Epitopc-specific effect of anti-CD45 mAb
Eur. J. Immunol. 1990. 20: 2801-2804
2801
Short paper Emili Alsinetv, Julia Inglks-Esteve, Ram& Vilella, Francisco Lozano, Jordi Mila, Isabel Rojo, Jaume Martorell, Jordi Vives and Antoni Gaya Servei d’Immunologia, Hospital Clinic i Provincial, Barcelona
Differential effects of anti-CD45 monoclonal antibody on human B cell proliferation: a monoclonal antibody recognizing a neuraminidase-sensitive epitope of the T200 molecule enhances anti-immunoglobulin-induced proliferation* We have generated seven monoclonal antibodies (mAb) that recognize the T200 molecule. These mAb have been classified by competitive binding assay in flow cytometry into three groups each reacting with a different epitope of the T200 molecule: (a) 136-4B5, that shows a sialic acid nature, (b) 135-4H9, 135-4C5, 144-2, 155-2 and (c) 72-5D3, 124-2H12b. A heterogeneous effect was observed when they were tested on an anti-immunoglobulin-induced B cell proliferation. Whereas 72-5D3 and 135-4H9mAb inhibited the proliferative response of B cells, 136-4B5 mAb greatly enhanced it, both effects being dose dependent. We can conclude that anti-CD45 mAb have a different and contrary functional behavior on anti-Ig-induced B cell proliferation, depending on the epitope recognized.The basis for such a difference could reside in the glucidic nature of the epitope recognized by the 136-4B5 mAb.
1 Introduction The leukocyte common antigen CD45 (LCA,T200 or Ly-5) is a family of high-molecular-weight glycoproteins expressed exclusively on the surface of cells of hematopoietic origin [l-31. CD45 antigen can be considered as a bifunctional molecule. The extracellular domain can be present in different forms [4] which can bind different ligands, whereas the intracellular one is common to all forms and shows a tyrosine phosphatase activity that can modulate the phosphorylation level of other membraneassociated proteins [5-71. Anti-CD45 mAb can affect a wide range of lymphocyte functions and sometimes in a contradictory way. It has been described that anti-CD45 mAb inhibit NK activity [8] and the cytotoxic activity ofTcells [9, lo], enhance or inhibit the proliferation of Tcells [11-121 and inhibit IgG production [13] and B cell proliferation [14, 151. One way to explain these functional effects is to consider that they could depend on the different epitopes recognized by the mAb used on such assays. A similar situation has been observed with anti-CD2 mAb, where mAb recognizing the Till epitope inhibit the proliferation of T cells to several mitogens, whereas mAb against T112 and Tl13 are mitogenic when used in combination (reviewed in [16]). [I 85511
* This work was supported by F.I.S.S. grant 88/0212 and 89/1083. Recipient of the grant “Iniciacibn a la Investigacion” 861249 from Fondo de Investigaciones Sanitarias de la Seguridad Social. Correspondence: Emili Alsinet, Servei d’Immunologia, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain Abbreviations: anti-Ig: Goat anti-human Ig F(ab)’z fragment LCA: Leukocyte common antigen 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1990
To test the hypothesis of an epitope-specific effect of anti-CD45 mAb we have tested the effects of several anti-CD45 mAb on proliferative assays. We describe seven anti-CD45 mAb that recognized three different epitopes. The analysis of their action on anti-Ig-induced B cell proliferation reveals a relationship between the functional effects of anti-CD45 mAb and the epitope recognized by them. Thus, 72-5D3 and 135-4H9 mAb, both recognizing two different epitopes on the CD45 antigen, inhibited the [3H]dThd incorporation. On the contrary, 136-4B5 mAb, recognizing a third epitope, which is neurarninidase sensitive, enhanced the proliferation in the same assay.
