amino acids

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The CD45 family contains protein tyrosine phosphatase (PTPase) activity and is expressed in one or more of its isoforms on all lymphohematopoietic cells. Considerable work has focused on CD45 expression by lymphoid cells, but minimal work has involved granulocytes. Granulocytic, or myeloid, cell differentiation is accompanied by a number of morphologic and immunophenotypic changes. This study used flow cytometric and immunocytochemical methods in conjunction with morphologic assessment to investigate the expression of CD4S isoforms during differentiation of normal and malignant granulocytic cells. On normal bone marrow cells, the quantity of surface CD45 did not change during earlier stages but did increase significantly at the terminal stages (bands and polymorphonuclear leukocytes |PMNs|). CD45RO (the low relative molecular mass [Mr] isoform) was very dimly expressed on immature cells but became increasingly brighter beginning at approximately the myelocyte stage. The high Mr isoform (CD45RA) was virtually absent from

181

CALDWELL ET AL. Expression of CD Antigen Isoforms

MATERIALS AND METHODS Specimens Peripheral blood and bone marrow samples were obtained by standard methods 8 in accordance with regulations of the Institutional Review Board of the University of Missouri School of Medicine. The specimens were obtained from patients undergoing evaluation for iron deficiency anemia. Additional blood samples were obtained from healthy consenting adult donors. Additional specimens were obtained from patients undergoing diagnostic evaluations for acute and chronic myeloid leukemias. These malignant specimens included AML classified according to the French-American-British (F.A.B) classification28 as M1, M2, M3, M4, and M5, as well as cases of chronic myelogenous leukemia (CML).

Cell Separation

Methods

Erythrocytes were removed from bone marrow and peripheral blood leukocytes by sedimentation in methylcellulose. Nine milliliters of heparinized blood (or bone marrow in RPMI 1640 tissue culture medium) was mixed with 3 mL of 1% (weight/volume [w/v]) methylcellulose (Sigma Chemicals, St. Louis, MO) in phosphate-buffered saline (PBS). The tube was then placed at a 45-degree angle and the red blood cells allowed to settle for 15-30 minutes at 37 °C. Leukocyte-rich plasma was then removed, washed twice in RPMI by centrifugation, and resuspended at 1 X 1010/L in the same medium. This left virtually all leukocyte populations intact, as documented by Wright-Giemsa staining. Platelets were removed by differential centrifugation. Monoclonal

Antibodies

The CD45 MoAb used in this study included HLe-1 (CD45) from Becton-Dickinson (Mountain View, CA), which reacts with epitopes present on at least four known M r isoforms of the antigen (180, 190, 200, 220 kD); the CD45RA MoAb 2H4 (Coulter Immunology, Hialeah, FL), which reacts with the high M r isoforms of 200-220 kD; and the CD45RO MoAb UCHL-1 (DAKO, Santa Barbara, CA), which reacts with the 180-kD isoform. In addition, an anti-CD l i b MoAb, Leu-15 (Becton-Dickinson), was used in some experiments. HLe-1 and 2H4 were used as direct fluorescein isothiocyanate (FITC) conjugates and Leu-15 as a direct phycoerythrin (PE) conjugate. UCHL 1 was unlabeled and used in an indirect immunostaining method. Immunostaining Analysis

of Cells and Flow Cytometric

Separated leukocytes were immunostained with appropriate MoAbs with the use of standard methods of dualcolor fluorescence and flow cytometric analysis as previously described.29 All MoAbs were used at saturating concentrations for the number of cells examined. A Becton-Dickinson FACScan* was used in these experiments. For the purpose of electronic cell separation, fluorescence measurements were made from the "myeloid" and "lymphoid" regions of the dual-parameter forward and perpendicular light scatter histograms. Earlier cell-sorting experiments (data not shown) and a previous report9 confirmed the cell populations within these light scatter regions. The "myeloid" region contained promyelocytes onward to mature PMNs, whereas the myeloblasts were found within the "lymphoid" region. In the case of the CD45 isoform MoAb, dual-parameter histograms were

