Eur. J. lmmunol. 1991. 21: 17-22 Differential splicing of CD4S in T1,l and T1,2 ccll cloncs 17

Mohammad Luqman, Pauline Johnsonon, Ian TrowbridgeO and Kim Bottomlyv

Section of lmmunobiology and the Howard Hughes Medical Institute, Yale University, School of Medicine, New Haven and Department of Cancer Biologyo, The Salk Institute, San Diego

Differential expression of the alternatively spliced exons of murine CD45 in Thl and Th2 cell clones*

Antigen-specific murine CD4+ T cell clones can be divided into functionally distinct subsets known as Thl and Th2. To date these cells have been indistin- guishable by surface phenotype. This report identifies two anti-CD45R mono- clonal antibodies (14.8 and C363.16A) that bind preferentially to Th2 cells. Further analysis of the CD45-specific mRNA in Thl and Th2 cells shows clear differences between these two cell types. Thl cell clones express mRNA for the two smallest forms of CD45 containing none or only one of the alternatively splices exons. I n contrast, Th2 cell clones express predominantly the high molecular weight isoforms of CD45 containing two or three of the alternatively spliced exons.

1 Introduction

Functionally distinct CD4+ T cell subsets have been de- scribed in human [ l , 21, rat [3, 41 and mouse [5, 61. In all three species, CD4+ Tcells can be subdivided on the basis of expression of CD45 isoforms. CD45 is a major cell surface glycoprotein expressed on all hematopoietic cells except mature erythrocytes; the antigen has been characterized in mouse [7, 81, rat [9], human [lo, 111 and chicken [12] and has similar properties in all species [13]. A key feature of CD45 is its heterogeneity in apparent molecular weight, glycosylation and antigenicity. This structural variation is cell type specific in that antibodies to CD45 distinguish betweenTcells and B cells [14], between CD4+ and CD8+ T cells [ 151, and between subsets of CD4+ Tcells [ 1-6, 161. Some of the heterogeneity is due to the presence of three variable exons which are differentially spliced in the RNA resulting in differences in the primary sequence of the extracellular domain 17-13]. Theoretically this differential exon usage could give rise to eight different mRNA and thus eight isoforms. The expression of different isoforms correlates with the function of the cell as has been demonstrated in rat [17] and human CD4+ Tcells 118-201, where naive cells and memory cells are distinguished by the expression of particular CD45 isoforms.

In the mouse, two major subsets of CD4+ Tcell, known as Thl and Th2 cells, have been defined by the analysis of clonal Tcell lines [16]. While Thl and Th2 cells have been shown to differ in the cytokines they release [21, 221, in their functional capabilities [23-271 and in their activation requirements [28, 291, to date theTh1 and Th2 cloned lines have not been shown to be phenotypically distinct. In this

report the CD45 phenotype of these functionally distinct cloned lines has been analyzed to determine if Thl and Th2 cells bear different isoforms of CD45. Two approaches have been utilized to identify CD45 isoforms. First, mAb with known specificity for particular CD45 isoforms were used to stain a panel of Thl and Th2 cell clones. Second, we have analyzed the expression of specific mRNA of CD45 in Thl and Th2 cell clones by amplification with the polymerase chain reaction (PCR). This method is extremely sensitive and has the capability of further defining closely related isoforms. The pattern of isoform expression suggests that Thl and Th2 cloned Tcells differ in their CD45 phenotype and that the expression of particular isoforms might be predictive of their functional capabilities.

2 Materials and methods

2.1 T cell lines

The production and maintenance of antigen-specific mouse Tcell clones used in these studies have been described elsewhere [30-321. These lines are of CD3+, CD4+ and CD8- phenotypes.Their phenotype and antigen specificity have been stable over the culture period of 2-8 years. For RNA isolation clones were allowed to rest for 15 days after antigen stimulation and the feeder cells were rigorously removed by two rounds of centrifugation on lymphocyte separation medium (LSM; OrganoTeknika, Durham, NC). The preparation of 3T3 fibroblast Vz cells expressing different individual isoforms of CD45 has been described previously*. Four transfectant lines individually expressing alternatively spliced exons ABC, BC, C and null (with all variable exons spliced out) are used in this study.

[I 81981

* This work was supported by NIH grants CA 38350, A1 26791, CA 163SA (Comprehensive Cancer Center Flow Cytome- try/Cell Sorting Core Facility), and CA 17733 and the Howard Hughes Medical Institute. Associate Investigator, Howard Hughes Medical Institute.