2 Materials and methods 2.1 mAb The rnAb used were produced in our laboratory as described elsewhere [17] and assigned to clusters by the Second, Third and Fourth International Workshop on Human Leucocyte Differentiation Antigens [ 18-21]: Cris7 (anti-CD3, IgG2,), Edu-2 (anti-CD4, IgGh), Cris-6 (anti-CD14, IgG,), 111-5A1 (anti-CD41, IgGI), 84-3C1 (anti-CD43, IgGI). The anti-CD45 mAb panel are: 72-5D3 (IgGz,), 124-2H12b (IgG2,), 135-4H9 (IgG2b), 135-4C5 (IgG2b), 136-4B5 (IgGI), 144-2 (IgM), 155-2 (IgM). mAb were partially purified from ascites fluid by precipitation with ammonium sulfate and used at a final concentration of 10 pg/ml (except when indicated). 2.2 Western blot analysis, immunofluorescence and neuraminidase treatment of PBMC
Western blot assay was performed using dense tonsillar B cell lysates as described elsewhere [22]. Imrnunofluorescence assays were performed by a standard indirect method
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E. Alsinet, J. InglCs-Esteve, R. Vilella et al.
as previously described [23] and FCM analysis was performed on a FACS Analyzer (Becton Dickinson, Mountain View, CA).To remove the sialic acid residues of cell surface glycoproteins, PBMC were treated with neuraminidase (Sigma Chemical Co., St. Louis, MO) as described [23].
Eur. J. Immunol. 1990. 20: 2801-2804 Blocking mAb 72 5D3
124 ZH12b
135 4C5
135 4H9
155 2
144 2
136 4B5
CD4
2.3 Competition binding assay Competition binding assay between unlabeled and biotinlabeled mAb was performed on PBMC, granulocytes and the CEM T cell line as described [24]. The percentage of inhibition was calculated as follows: ( Y maximum fluorescence - YO test fluorescence)/( % maximum fluorescence). Those mAb giving >50% inhibition were considered as recognizing the same epitope.
2.4 Cell cultures Tonsillar B cells were obtained and cultured as previously described [25]. Cells were separated into different density fractions on discontinuous Percoll gradients (Pharmacia, Uppsala, Sweden) and cells pelleted below 60% Percoll were used as dense B cells.
Figure 1. Epitope mapping of anti-CD45 mAb. PBMC, granulocytes and theTcell line CEM were incubated with a saturating dose of either anti-CD45 mAb or control EDU-2 (anti-CD4) (listed on the top). Afterwards, cells were incubated with biotinylated anti-CD45 mAb (listed on the left).The resulting fluorescence was measured by FCM analysis and expressed as a % of inhibition with respect to the maximum fluorescence. obtained when the cells were preincubated without mAb. CD43 (84-3C1)
CD45 (72-5D3)
CD45 (135-4H9)
CD45 (136485)
3 Results 3.1 Molecular characterization of seven mAb against the T200 molecule 72-5D3, 124-2H12b, 135-4H9, 135-4C5, 144-2, 155-2 and 136-4B5mAb were obtained in our laboratory by standard hybrydoma techniques [17]. All of them were classified as anti-CD45 mAb in different International Workshops on Human Leucocyte Differentiation Antigens [ 18-21]. Western blot analysis on PBMC cell lysate showed that all the mAb recognized the four band pattern characteristic of the CD45 antigen (data not shown).
3.2 The seven anti-CD45 mAb recognize three different epitopes To assess whether the mAb used in this study recognized the same epitope on the CD45 molecule, a competitive binding assay was performed. Fig. 1 show the results obtained by using PBMC although the same results were obtained by using granulocytes and the CEM T cell line (data not shown). All mAb were biotinylated and tested against unbiotinylated mAb. We considered that two mAb recognized the same epitope when > 50% of maximum fluorescence was inhibited. The inhibition specificity was controlled by the inclusion of unrelated mAb, as well as by the bidirectionality of the inhibition. As observed in Fig. 1, each mAb was completely inhibited by itself. Unrelated mAb, or those recognizing other epitopes, were unable to inhibit > 15%-20%, whereas those classified in the same group were usually able to inhibit >75% of maximum binding. The results obtained using the three cell types mentioned above were similar (data not shown). From these results, the seven anti-CD45 mAb can be classified into three groups, each one recognizing a different epitope on the CD45 molecule. One group is formed by the 136-4B5
LOG FLUORESCENCE INTENSITY
Figure 2. Stability of CD45 epitopes to neuraminidase treatment. PBMC were treated with neuraminidase prior to the addition of anti-CD45 mAb (72-5D3, 135-4H9 and 136-4B5) or the positive control anti-CD43 mAb (84-3C1). Reactivity was measured by FCM. Solid line represents the fluorescence level without neuraminidase treatment and dotted line represents the fluorescence level after neuraminidase treatment.
mAb. A second one by mAb 135-4H9,135-4C5,144-2 and 155-2. The third group is comprised of mAb 72-5D3 and 124-2H12b.