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within the cytoplasmic domain of various cell types, but sequence variation of the 400-550 amino acids in the distal part of the extracellular domain, as well as variable levels of glycosylation, accounts for the differences in Mr ofCD45. The functions of the CD45 antigen are unknown. Most reports have discussed possible roles in T- or B-cell differentiation, natural killer (NK) cell or cytotoxic T-lymphocyte (CTL) activities, or other T-cell functions.16"22 However, recent reports of protein tyrosine phosphatase (PTPase) activity associated with the cytoplasmic domain of CD45 suggest a role in feedback regulation of protein phosphorylation.23"25 In T cells, CD45 has been shown to function as a signal transducing molecule24'26 and is necessary for activation of the pp56'ck member of the src family during T-cell activation.27 Expression of CD45 isoforms changes during lymphohematopoietic cellular maturation. Different isoforms of CD45 appear on the surface of T cells at various stages during cell activation and differentiation.3 During normal B-cell differentiation, there is a quantitative relationship between CD45 expression and stages of cellular maturation.8 Shah and associates9 reported no changes in CD45 expression during bone marrow myeloid differentiation. However, their studies failed to account for the various isoforms of CD45 or for mature neutrophils. The current work documents that, although overall surface CD45 expression does not change significantly during the earlier stages of granulocytic differentiation, that of both cytoplasmic and surface isoforms does change systematically near the terminal stages. In addition, although normal early myeloid cells do not express surface CD45RA, the high M r isoform, malignant acute myeloid leukemia (AML) cells frequently express this isoform, with or without CD45RO, the low M r isoform.

HEMATOPATHOLOGY

182

Original Article collected from cells immunostained with CD1 lb-PE (yaxis) and each of the indicated CD45 MoAbs (x-axis). Appropriate isotypic negative control MoAbs were included to determine positivity and negativity. Immunocytochemical Staining for Cytoplasmic Antigen Cytocentrifuge preparations of cell suspensions were air dried overnight, chemically fixed with 5% acetic acid/ 95% methanol (volume/volume [v/v]) for 30 seconds at -20 °C, and immunostained with the use of MoAbs and the Vectastain* immunoalkaline phosphatase system (Vector Laboratories, Burlingame, CA) under conditions recommended by the manufacturer.

r

B

HLe-1

Surface Antigen Expression of Normal Cells

to"

Flow cytometric analysis under standardized conditions was used to determine the cell surface expression of the CD45 family of antigens. These conditions of immunostaining on viable cells obviated cytoplasmic staining. Cell surface expression of the CD45 family of antigens in relation to the stage of normal myeloid cell differentiation is shown in Figure 1. In all cases, the y-axis represents the fluorescence intensity (FI) of PE-labeled antiCD 1 lb (Leu-15) immunostaining (or the negative control MoAbs), whereas the x-axis represents the FI of FITClabeled CD45 MoAb (CD45, CD45RO, CD45RA) as indicated. The dual-parameter histogram of PE- and FITClabeled negative control MoAbs is shown in Figure \A. Combined immunostaining with CD45 (HLe-1) and CD1 lb (Leu-15), an antigen quantitatively related to the stage of granulocytic cell differentiation,9 produced the pattern in Figure IB. Immature granulocytic cells expressed relatively constant amounts of CD45, even though CD1 lb increased quantitatively with the stage of differentiation. Myeloblasts were negative for CD1 lb. Only at the most mature stages of granulocyte differentiation (bands and PMN forms) was there an increase in CD45 surface expression (as well as CD 11 b) compared with more immature cells. This was confirmed by flow cytometric analysis of isolated peripheral blood granulocytes, as well as density gradient-separated mononuclear (less mature) granulocytes and morphologic confirmation by WrightGiemsa staining (not shown). CD45RO (UCHL 1) expression was very dim on immature granulocytic cells but began a progressive increase in FI at about the myelocyte/metamyelocyte stage of differentiation (Fig. IC). In fact, antigen expression was so dim that some cells could be considered negative. Mature

ia*

10"

I0 a

10

FITC FLUORESCENCE INTENSITY FIG. 1. Dual-color immunostaining of bone marrow cells with PElabeled anti-CD 1 lb (y-axis) and the indicated FITC-labeled anti-CD45 family member (x-axis). Panel A is the isotypic negative control for the direct staining of panels B and D. Positivity and negativity are indicated by the quadrant markers. Panel C illustrates the same cells immunostained with anti-CD45RO in an indirect procedure. The quadrant markers indicate the appropriate level of cutoff for positive and negative staining. All histograms were collected using logarithmic amplification of fluorescence signals.