A Recipient of a Cancer Research Institute fellowship.

Correspondence: Mohammad Luqrnan, Section of Immunobiolo- gy, Yale University School of Medicine, New Haven, CT 06510, USA

Abbreviations: LT: Lymphotoxin PCR: Polymerase chain reae- tion

2.2 mAb

TlB122 (MU9.3.44HL2) andTlB164 (14.8) were obtained from the American Type Culture Collection, Rockville, MD, and have been characterized previously 133, 341. C363.16A was produced in our laboratory, and its produc- tion and characterization has been reported elsewhere [5]. Ig from hybridoma SN were precipitated in 45% saturated ammonium sulfate, dissolved in PBS and dialyzed against

* Johnson. P., Greenbaum, L., Bottomly, K. and Trowbridge, I. S., J. Exp. Med. 1989. 169: 1179.

0 VCH Verlagsgesellschaft mbH. D-6940 Weinheim, 1991 OO14-2980/9 1/01Ol-OO17$3.S0 + .25/0

18 M. Luqman. P. Johnson. I . Trowbridge and K. Bottomly Eur. J. Immunol. 1901. 21: 17-22

PBS to remove traces of ammonium sulfate. For certain experiments, mAb were purified by affinity chromatogra- phy using protein G columns (Pharmacia. Piscataway, NJ).

2.3 Exon-specific probes

A cDNA clone, p70Z/3 [7]. containing all three alternate exons was used to generate radiolabeled exon-specific probes by blunt-end subcloning of the following restriction fragment into the Sma I site of pGEM-3 (Promega Biotech, Madison, WI): for exon A, the 133-bp DraIII/BstNI fragment: for exon B, the 147-bp Bst NI/Dra I11 fragment; and for exon C, the 133-bp Dra III/Eco RV fragment. To obtain a cDNA probe for null isoform message which contains no alternate cxons, primers complementary to sequences flanking the alternate exons were used to generate a number of CD45-specific products by PCR treatment of total RNA from a Thl cell clone. A 167-bp product predicted to correspond to null isoform sequences was blunt-end subcloned into the Sma I msite of pGEM-3 and its identity confirmed by restriction mapping. 32P- labeled anti-sense RNA probes were generated by in vitro transcription according to the manufacturer's instructions (Promega) and the lack of cross-reactivity between exon- specific probes was confirmed by DNA-RNA hybridization (data not shown).

0.5 mg/ml of salmon sperm DNA and 30 x loh of exon- specific "P-labeled antisense RNA probes overnight at 45 "C. Blots were washed vigorously at room temperature in 2 x SSC/0. 1% SDS, 0.5 x SSC/O. 1% SDS and 0.1 x SSC/0.1% SDS successively, followed by a final wash in 0.1 x SSCIO. 1% SDS at 65 "C for 1 h. Nonspecifically bound RNA probe was removed by treating the blots at 37°C in solution containing 0.3 M NaCl, 10 mM Tris-HCI, pH 7.5, 1 mM EDTA and 10 pg of RNase A for 30 min. Washed filters were subjected to autoradiography at - 70 "C.

2.6 FCM

Phenotypic analysis of the clones was performed by indirect immunofluorescence. Cells were washed in staining buffer (PBS/S% FCS/O. 1% sodium azide) and incubated with biotinylated antibodies, MU9.3.3 4HL2 (M1/9), C363.16A (16A) or 14.8 for 30 min at 4°C. After washing extensively with staining buffer the cells were incubated with secondary reagent either avidin fluorescein or fluoresceinated goat anti-rat Ig at 4°C for 20 min. After washing extensively to remove excess antibody the cells were analyzed on FACScan (Becton Dickinson, Mountain View, CA). Cells stained only with secondary fluoresceinated antibody or isotype-matched control antibodies were used as negative controls.

2.4 RNA isolation, cDNA synthesis and PCR 3 Results

RNA from 3- x 1O"-S x 10" cells from each clone was isolated by the guanidinium thiocyanate-phenol chloro- form extraction procedure as described [ 3 5 ] . First-strand cDNA was synthesized in a reaction volume of 50 pi containing 50 mM Tris-HCI, pH 8.3, 75 mM KCI, 3 mM MgCI?, 10 mM DTT. 1 mM dNTR 50 U of RNAsin (Prom- ega). 1 pg of anti-sense primer P2 and 200 U of Moloney murine leukemia virus reverse transcriptase by incubating the reaction mixture at 40°C for 2 h. One twentieth of the product of cDNA synthesis was used for PCR in a reaction volume of SO PI containing 10 mM Tris-HCI, pH 8.3,50 mM KCI, 3 mM MgC12. 0.lY0 gelatin, 0 . 5 wg each of oligonu- cleotide primers PI and P2 and 2.5 U of Taq polymerase. Each amplification cycle consisted of a denaturation tem- perature of 94"C.an annealing temperature of 65 "C and an extension temperature of 72°C. A total of 20-25 cycles were used to amplify the CD4S-specific sequences in each clone. The sequences of the oligonucleotide primers are described below:

PrimerP' ACCATG G GTTTGTG GCTCA A ACT""-' 5o

Pri merPZ G CTATGGTTGTGCTTG GAGGGTChx3-71 -' ant i -

\en\c

\L l l \C

2.5 Southern transfer and hybridization

After electrophoresis o n a 20/0 agarose gel, PCR products were transferred onto zeta-probe membrane (Bio-Rad, Richmond. CA) in 0.4 M NaOH as described by Mann and Reed [%I. and hybridized in a solution containing 50% forniamidc. 2 x SSC. 0.5% blotto (nonfat powdered milk),

3.1 Characteristics of functional subsets of CD4+ T cell clones

The CD4+, antigen-specific, class 11-restricted cloned T cells used for these experiments can be divided into two groups typical of Thl and Th2 cells. One panel of cloned CD4 T cells. referred to as Th2, is effective at helping antigen-specific B cells, but does not activate cytostatic activity from MO, kill B lymphoma lines, or mediate DTH responses. The other panel of CD4+ clones, referred to as T h l , is effective at activating MO, killing B lymphoma cells and mediating DTH [23-271. Further, it has been shown that Thl lines produce IL2, IFN-y and lymphotoxin (LT) while Th2 cloned lines produce IL4 and IL5 [21-23, 371. These studies have been summarized in Table 1.

3.2 Analysis of CD45 expression in clones using mAb

The objective of these studies was to analyze the expression of various isoforms of CD4S molecules in functionally different CD4+ cloned Tcells.To identify the isoforms, two types of mAb recognizing CD4S molecules have been utilized. One group of antibodies recognizes all isoforms of CD4S and is referred to as anti-CD4S. The other group of antibodies recognizes only restricted sets of isoforms and are referred to as CD4SR. The different isoforms of CD4S molecule are generated in part by differential splicing of three variable exons A, B and C (see Fig. 2). Within the anti-CD45R group of antibodies, some require the expres- sion of exon A (anti-CD4SRA) for binding, some require the expression of exon B (anti-CD45RB) for binding while others recognize a null isoform with all variable exons

Eur. J. Imrnunol. 1091. 21: 17-22

Table 1. Cloned CD4+ Tcells can be divided into two groups on the basis of functional analysis and cytokincs relcascd

Differential splicing of CD4S in Thl and Th2 cell clones I Y

Clones.'' Ag specificityh' Clone Hclp for specific Cyto- DTH MO IL1 IL2 IFN-.; 111' t Y P C ;in t ibod y toxicity activation

TAKR (010) A KS D8 6.2 5.2 5.C) PR3 A E 10.7

Conalbumin Conalbumin Ovalbumin MB protein Ovalbumin Ovalbumin

Autoreactive Autoreactive

-

-

-

ND + +

ND ND

-

-

-

ND + +

ND ND

- - + - + - + - - ~

+ - - SD - + + + - + + + - + + + - + + +

- -

a) D10 is a subclonc of TAKR. b) The antigen-specific cloned Tcells were selected from various MHC backgrounds including LAk, I-A", I-A" and I-Aho": MB = niyclin

basic: NOD = non-obese diabetic.

spliced out (anti-CD45RO) in the human. The three anti- CD45 mAb, 14.8, C363.16A and M1/9.3.4HL.2, chosen for this study have been previously characterized to determine which exons are recognized by each for binding [38]. Antibody 14.8 (anti-CD45RA) has been shown to require the expression of exon A and is known to bind the large molecular weight isoform utilizing all three variable exons (ABC). C363.16A (anti-CD45RB) requires the expression of exon B and binds to the isoforms expressing all three variable exons (ABC) and the isoforms expressing two variable exons (BC). MU9.3.4HL.2 (anti-CD45) recog- nizes all isoforms of CD45. From these studies, it is not yet clear whether the expression of exon A or exon B alone is sufficient for binding of the isoform to antibodies 14.8 and C363.16A, respectively, o r whether these exons have to be