3.3 136-4B5 mAb recognizes an epitope formed by carbohydrate residues There is increasing evidence that the carbohydrate structures are an important functional characteristic of the T200 glycoprotein [26, 271, so we were interested in verifying whether some of our anti-CD45 mAb recognized any glucidic epitope on this antigen. As is shown in Fig. 2, pretreatment of PBMC with neuraminidase completely inhibits the fluorescence yielded by 136-4B5 mAb, whereas the other anti-CD45 mAb retain their reactivity. The 84-3C1 mAb (anti-CD43) represents a positive control to the neuraminidase treatment [23].
3.4 136-4B5 mAb enhance the anti-human Ig-induced proliferation on B cells An inhibitory effect of anti-CD45 mAb on anti-Ig-induced B cell proliferation has been demonstrated [ 14, 151. Thus,
Epitope-specific effect of anti-CD4S mAb
Eur. J. Immunol. 1990. 20: 2801-2804
2803
addition, a sequential immunoprecipitation between 725D3 and 136-4B5 showed that after preclearing of a cell lysate of the EBV-tranformed WT 47 cell line with 72-5D3, 136-4B5 was unable to precipitate any molecule (data not shown), thus confirming that 136-4B5did not react with any other molecule other than CD45.
4 Discussion
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Figure 3. Dose-response effect of anti-CD45 mAb on anti-Iginduced B cell proliferation. Tonsillar B cells were stimulated with 50 pglml of anti-Ig and doses ranging from 1 nglml to 1 x 104 nglml of anti-CD45 mAb [72-SD3, (A)135-489, (W) and 136-4B5 (V)]or anti-CD41 control mAb [lll-5A1, ( O ) ] .After 48 h of culture at 37 "C in a 5% C 0 2 humidified atmosphere, 1 pCilwell of [3H]dThd was added and uptake of [3H]dThd was measured 18 h later. The proliferative level of B cells in medium (*) or when stimulated with anti-Ig alone (+)is also shown. Each point shows the mean of ["H]dThd incorporation of triplicate determinations. SD was < 15% of the mean.
we were interested in assessing the functional effect of our mAb on this B cell proliferation assay. As can be observed in Fig. 3 the presence of 50 pg/ml of anti-Ig in the culture medium yielded a clear proliferative response. As expected, the addition of 72-5D3 or 135-4H9 mAb greatly inhibited the proliferative response of B cells, lowering the ['HIdThd incorporation to 75-99% of the control response. In contrast, the addition of 136-4B5 mAb enhanced two-to fourfold the proliferative response of B cells. As can be observed in Fig. 3, both the enhancing as well as the inhibitory activity effect were dose dependent. However, the enhancing effect of 136-4B5mAb was not detected in a co-stimulatory assay with anti-Ig plus IL 2, IL 4 or low molecular weight B cell growth factor whereas 72-5D3 and 135-4H9 mAb still inhibited such proliferation (data not shown). The control 111-5A1 mAb (anti-CD41, IgGI) did not affect the level of B cell proliferation at any concentration tested, ruling out an isotype-specific effect.