PMNs expressed the highest amounts of the antigen, although this was still less than the amount of CD45 (HLe-1). CD45RA (2H4) expression was dramatically different than that of CD45 or CD45RO (Fig. ID) in that there was virtually no detectable surface antigen from the blast stage onward to mature PMNs. The peripheral blood of various donors contained 3-15% PMNs, with very dim expression of 2H4 (linear channel 8 ± 5 [2 SD]). This FI was much less than that of either of the other two CD45 MoAbs and was in fact difficult to discern from the negative control. Cytoplasmic Antigen Expression in Normal Cells Immunoalkaline phosphatase (IAP) staining of chemically fixed and permeabilized cells was used to demonstrate the cytoplasmic antigen. This method revealed both surface and cytoplasmic staining under the conditions used. However, surface membrane staining could usually be visually separated from cytoplasmic staining because surface staining was primarily a thin rim of positivity,

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RESULTS

CALDWELL ET AL. Expression of CD Antigen Isoforms

The low Mr isoform, CD45RO (UCHL 1), was essentially absent from blasts, as determined from immunocytochemical staining, although flow cytometric analysis revealed very dim surface positivity. This is probably a result of the level of sensitivity of the methods. The surface expression of this antigen first appeared at the promyelo-

ft CONTROL

cyte stage of differentiation and increased significantly in the cytoplasm, but not necessarily on the surface, of myelocytes, bands, and PMNs. CD45RO was also present in a granule-associated pattern similar to that of HLe-1 (Fig. 3). Expression of CD45RA (2H4) began at the least mature myeloblast stage of differentiation and persisted throughout the full spectrum including mature PMNs. IAP staining with 2H4 was dim compared with that of HLe-1 and UCHL1 but was still greater than background. Of interest, this isoform was absent from the surface but remained as a cytoplasmic pool of antigen in mature PMNs. Surface Antigen Expression by Malignant Cells

Myeloid

In contrast to the surface and cytoplasmic expression of both CD45 and CD45RO and the absence of surface CD45RA expression by normal granulocytic precursors, malignant cells from most cases of AML expressed surface CD45RA in the presence or absence of CD45RO. Tables 1 and 2 contain flow cytometric data obtained by immunostaining normal and malignant myeloid cells from AMLs of various FAB classifications.28 All data are expressed in linear units of FI after immunostaining with saturating concentrations of the appropriate MoAbs. In

urn HLe-1

UCHL 1

2H4

FlG. 2. Composite photograph of bone marrow cells immunostained by the IAP method with an isotypic negative control or each of the indicated MoAbs (X500). The indicated cells are: Bl = blasts; P = promyelocyte;;; M = myelocytes; Me = metamyelocytes; B = band neutrophils; PMN = polymorphonuclear neutrophils; E = eosinophilic myelocyte.

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whereas cytoplasmic staining was more diffuse and mainly granule associated. As shown in Figure 2, bone marrow granulocytic cells at all stages of differentiation expressed CD45 (HLe-1). This antigen was expressed in the cytoplasm as well as on the surface of the cells. Immature blasts (Bl) contained scant cytoplasm, thus making evaluation of cytoplasmic staining difficult, but, as shown, there was dim staining associated with blast cells. It was not possible to determine unequivocally whether this was only surface staining or whether some cytoplasmic staining was also present. However, the pattern was strongly suggestive of surface, but not cytoplasmic, expression. Progranulocytes provided essentially the same pattern and intensity of staining as blasts. At the myelocyte stage, cytoplasmic (and surface) expression increased over less mature forms. Band forms and mature PMNs contained significantly increased cytoplasmic and surface expression. As shown in Figure 3, much of this immunostaining appeared to be associated with cytoplasmic granules.