stainingantibodies

t

AE 103 '# . -<- I - -I& - .-LL----LLL-

Th' Th2

Figure I. Analysis of CD4S expression in T h l and Th2 cloned T cells using mAb. Staining profiles of Thl and Th2 cloned lines with 14.8 (anti-CD45RA). 16A (anti-CD45RB) and MU9 (anti-CD45) mAb are shown. Binding of 14.8 to these lines was assayed by indirect immunofluorescence with 14.8 as the first step and fluoresceinated anti-rat Ig as the second step (solid lines). Staining with fluoresceinated anti-rat Ig only is a negative control (dotted lines). To assay the binding of 16A and M119, antibodies were biotinylated and detected with avidin-fluorescein. Staining with avidin-fluorescein only is a negative control (dotted lines). Solid lines show staining with MU9 and stippled lines represent staining with MA. Dotted lines in all cases represent the negative controls.

expressed in combination with other exons of the CD45 molecule.

As expected, all the cloned cells express cell surface CD45 as seen by staining with MU9.3.4HL.2 (Fig. 1). Staining with 14.8 (anti-CD45RA), however, revealed an interesting differential pattern. All Th2 clones tested in these studies (TAKR, AK8, D8 and 6.2) expressed isofoms bound by anti-CD45RA, whereas Thl clones (5.2, 5.9, PR3 and AE103) did not. A similar pattern of reactivity was observed with C363.16A (anti-CD45RB) antibody in that all Th2 clones were stained strongly while T h l clones were either totally negative o r low in expression of the relevant isoforms. Since it is not known if the single variable exon isoforms A and B are bound by the anti-CD45RA and anti-CD45RB antibodies, respectively, one may conclude only that the expression of exon A and/or exon B in single-exon isoform, double-exon isoforms or triple-exon isoforms is different in the two subsets of CD4+ cloned T cells.

3.3 Analysis of CD45 mRNA using PCR

Since it is not possible to determine, using available mAb, what combination of exons A and B create the isoforms that are differentially expressed on the two subsets, a different approach was utilized to further define the isoforms present on Thl and Th2 cells. CD45 mRNA was amplified by the PCR using 23-base oligonucleotide prim- ers flanking the region encoding three variable expressed exons. Purified mRNA from each clone was used to synthesize first-strand cDNA.The sequences of primers are described in Sect. 2.4 and the predicted sizes of amplified products containing no-exon, single-exon. double-exon and all three-exons are given in Fig. 2.

To establish the identity of CD45-specific PCR products, the amplified material was transferred onto a nylon mem- brane and analyzed for hybridization to CD45-exon- specific probes. Three probes specific for exon A, exon B, exon C, and the fourth containing sequences complemen- tary to null isoform mRNA (all variable exons spliced out) were used for this analysis. The construction and specificity of these probes have been described in Sect. 2.3.To further emphasize the specificity of polymerase chain reaction and

20 M. Luqman. €? Johnson. I. Trowbridge and K. Bottomly

Predicted sizesof

PCR products in bp

Eur. J. lmmunol. 1991. 21: 17-22

- 584 I A I B I c I B

443 I A I B l a 455 I B I c l a

437 -a 296 '@ I A

314 m-fl 308

I I A I B I c 1q-I

128 222 351 498 639 711 bp

Figurr2. A schematic o f the PCR assay for CD4S isoform exprcssion.The CD4S cDNA is4991 bp long excluding thc poly(A) tail and is composed of 34 exons encoding 1291 amino acids. The thick bar represents the 71 I-bp region containing the thrce variably expressed exons A. B and C.Thc positions o f the PCR primers PI. P2 and the exon boundaries within the amplified region are indicated: bp are numhercd according toThomas et al. [7] where protein sequence is initiated at a methionin corresponding to nucleotide position 131. Thc striped bars represent the sequences complementary t o primers. The sizes and composition of eight predicted amplified products are iilso shown.

hybridization, we have included fibroblast cell lines V? expressing a defined single isoform of CD45, namely ABC, BC, C or null, for PCR amplification and southern blot hybridization. A single product of predicted size is amplif- ied from each of these lines, 584 bp from V? ABC, 455 bp from Y, BC, 308 bp from Yz C and 167 bp from V? Null. As shown in Fig. 3, probe A hybridizes to the PCR products of W, ABC only and probe B to W2 ABC and Y? BC only, whereas probe C hybridizes to the amplification products of Y2 ABC, Y2 BC and Y, C tranfectants. These controls provide the support for the accuracy of identification of CD45-specific PCR products. The results of hybridization with exon A-, exon B- and exon C-specific probes are shown in Fig. 3a, b, c. All clones of both Thl and Th2 types express CD45 isoforms containing only single variable exons: single exon A. single exon B, or single exon C (300 bp in Fig. 3). By this technique it is not possible,