The aim of this study was to characterize the functional properties of the extracellular domain of the CD45 molecule through an epitope analysis. For this purpose seven anti-CD45 mAb produced in our laboratory and studied in the International Workshops on Human Leukocyte Differentiation Antigens were used. By competitive binding analysis, we have shown that these mAb recognized three different epitopes (Fig. 1). From all of them only the one recognized by the 136-4B5 mAb was neuraminidase sensitive. In functional assays we have observed that all these anti-CD45 mAb were able to induce Tcell proliferation on anti-CD3-Sepharose-activated cells highly depleted of monocytes (MHDC) (data not shown) as already described for anti-CD45 mAb [ ll] . Moreover, all these anti-CD45 mAb, except 136-4B5 mAb, also inhibited the anti-Iginduced B cell proliferation (Fig. 3) as already reported for the anti-CD45 mAb [14, 151. Surprisingly, 136-4B5 rnAb not only did not inhibit anti-Ig-induced B cell proliferation but greatly enhanced it (Fig. 3). Until now, 136-4B5mAb is
kDa 200-
11592-
62-
3.5 136-4B5 mAb recognizes only the CD45 antigen on B cells 45-
Taking into account that the effect of 136-4B5 on anti-Ig stimulated B cell proliferation is contrary to the effect of all the other anti-CD45 antibodies tested, we were interested in verifying that 136-4B5 does not recognize any other molecule than CD45 on the surface of B cells. In order to test this point 136-4B5, 72-5D3 and 135-4H9, that each react with different epitopes on CD45, were subjected to a Western blot analysis. Dense tonsillar B cell lysates were run on an 8% SDS-PAGE gel under nonreducing conditions. As shown in Fig. 4, all the mAb recognized the four-band pattern characteristic of the CD45 antigen. In
Figure 4. Western blot analysis of 136-4BS mAb from tonsillar B cell lysate. Samples of tonsil B cell lysates were run on an 8%
SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose paper. Each strip was incubated with the appropriately diluted purified mAh listed in the figure for 1 h. Peroxidase-labeled rabbit anti-mouse Ig were used as a second antibody.
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Eur. J. Immunol. 1990. 20: 2801-2804
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the only anti-CD45 mAb known to induce an increase in the proliferative response of B cells stimulated by anti-Ig. Due to this special feature of 136-4B5mAb, as opposed to all the anti-CD45 mAb tested by different authors [14, 151, it was important to assess whether 136-4B5 mAb really belonged to the antLCD45 mAb family. That this was the case was confirmed in four different ways. First, the cellular and tissue distribution data obtained by us (data not shown) together with that reported in the Fourth International Workshop on Human Leukocyte Differentiation Antigens confirm that 136-4B5 mAb belongs to the anti-CD45 mAb family [21]. Furthermore, the results obtained from the proliferative studies on MHDC (data not shown) agree with the ones described for anti-CD45 mAb [ll].Finally, the Western blot analysis of B cell lysates (Fig. 4) and sequential immunoprecipitation (data not shown) confirms that 136-4B5 recognizes only the CD45 antigen on B cells. The studies reported in the literature on the role of anti-CD45 mAb on the proliferative response are conflicting. In some cases they inhibit Tand B cell proliferation [12, 14, 151. In other situations, anti-CD45 mAb enhanceTcell proliferation [ l l ] . The peculiar behaviour of 136-4B5 m Ab described in this report could be considered as a new conflicting element to the ones already existing in this field. However, it should be taken into account that, in contrast to the results published in which different experimental systems are used, our results were obtained using the same cells and the same stimulations. The only difference in our system was the anti-CD45 mAb used recognized different epitopes.
It is also very surprising that 136-4B5 mAb has an opposite effect on B cells to that of the rest of the anti-CD45 mAb used in this study and to that of the ones described in the literature, although it has the same effect as the rest of anti-CD45 mAb on T cells. Since mAb against different epitopes can mimic the physiological ligands of cell receptors, the results obtained in this study raise again the question of the natural ligands of the CD45 molecule. The extracellular diversity of this molecule on different cell types allows us to assume that they are able to bind different ligands. Moreover, the fact that CD45 is a highly glycosylated molecule the glycosylation pattern of which changes according to the cell lineages and the activation state of the cell induces us to postulate that the carbohydrate structures play a role in the function of the CD45 molecule. The recently described inhibition of binding between NK cells and their targets by anti-CD45 mAb recognizing the carbohydrate residues of the CD45 molecule [27] supports the hypothesis that they are involved in the cell to cell interaction as has been recently suggested [28].The peculiar effect of the 136-4B5mAb on B cells described in our study also favors this suggestion. Taking into account that CD45 has a tyrosine phosphatase activity and the recent direct involvement of the CD45 molecule in T cell proliferation [28], it could be assumed that the effects of the different anti-CD45 mAb on B cell proliferation are related in some way with its phosphatase activity. The fact that in T cells, CD45 phosphotyrosine phosphatase acts on the T cell tyrosine protein kinase pp56ICk[7], which is not present on B cells, allows us to assume that CD45 phosphatase activity on B cells should act on other tvrosine Drotein kinase. In anv case. it can not be ruled out' that [he mechanisms thaf inhibit B cell
proliferation could be different from the ones inducing an increase of the proliferation. We thank Dr. 0.Viiias and J. J. Barcelo for their advice and practical help on flow cytometric analysis. Received May 7, 1990; in revised form July 16, 1990.