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HEMATOPATHOLOGY Original Article

HLe-1

CONTROL

2H4

UCHL1

FIG. 3. Higher magnification (XI ,000) photograph of mature PMNs immunostained by the IAP method with each of the indicated MoAbs to illustrate the granule-associated pattern of antigen.

expression at relapse closely approximated that at diagnosis. Therefore, this aberrant CD45 expression may be a consistent feature of the individual malignancy. DISCUSSION A number of morphologic and biochemical changes occur programmatically during differentiation of hematopoietic cells. In the case of granulocytic cells, characteristic morphologic changes accompany differentiation from immature myeloblasts to become promyelocytes, myelocytes, metamyelocytes, and, finally, mature neutrophils. Morphologic examination remains the "gold standard" for monitoring granulocytic cell differentiation. Thus, we used morphologic examination in conjunction with immunologic probes to determine the relative surface and cytoplasmic antigen expression of certain CD45 isoforms during differentiation. As determined by flow cytometry, there was no quantitative change in surface CD45 (HLe-1) expression during the earlier stages of granulocytic differentiation. This is in agreement with previous reports.9 However, at the terminal stages of differentiation, there was an obvious in-

TABLE 1. POSITIVITY OF AML SUBTYPES, CML, AND NORMAL BONE MARROW CELLS FOR CD45 ISOFORM EXPRESSION Cases Positive (number poslnumber

tested)*

MoAbs Used

M/f

M2

M3

M4

M5

CML

Normal

CD45 (HLe-1) CD45RO(UCHL 1) CD45RA (2H4)

4/4 2/4 3/4

7/7 4/7 7/7

3/3 3/3* 3/3*

9/9 5/9 9/9

2/2 1/2* 2/2

6/6 6/6 0/6

8/8 8/8 0/8

* Cases were considered positive if >20% of the malignant cells were positive. t AML subtypes M1-M5 were classified according to the FAB.28 $ Only approximately 40% of cells were positive.

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all cases, the malignant cells were readily separable from normal cells on the basis of flow cytometric light scatter patterns and/or the large number of malignant versus nonmalignant cells in the specimen. CD45RA was expressed on the malignant cells of most cases of AML, whereas none of the cases of CML or normal bone marrow myeloid cells expressed this isoform. The FAB classification of the AML cases did not relate to CD45RA or RO expression. For measurements of FI, the FI of cells treated with isotypic negative control MoAbs was subtracted from specific FI. In that CD45RA expression on normal granulocytic cells was absent, or below detection limits of the flow cytometer, it was not possible to directly compare quantitative expression of CD45RA. However, as a bench mark, the FI of normal monocytes was 6 ± 4 linear units and that of the small percentage of PMNs expressing the antigen was 8 ± 5 units, whereas that of AML blasts was two to four times this level. The FI of all positive cases of AML was significantly greater than that of normal cells, thus precluding confusion with normal monocytes and granulocytes in patients with AML. Seven patients with AML were tested at initial diagnosis and at relapse 3-11 months later. In all cases, the CD45 isoform

CALDWELL ET AL. Expression ofCD45 Antigen Isoforms TABLE 2. FLUORESCENCE INTENSITY OF IMMUNOSTAINED CD45RA-POSITIVE AML CELLS MoAb Used

Ml*

M2

M3

M4

M5

CML

Normal

CD45RA (2H4) SDofFI

19 6

26 9

17 7

26 9

29 6

Negt Negt

6t 4t

• AML subtypes MI-M5 were classified according to the FAB.21 t CML cells and normal marrow granulocytic cells were CD45RA negative. Normal monocytes were positive with an average Fl of 6 ± 4 (2 SD) linear units.