Thl Controls , \/- - \

Th2 A - . ,~ A

587-

(a) 458-

2 98-

5 8 7 -

(b) 458-

298- -

58 7-

(c) 458-

298-

(dl

Exon A

Exon 6

Exon c

Null

Figure 3. Exprcssion o f CD4S mRNA inTI,l and T1,2 clones. RNA isolated from TI,l , Th2 clones and fibroblast lines vJz transfccted with individual CD45 isoforms were uscd to synthesize first strand cDNA. Sequences containing alternatively spliced exons werc amplified by PCR. fractionated on agarosc gels. transferred to Nylon mernbranc and hybridized with probes specific for exon A (a). exon B (b). exon C (c) and null isoform mRNA (d). Size markers in bp are indicated on the left side. Prcdictcd sizes for PCR productsare: 167 bp for null. 2Yh-314 bp foronc-cxon. 437-455 bp for two-exon and 584 bp for three-exon isoforms.

however, to quantitatively compare the relative expression of the isoforms in the two subsets of clones. In contrast, expression of double exon forms is limited to the clones of the Th2 type (see bands at 450 bp). The two exon forms, A + B and A + C, will hybridize to the exon A-specific probe, while the two exon forms, A -t B and B + C, will hybridize to the exon B-specific probe.The exon C-specific probe will hybridize to the forms containing either exons A + C or B + C. However, the sizes of amplified products of

Table 2. Differential expression o f CDJS on T,,1 and T,,2 cloned cells

C'D-1.5 mRNA cxprcssion A n t i hod! hi ndi n p C'lonc TI, I ! ~ C N o cxtm Singlr exon Two exon Thrcc exon 14,s I hA M 111)

fo r m form form forni snli-CD-lSRA anti-CD4SRB ;inti-C'D-IS

+ t t t +

+ + t +++ t + + -t t i

++ + + ++ + + + + + +

+ + + + + + + + + + ++ ++ + + - -

- -

- -

~ -

+ + + +++ + + + +++

-

-

-

--/+

+ + + 1 t + + + + + + t + + + + + + + + + + t +

Eur. J. Immunol. 1991. 21: 17-22 Diffcrcntial splicing o f CD45 in Thl and Th2 cell clones 21

CD45 isoforms containing two exons are such that they cannot be easily resolved to establish whether all three two-exon forms are expressed in Th2 clones or only two of them are utilized. However, none of the two-exon forms are expressed in the T h l clones. Furthermore, there exists a clear distinction between T h l and Th2 clones in terms of expression of the three-exon form (see band at 584 bp in Fig. 3). This CD45-isoform is expressed in al Th2 clones, although the relative ratio of the expression of the three- exon isoform to that of other isoforms varies among these clones as exemplified by the low expression of the three- exon isoform in the D8 clone (stronger band visible at longer exposure). None of the Thl clones was found to express RNA for this large forms of the CD45 molecule. Thus, as indicated by antibody binding, the high molecular weight isoforms (those containing exon A and B) are not present inThl clones. Results of the hybridization with the null form probe indicate that the CD45RO mRNA is expressed at significantly higher levels in Thl clones than in Th2 clones. Although quantitative comparison of each isoforms in one cell type cannot be made with that expressed in another cell type, relative expression of the mRNA encoding single, double, triple exons and the null form within a single cell type can be compared. By this analysis, it is clear that there is dominant expression of CD45RO mRNA in the T h l subset. Among the Th2 cell types, only 6.2 clone expresses significant amounts of null isoform message.

Taken together, the protein and mRNA data suggest that Th 1 cloned lines express predominantly the low molecular weight isoforms of CD45, including the single exon forms and the null isoform containing no variable exons. By contrast theTh2 cloned lines express the three-exon form of CD45 as well as some if not all 2 exon isoforms of CD45. These data are summarized in Table 2.

4 Discussion

These studies were undertaken to analyze the pattern of expression of CD45 isoforms in CD4+ cloned Tcells which possess distinct functional activities. Using the PCR ampli- fication technique, we have shown that a single Tcell clone expresses multiple CD45 isoforms. Cloned CD4+ Tcells of the Th2 subset express the two-exon and three-exon isoforms while these are absent from cells of theThl clones in which the CD4SRO isoform dominates. These results have been confirmed with mAb which recognize restricted isoforms of CD45 on the cell surface. The two mAb which recognize restricted epitopes on high molecular weight isoforms react well with the Th2 subset of CD4+ cloned lines, but not with the Thl subset.