5 References 1 Dalchau, R., Kirkley, J. and Fabre, J. W., Eur. J. Imrnunol. 1980. 10: 737. 2 Omary, M. B.,Trowbridge, 1. S. and Battifora, H. A., J. Exp. Med. 1980. 152: 842. 3 Dalchau, R. and Fabre, J., J. Exp. Med. 1981. 153: 753. 4 Streuli, M., Hall, L. R., Saga,Y., Schlossman, S. F. and Saito, H., J. Exp. Med. 1987. 166: 1548. 5 Tonks, N. K., Charbonneau. H.. Diltz, C. D., Fischer, E. H. and Wlash, K. A., Biochemistry 1988. 27: 8695. 6 Clark. E. A. and Ledbetter, J. A.. Immunol. Today 1989. 10: 225. 7 Mustelin, T.. Coggeshall, K. M. and Altman, A., Proc. Natl. Acad. Sci. USA 1989. 86: 6302. 8 Newman, W., Fast. L. D. and Rose, L. M., . I Immunol. . 1983. 131: 1742. 9 Lefranqois, L. and Bevan, M. J., Nature 1985. 314: 449. 10 Demm. R. L., Shanahan, E , Niederlehner, A. and Targan. S. R.. Cell. Immunol. 1988. 117: 99. I1 Martorell, J..Vilella, R., Borche. L., Rojo. 1. and Vives, J.. Eur. J. Immunol. 1987. 17: 1447. 12 Bernabcu. C., Carrera, A. C., Landazuri, M. 0. and SanchezMadrid. F., Eur. J. Imrnunal. 1987. 17: 1461. 13 Yakura. H., Kawabata. I., Ashida. T. and Katadiri. M., J. Immunol. 1988. 141: 87.5. 14 Mittler, R. S., Greenfield, R. S., Schacter, B. Z . , Richard, N. E and Hoffman, M. K.. J. Immunol. 1987. 138: 3150. 15 Gruber, M. F., Bjorndahl. J. M.. Nakamura, S. and Fu, S. M.. J. Immunol. 1989. 142: 4144. 16 Linch, D. C., Wallace, D. L. and O'Flynn. K.. Irnrnunol. Rev. 1987. 95: 137. 17 Vilella, R..Yagiie, J. and Vives, J., Human Immunol. 1983.6: 53. 18 Haynes, B. F.. in Reinherz, E. L., Haynes. B. F.. Nadler, L. M. and Berstein, I. D. (Eds.), Leucocyte Typing 11, SpringerVerlag, New-York 1986, I : 3. 19 Berstein, I. D. and Self, E., in Reinherz. E . L., Heynes, B. F., Nadler, L. M. and Berstein. I. D. (Eds.), Leucocyte Typing 11, Springer-Verlag, New-York 1986, Ill: 1. 20 Cobbold, S., Halc, G. and Waldman, H., in McMichel, A . J.. Beverley, €? C. L., Cobbold, S., Crumpton, M. J., Gilks, W.,
21
22 23 24 25
Gotch, F. M., Hogg, N., Horton, M., Ling. N., MacLennan, I. C. M.. Mason. D. Y., Milstein. C., Spiegelhalter. D. and Waldman, H. (Eds.). Leucocyte Typing IIZ, Oxford University Press, Oxford 1987. p. 788. Schmidt, R. E., in Knapp,W., Dorken, B., Gilks,W. R., Rieber. Schmidt, R. E., Stein, H. and Von dem Borne, A. E. G. E. €?. (Eds.). Leucocyte Typing Iq Oxford University Press. Oxford 1989, p. 517. Lozano, F., Alberola-Ila. J., Places. L. and Vives, J., Mol. Imrnunol. 1989. 26: 1187. Borche. L., Lozano, F., Vilella, R. and Vives, J., Eur. J. Immunol. lY87. 17: 1523. Lozano. F., Borche, L., Places, L., Alberola-Ila, J., Gaya, A., Vilella, R. and Vives, J., Tissue Antigens. 1990. 35: 193. InglCs, J., Engel, P., Dc la Calle. O., Gal1art.T.. immunology
1989. 67: 359. 26 Cook, R. G., Landolfi, N. F.. Mehta. V.. Leone. J. and Hoyland, D., J. Irnmunol. 1987. 1.39: 991. 27 Gilbert, C. W., Zaroukian, M. H. and Essclman. W. J., J. Imrnunol. 1988. 140: 2821. 28 Pingel, J. T. and Thomas, M. L., Cell 1989. 58: 1055.