the same restricted epitope as F8.11.13. 35 Therefore, our data support earlier reports in that 2H4 was absent from the surface of normal immature myeloid cells but present on the surface of most of the myeloid leukemias. In contrast to the absence of surface CD45RA from normal myeloid cells, we were able to demonstrate a cytoplasmic pool of this antigen. It should be pointed out that CD45RA may exist on myeloid "stem cells." In a study using various strains of mice,36 preincubation of bone marrow cells with certain T200 (CD45) MoAbs before reinfusion into irradiated hosts was capable of inhibiting splenic colony formation. Therefore, it is possible that CD45 and CD45RA may be present on morphologically unidentifiable "stem cells." In the current study, only myeloid cells from the recognizable myeloblast stage onward were evaluated. The low M r isoform CD45RO (UCHL1) first appeared at the blast/promyelocyte stage, then increased consistently during differentiation, with a remarkable cytoplasmic pool of granule-associated antigen in more mature cells. Studies of cell lines demonstrated mutually exclusive expression of UCHL-1 and 2H4 on T- and B cells but coexpression on two myeloid lines, HL60 and U937.31 This is not supported by our data from normal cells at the blast/progranulocyte stage. UCHL-1 was expressed at very low levels on the surface of myeloblasts, then increased significantly as cells differentiated from myelocytes onward to mature PMNs. This expression was observed to some degree on the surface and, to a remarkable degree, in the cytoplasm. The cytoplasmic antigen expression, particularly of 2H4 (CD45RA), is of interest. Some surface antigens, such as fi heavy chains on B cells, CD3 on T cells, and CD33 on granulocytic cells, show a strong cytoplasmic antigenic component before surface expression of the antigen.37"39 The current study demonstrates a similar process in granulocytes with members of the CD45 family. The cytoplasmic antigen pool of CD45 and CD45RO appears at about the same time as the surface antigen but increases dramatically at terminal differentiation. In contrast, CD45RA expression is mainly in the cytoplasm, with only a minor fraction of normal mature PMNs expressing this antigen very dimly on the surface. In mature PMNs, this cytoplasmic pool of CD45RA can be translocated to the cell surface by the calcium ionophore A23187 (Caldwell and associates, unpublished observations). This finding is in contrast to a similar system in which the investigators were unable to demonstrate CD45RA in mature PMNs, even under stimulatory conditions.40 Their CD45RA MoAb was different than 2H4, and thus the potential MoAb differences in reactivity may be responsible for the different observations.

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crease in surface expression of both CD45 and CD45RO but not of CD45RA. The most likely reason for the previous report that CD45 expression was unchanged throughout granulocytic differentiation9 is that cells were density gradient separated from bone marrow, and thus mature cells were lost. When we used density gradientseparated cells, we obtained the same expression pattern previously reported (data not shown). These same investigators also reported early myeloid cells to be CD45RA". HLe-1 is reactive with at least four M r isoforms of the human CD45 antigen. The pattern of expression on granulocytic cells is in contrast to the expression on B cells during differentiation. HLe-1 expression is very dim on B-cell precursors and increases systematically during differentiation until it disappears at the plasma cell stage.8 A similar process occurs in T-cell differentiation (data not shown). Monocytic cells show an increase in HLe-1 expression during differentiation, thus behaving more like lymphoid cells.9 However, granulocytic cells expressed relatively constant amounts of CD45 from immature stages until about the band or PMN stage, at which time surface expression increased significantly. The MoAb 2H4 (CD45RA), reactive mainly with the higher M r isoforms of 200-220 kD, was virtually nonreactive on normal immature myeloid cells and, for the most part, remained absent from the surface during differentiation. A cytoplasmic CD45RA antigen pool first became apparent at about the myelocyte stage and persisted to mature PMNs. The cell surface of most mature neutrophils was negative for this marker. However, a small, variable percentage (3-15%) of mature PMNs from some donors were dimly positive for CD45RA. A similar pattern of staining on myeloid cells has been observed with another CD45RA MoAb, GRT22. 30 However, GRT22 may mark a different epitope than 2H4, since Huelin and associates30 reported HL-60 cells to be negative for GRT22, whereas Terry and associates31 found the same cells to be positive for 2H4. Another CD45RA MoAb (F8.11.13) was found to be nonreactive on normal monocytes, granulocytes, and CFU-C 3233 but present on approximately one-third of AMLs and acute myelomonocytic leukemias (AMMLs).34 2H4 is thought to recognize