Human CD4+ Tcells can be subdivided on the basis of the expression of CD45 isoforms. High-molecular weight iso- forms of the CD45 molecule which utilize exon A [39] are expressed on a subset of CD4 cells termed suppressor- inducer (naive) and are absent from helper-inducer (me- mory) cells [2, 181. Similarly, in rat exon B-dependent high-molecular weight isoforms [9] are expressed on a subpopulation of CD4+ Tcells that mediate GvH reaction and are either absent or expressed at very low levels on the CD4+ Tcells that provide help for antibody response [4]. I n

the mouse, exon B-dependent high molecular weight iso- forms [38] were found to be expressed on the subpopulation of CD4+ Tcells that produce IL2 upon polyclonal activa- tion and were expressed only at low levels on the cells that produce IL4 [6]. At this point, the relationship between these two subpopulations of freshly isolated CD4+ T cells and the Thl and Th2 clones is not clear. These studies suggest, however, that the expression of different isoforms of the CD45 molecule may be associated with functional specialization of CD4+ Tcells. Studies of CD45 in human, rat and mouse have shown that the subpopulations of CD4 cells are not different lineages of cells but correspond instead to maturational stages [17, 19, 20, 401. Thus, high-molecular weight CD45 isoforms are lost from the surface of naive CD4+ T cells following activation. One might predict that long-term antigen-stimulated clones would express only low-molecular weight isoforms. The results of these studies reported in this article suggest that Thl and Th2 cells are phenotypically distinct in that the expression of large-molecular weight isoforms (three-exon and two-exon forms) is maintained (or perhaps regained) in Th2 cloned Tcells but not in Thl cells. While studies with normal murine CD4+ Tcells have shown that CD45 isoform expression changes after activation (401. once cloned, the cells appear to maintain a characteristic profile of isoforms after stimulation (data not shown). Other studies in mouse have also shown that large-molecular weight CD4S iso- forms identified by anti-CD45RA mAb, 14.8 are expressed on subset of CD45+ Tcells and play a role in their activation [421.

To understand the role of CD45 in the differentiation and function of lymphoid cells, it is important to identify all the isoforms of this molecule expressed in cells of different lineages and functions. However, this task is complicated by a number of technical considerations. Immunoprecipi- tation with available antibodies in combination with SDS- PAGE is useful for the identification of some isoforms while others cannot be distinguished. Also, isoforms that are expressed at very low levels are difficult to detect with this approach. Identification of cell surface CD45 expres- sion using mAb is complicated in that most antibodies bind to more than one isoform and that the precise requirement for individual exon expression needed for binding is undefined. For example 14.8 and 16A are known to bind to double and triple exon forms of CD45. 14.8 requires the presence of exon A and 16A requires the presence of exon A and 16A requires the presence of exon B, yet it is not known if they will bind to the single exon forms of CD45 or if their binding depends on the expression of more than one variable exon. Our data comparing antibody staining and mRNA expression would suggest that the single exon A and B isoforms are present onThl cells but are not bound by the antibodies 14.8 or 16A. Further analysis to determine the antibody’s fine specificities is underway. Therefore, the method of primer-directed enzymatic amplification, which uses only small amounts of mRNA, is uniquely suited for the analysis of the complex pattern of expression of the CD45 family members. Recently this approach has been successfully used to determine the expression of CD45 family members in the mouse [43]. Our data clearly demonstrate that this method, in combination with battery of mAb that recognize restricted epitopes, is an effective and powerful tool that will help us to understand the role of various isoforms in the function of lymphoid cells.

22 M. Luqman. l? Johnson. I. Trowbridge and K. Bottomly Eur. J. Immunol. 1991. 21: 17-22

Finally, recent studies have revealed that CD45 is a tyrosine-specific phosphatase [44, 451. Regulation of Tcell activation through enzymatic activity of this tyrosine- specific phosphatase has been suggested. It is conceivable that the putative ligand for CD4.5 is changed or its affinity altered by selective expression of certain isoforms of this molecule. This provision may allow the cells to regulate tyrosine-specific phosphorylation and thus, in turn, modu- late effector function.

Received December 11 . 1989: in final revised form October 22. 1990.

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