Eur. J. Immunol. 1990. 20: 2801-2804
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Short paper Emili Alsinetv, Julia Inglks-Esteve, Ram& Vilella, Francisco Lozano, Jordi Mila, Isabel Rojo, Jaume Martorell, Jordi Vives and Antoni Gaya Servei d’Immunologia, Hospital Clinic i Provincial, Barcelona
Differential effects of anti-CD45 monoclonal antibody on human B cell proliferation: a monoclonal antibody recognizing a neuraminidase-sensitive epitope of the T200 molecule enhances anti-immunoglobulin-induced proliferation* We have generated seven monoclonal antibodies (mAb) that recognize the T200 molecule. These mAb have been classified by competitive binding assay in flow cytometry into three groups each reacting with a different epitope of the T200 molecule: (a) 136-4B5, that shows a sialic acid nature, (b) 135-4H9, 135-4C5, 144-2, 155-2 and (c) 72-5D3, 124-2H12b. A heterogeneous effect was observed when they were tested on an anti-immunoglobulin-induced B cell proliferation. Whereas 72-5D3 and 135-4H9mAb inhibited the proliferative response of B cells, 136-4B5 mAb greatly enhanced it, both effects being dose dependent. We can conclude that anti-CD45 mAb have a different and contrary functional behavior on anti-Ig-induced B cell proliferation, depending on the epitope recognized.The basis for such a difference could reside in the glucidic nature of the epitope recognized by the 136-4B5 mAb.
1 Introduction The leukocyte common antigen CD45 (LCA,T200 or Ly-5) is a family of high-molecular-weight glycoproteins expressed exclusively on the surface of cells of hematopoietic origin [l-31. CD45 antigen can be considered as a bifunctional molecule. The extracellular domain can be present in different forms [4] which can bind different ligands, whereas the intracellular one is common to all forms and shows a tyrosine phosphatase activity that can modulate the phosphorylation level of other membraneassociated proteins [5-71. Anti-CD45 mAb can affect a wide range of lymphocyte functions and sometimes in a contradictory way. It has been described that anti-CD45 mAb inhibit NK activity [8] and the cytotoxic activity ofTcells [9, lo], enhance or inhibit the proliferation of Tcells [11-121 and inhibit IgG production [13] and B cell proliferation [14, 151. One way to explain these functional effects is to consider that they could depend on the different epitopes recognized by the mAb used on such assays. A similar situation has been observed with anti-CD2 mAb, where mAb recognizing the Till epitope inhibit the proliferation of T cells to several mitogens, whereas mAb against T112 and Tl13 are mitogenic when used in combination (reviewed in [16]). [I 85511
* This work was supported by F.I.S.S. grant 88/0212 and 89/1083. Recipient of the grant “Iniciacibn a la Investigacion” 861249 from Fondo de Investigaciones Sanitarias de la Seguridad Social. Correspondence: Emili Alsinet, Servei d’Immunologia, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain Abbreviations: anti-Ig: Goat anti-human Ig F(ab)’z fragment LCA: Leukocyte common antigen 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1990
To test the hypothesis of an epitope-specific effect of anti-CD45 mAb we have tested the effects of several anti-CD45 mAb on proliferative assays. We describe seven anti-CD45 mAb that recognized three different epitopes. The analysis of their action on anti-Ig-induced B cell proliferation reveals a relationship between the functional effects of anti-CD45 mAb and the epitope recognized by them. Thus, 72-5D3 and 135-4H9 mAb, both recognizing two different epitopes on the CD45 antigen, inhibited the [3H]dThd incorporation. On the contrary, 136-4B5 mAb, recognizing a third epitope, which is neurarninidase sensitive, enhanced the proliferation in the same assay.