185

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HEMATOPATHOLOGY Original Article

The functions of CD45 from neutrophils are not clear at this time. However, a very interesting recent finding is the enzymatic activity associated with CD45. The cytoplasmic portion of CD45 contains two repeating domains that have high sequence homology with a protein tyrosine phosphatase (PTPase) of human placenta, and the CD45 from splenic lymphocytes contains PTPase activity.23,25 It is therefore possible that some or all of the CD45 family of antigens may modulate the state of phosphorylation of tyrosine phosphoproteins through their phosphatase activity. It is known that protein tyrosine phosphorylation accompanies cellular activation,35,41 but dephosphorylation can also activate protein tyrosine kinases of the src gene family.27,42 Of interest, CD45 itself is phosphorylated on cellular stimulation. 43,44

Note added in proof: Since submission of this article for publication, Lansdorp and colleagues (J Exp Med 1990; 172:363366) demonstrated that bone marrow cells sorted on the basis of CD34 and CD45RO or CD34 and CD45RA expression are capable of forming CFU-GM. Thus, it is probable that CD45RA and CD45RO are present on immature myeloid cells prior to the morphologically identifiable myeloblast stage. This may help explain why CD45RA is present on many of our myeloid leukemia cases. Acknowledgments. The authors thank Dr. Hattie Gresham for her helpful suggestions and review of the manuscript. They also thank Susan Hartley, MT(ASCP), Nancy Case, MT(ASCP), Jim Ford, MT(ASCP), Ann Wilson, MT(ASCP), and Pam Puig, MT(ASCP), all of the Flow Cytometry Laboratory, Department of Pathology.

1. Thomas ML. The leukocyte common antigen family. Annu Rev Immunol 1989;7:339-369. 2. Thomas ML, Lefrancois L. Differential expression of the leucocytecommon antigen family. Immunology Today 1988;9:320-326. 3. Serra HM, Krowka JF, Ledbetter JA, Pilarski LM. Loss of CD45R (Lp220) represents a post-thymic differentiation event. J Immunol 1988;140:1435-1441. 4. Akbar AN, Terry L, Timms A, Beverley PCL, Janossy F. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J Immunol 1988;140:2171-2178. 5. Newman W, Targen SR, Fast LD. Immunobiological and immunochemical aspects of the T-200 family of glycoproteins. Mol Immunol 1984;21:1113-1121. 6. Pulido R, Cebrain M, Acevedo A, De Landazuri MO, SanchezMadrid F. Comparative biochemical and tissue distribution study of four distinct CD45 antigen specificities. J Immunol 1988,140: 3851-3857. 7. Caldwell CW, Patterson WP, Hakami N. Alterations of HLe-1 (T200) fluorescence intensity on acute lymphoblastic leukemia cells may relate to therapeutic outcome. Leuk Res 1987,11:103-106. 8. Caldwell CW, Patterson WP. Relationship between T200 antigen expression and stages of B cell differentiation in resurgent hyperplasia of bone marrow. Blood 1987;70:1165-1172. 9. Shah VO, Civin CI, Loken MR. Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis. J Immunol 1988;140:1861-1867. 10. Lacal P, Pulido R, Sanchez-Madrid F, Mollinedo F. Intracellular location of T200 and Mol glycoproteins in human neutrophils. J Biol Chem 1988;263:9946-9951. 11. Barclay AN, Jackson DI, Willis AC, Williams AF. Lymphocyte specific heterogeneity in the rat leukocyte common antigen (T200) is due to differences in polypeptide sequences near the NH2-terminus. EMBOJ 1987;6:1259-1264. 12. Ralph SJ, Thomas ML, Morton CC, Trowbridge IS. Structural variants of human T200 glycoprotein (leukocyte-common antigen). EMBOJ 1987;6:1251-1257. 13. Saga Y, Tung J-S, Shen F-W, Boyse EA. Alternative use of 5' exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell lineages. Proc Natl Acad Sci (USA) 1987;84:5364-5368. 14. Streuli M, Hall LR, Saga Y, Schlossman SF, Satio H. Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens. J Exp Med 1987; 166: 1548-1566. 15. Thomas ML, Reynolds PJ, Chain A, Ben-Neriah Y, Trowbridge IS. B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing. Proc Natl Acad Sci USA 1987;84:5360-5363. 16. Bernabeu C, Carrera AC, De Landazuri MO, Sanchez-Madrid F. Interaction between the CD45 antigen and phytohemagglutinin. Inhibitory effect of the lectin-induced T cell proliferation by antiCD45 monoclonal antibody. Eur J Immunol 1987;17:1461-1466. 17. Harp JA, Davis BS, Ewald SJ. Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera. J Immunol 1984;133:10-15. 18. Ledbetter JA, Rose LM, Spooner CE, Beatty PG, Martin PH, Clark EA. Antibodies to common leukocyte antigen p220 influence human T cell proliferation by modifying IL2 receptor expression. J Immunol 1985;135:1819-1825. 19. Matorell J, Vilella R, Borche L, Rojo I, Vives J. A second signal for T cell mitogenesis provided by monoclonal antibodies CD45 (T200). Eur J Immunol 1987;17:1447-1451. 20. Mittler RS, Greenfield RS, Schacter BZ, Richard NF, Hoffman MK. Antibodies to the common leukocyte antigen (T200) inhibit an early phase in the activation of resting human B cells. J Immunol 1987;138:3159-3166. 21. Newman W, Fast LD, Rose LM. Blockade of NK. cell lysis is a

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The effect of phosphorylation on PTPase activity of CD45 is not known. It is known that many malignancies are characterized by increased expression of oncogene products possessing tyrosine kinase activity.45 Myeloid cells contain a member of the src family of tyrosine protein kinases (TPKs), hck, which appears to be related to cell differentiation.46,47 In vivo, members of the src family are heavily phosphorylated,48 and the level of phosphorylation correlates inversely with catalytic activity.42 However, on dephosphorylation of a specific tyrosine residue, activation of TPK activity occurs.42 In T cells, CD45 plays a significant role in control of pp56lck (another member of the src family) TPK activity.27 The absence of CD45RA from the surface of normal myeloblasts and promyelocytes and its expression on most leukemic blasts raises questions about the possible contribution of CD45RA in the maintenance of the undifferentiated state of the leukemic blasts through the activation of TPK produced by src family members such as hck. It is tempting to speculate that the surface and cytoplasmic pool of CD45 antigens may help regulate cell differentiation functions by regulating protein phosphorylation.

REFERENCES

CALDWELL ET AL.

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Expression of CD Antigen Isoforms

22. 23.

24.

25. 26. 27.

29. 30.

31.

32.

33. 34.

35.

36.

37.

38. 39.

40.

41. 42. 43. 44. 45. 46. 47. 48.