2 Materials and methods 2.1 mAb The rnAb used were produced in our laboratory as described elsewhere [17] and assigned to clusters by the Second, Third and Fourth International Workshop on Human Leucocyte Differentiation Antigens [ 18-21]: Cris7 (anti-CD3, IgG2,), Edu-2 (anti-CD4, IgGh), Cris-6 (anti-CD14, IgG,), 111-5A1 (anti-CD41, IgGI), 84-3C1 (anti-CD43, IgGI). The anti-CD45 mAb panel are: 72-5D3 (IgGz,), 124-2H12b (IgG2,), 135-4H9 (IgG2b), 135-4C5 (IgG2b), 136-4B5 (IgGI), 144-2 (IgM), 155-2 (IgM). mAb were partially purified from ascites fluid by precipitation with ammonium sulfate and used at a final concentration of 10 pg/ml (except when indicated). 2.2 Western blot analysis, immunofluorescence and neuraminidase treatment of PBMC
Western blot assay was performed using dense tonsillar B cell lysates as described elsewhere [22]. Imrnunofluorescence assays were performed by a standard indirect method
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as previously described [23] and FCM analysis was performed on a FACS Analyzer (Becton Dickinson, Mountain View, CA).To remove the sialic acid residues of cell surface glycoproteins, PBMC were treated with neuraminidase (Sigma Chemical Co., St. Louis, MO) as described [23].
Eur. J. Immunol. 1990. 20: 2801-2804 Blocking mAb 72 5D3
124 ZH12b
135 4C5
135 4H9
155 2
144 2
136 4B5
CD4
2.3 Competition binding assay Competition binding assay between unlabeled and biotinlabeled mAb was performed on PBMC, granulocytes and the CEM T cell line as described [24]. The percentage of inhibition was calculated as follows: ( Y maximum fluorescence - YO test fluorescence)/( % maximum fluorescence). Those mAb giving >50% inhibition were considered as recognizing the same epitope.
2.4 Cell cultures Tonsillar B cells were obtained and cultured as previously described [25]. Cells were separated into different density fractions on discontinuous Percoll gradients (Pharmacia, Uppsala, Sweden) and cells pelleted below 60% Percoll were used as dense B cells.
Figure 1. Epitope mapping of anti-CD45 mAb. PBMC, granulocytes and theTcell line CEM were incubated with a saturating dose of either anti-CD45 mAb or control EDU-2 (anti-CD4) (listed on the top). Afterwards, cells were incubated with biotinylated anti-CD45 mAb (listed on the left).The resulting fluorescence was measured by FCM analysis and expressed as a % of inhibition with respect to the maximum fluorescence. obtained when the cells were preincubated without mAb. CD43 (84-3C1)
CD45 (72-5D3)
CD45 (135-4H9)
CD45 (136485)
3 Results 3.1 Molecular characterization of seven mAb against the T200 molecule 72-5D3, 124-2H12b, 135-4H9, 135-4C5, 144-2, 155-2 and 136-4B5mAb were obtained in our laboratory by standard hybrydoma techniques [17]. All of them were classified as anti-CD45 mAb in different International Workshops on Human Leucocyte Differentiation Antigens [ 18-21]. Western blot analysis on PBMC cell lysate showed that all the mAb recognized the four band pattern characteristic of the CD45 antigen (data not shown).