framework and restricted antigenic determinants. Leuk Res 1985;9:1249-1254. Cobbold S, HaleG, Waldmann H. Non-lineage, LFA-1 family, and leukocyte common antigens: new and previously defined clusters. In: McMichael AJ, Beverley PCL, Cobbold S, et al, eds. Leukocyte typing, vol 3. New York: Oxford University Press, 1987:788— 803. Ralph SJ, Berridge MV. Expression of antigens of the "T200" family of glycoproteins on hemopoietic stem cells: evidence that thymocyte cell lineage antigens are represented on "T200." J Immunol 1984;132:2510-2514. Campana D, Janossy G. Analysis of myeloid antigens on the cell surface and in the cytoplasm. In: McMichael AJ, Beverley PCL, Cobbold S, et al, eds. Leukocyte typing, vol 3. New York: Oxford University Press, 1987:661-663. Furley AJ, Mizutani S, Weilbaecher K, et al. Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex. Cell 1986;46:75-87. Gathings WE, Lawton AR, Cooper MD. Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans. Eur J Immunol 1977;7: 804-810. Pulido R, Lacal P, Mollinedo F, Sanchez-Madrid F. Biochemical and antigenic characterization of CD45 polypeptides expressed on plasma membrane and internal granules of human neutrophils. FEBS Lett 1989:249:337-342. Cohen P. The structure and regulation of protein phosphatases. Ann RevBiochem 1989;58:453-508. Marth JD, Cooper J A, King CS, et al. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol 1988:8:540-550. Autero M, Gahmberg CG. Phorbol diesters increase the phosphorylation of the leukocyte common antigen CD45 in human T cells. Eur J Immunol 1987;17:1503-1506. Shackelford DA, Trowbridge IS. Identification of lymphocyte integral membrane proteins as substrates for protein kinase C. J Biol Chem 1986;261:8334-8341. Perlmutter RM, Marth JD, Ziegler SG, et al. Specialized protein tyrosine kinase protooncogenes in hematopoietic cells. Biochim BiophysActa 1988;948:245-262. Quintrell N, Lebo R, Varmus H. et al. Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells. Mol Cell Biol 1987;7:2267-2275. Ziegler SF, Marth JD, Lewis DB, Perlmutter RM. Novel proteintyrosine kinase gene (hck) preferentially expressed in cells of hematopoietic origin. Mol Cell Biol 1987;7:2276-2285. Veillette A, Horak ID, Horak EM, Bookman MA, Bolen HB. Alternations of the lymphocyte-specific protein tyrosine kinase (p56kk) during T-cell activation. Mol Cell Biol 1988:8:4353-4361.

Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016

28.

property of monoclonal antibodies that bind to distinct regions of T-200. J Immunol 1983;131:1742-1747. Targen SR, Newman W. Definition of a "trigger" stage in the NK cytolytic reaction sequence by a monoclonal antibody to the glycoprotein T-200. J Immunol 1983;131:1149-1153. Charbonneau H, Tonks NK, Walsh KA, Fischer EH. The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase. Proc Natl Acad Sci (USA) 1988;85:71827186. Ledbetter JA, Tonks NK, Fischer EH, Clark EA. CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells. Proc Natl Acad Sci (USA) 1988;85:8628-8632. Tonks NK, Charbonneau H, Diltz CD, Fischer EH, Walsh KA. Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase. Biochemistry 1988,27:8695-8701. Deans JP, Shaw J, Pearse MJ, Pilarski LM. CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8- thymocytes. J Immunol 1989:143:2425-2430. Mustelin T, Coggeshall KM, Altman A. Rapid activation of the Tcell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. Proc Natl Acad Sci (USA) 1989;86:6302-6306. Bennett JM, Catovsky K, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloblasts leukemia. A report of the French-American-British cooperative group. Ann Intern Med 1985;103:620-626. Caldwell CW, Maggi J, Henry LB, Taylor HM. Fluorescence intensity as a quality control parameter in clinical flow cytometry. Am J Clin Pathol 1987;88:447-456. Huelin C, Gonzalez M, Pedrinaci S, et al. Distribution of the CD45R antigen in the maturation of lymphoid and myeloid series: the CD45R negative phenotype is a constant finding in T CD4 positive lymphoproliferative disorders. Br J Haematol 1988;69:173-179. Terry LA, Brown MJ, Beverley PCL. The monoclonal antibody, UCHL-I, recognizes a 180,000 MW component of the human leucocyte-common antigen, CD45. Immunology 1988;64:331336. Dalchau R, Fabre JW. Identification with a monoclonal antibody of a predominantly B lymphocyte-specific determinant of the human leukocyte common antigen: evidence for structural and possible functional diversity of the LC molecule. J Exp Med 1981; 153: 753-765. Morstyn G, Metcalf D, Burgess A, Fabre JW. Surface antigens on normal and leukemic human cells detected by monoclonal antibodies. Scand J Haematol 1981;26:19-30. Ritter MA, Sauvage CA, Pecram SM, Myers CD, Dalchau R, Fabre JW. The human leukocyte-common (LC) molecule: dissection of leukemias using monoclonal antibodies directed against