3.2 The seven anti-CD45 mAb recognize three different epitopes To assess whether the mAb used in this study recognized the same epitope on the CD45 molecule, a competitive binding assay was performed. Fig. 1 show the results obtained by using PBMC although the same results were obtained by using granulocytes and the CEM T cell line (data not shown). All mAb were biotinylated and tested against unbiotinylated mAb. We considered that two mAb recognized the same epitope when > 50% of maximum fluorescence was inhibited. The inhibition specificity was controlled by the inclusion of unrelated mAb, as well as by the bidirectionality of the inhibition. As observed in Fig. 1, each mAb was completely inhibited by itself. Unrelated mAb, or those recognizing other epitopes, were unable to inhibit > 15%-20%, whereas those classified in the same group were usually able to inhibit >75% of maximum binding. The results obtained using the three cell types mentioned above were similar (data not shown). From these results, the seven anti-CD45 mAb can be classified into three groups, each one recognizing a different epitope on the CD45 molecule. One group is formed by the 136-4B5
LOG FLUORESCENCE INTENSITY
Figure 2. Stability of CD45 epitopes to neuraminidase treatment. PBMC were treated with neuraminidase prior to the addition of anti-CD45 mAb (72-5D3, 135-4H9 and 136-4B5) or the positive control anti-CD43 mAb (84-3C1). Reactivity was measured by FCM. Solid line represents the fluorescence level without neuraminidase treatment and dotted line represents the fluorescence level after neuraminidase treatment.
mAb. A second one by mAb 135-4H9,135-4C5,144-2 and 155-2. The third group is comprised of mAb 72-5D3 and 124-2H12b.
3.3 136-4B5 mAb recognizes an epitope formed by carbohydrate residues There is increasing evidence that the carbohydrate structures are an important functional characteristic of the T200 glycoprotein [26, 271, so we were interested in verifying whether some of our anti-CD45 mAb recognized any glucidic epitope on this antigen. As is shown in Fig. 2, pretreatment of PBMC with neuraminidase completely inhibits the fluorescence yielded by 136-4B5 mAb, whereas the other anti-CD45 mAb retain their reactivity. The 84-3C1 mAb (anti-CD43) represents a positive control to the neuraminidase treatment [23].
3.4 136-4B5 mAb enhance the anti-human Ig-induced proliferation on B cells An inhibitory effect of anti-CD45 mAb on anti-Ig-induced B cell proliferation has been demonstrated [ 14, 151. Thus,
Epitope-specific effect of anti-CD4S mAb
Eur. J. Immunol. 1990. 20: 2801-2804
2803
addition, a sequential immunoprecipitation between 725D3 and 136-4B5 showed that after preclearing of a cell lysate of the EBV-tranformed WT 47 cell line with 72-5D3, 136-4B5 was unable to precipitate any molecule (data not shown), thus confirming that 136-4B5did not react with any other molecule other than CD45.
4 Discussion
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4
Log (ng/ml) of MoAb
Figure 3. Dose-response effect of anti-CD45 mAb on anti-Iginduced B cell proliferation. Tonsillar B cells were stimulated with 50 pglml of anti-Ig and doses ranging from 1 nglml to 1 x 104 nglml of anti-CD45 mAb [72-SD3, (A)135-489, (W) and 136-4B5 (V)]or anti-CD41 control mAb [lll-5A1, ( O ) ] .After 48 h of culture at 37 "C in a 5% C 0 2 humidified atmosphere, 1 pCilwell of [3H]dThd was added and uptake of [3H]dThd was measured 18 h later. The proliferative level of B cells in medium (*) or when stimulated with anti-Ig alone (+)is also shown. Each point shows the mean of ["H]dThd incorporation of triplicate determinations. SD was < 15% of the mean.
we were interested in assessing the functional effect of our mAb on this B cell proliferation assay. As can be observed in Fig. 3 the presence of 50 pg/ml of anti-Ig in the culture medium yielded a clear proliferative response. As expected, the addition of 72-5D3 or 135-4H9 mAb greatly inhibited the proliferative response of B cells, lowering the ['HIdThd incorporation to 75-99% of the control response. In contrast, the addition of 136-4B5 mAb enhanced two-to fourfold the proliferative response of B cells. As can be observed in Fig. 3, both the enhancing as well as the inhibitory activity effect were dose dependent. However, the enhancing effect of 136-4B5mAb was not detected in a co-stimulatory assay with anti-Ig plus IL 2, IL 4 or low molecular weight B cell growth factor whereas 72-5D3 and 135-4H9 mAb still inhibited such proliferation (data not shown). The control 111-5A1 mAb (anti-CD41, IgGI) did not affect the level of B cell proliferation at any concentration tested, ruling out an isotype-specific effect.
The aim of this study was to characterize the functional properties of the extracellular domain of the CD45 molecule through an epitope analysis. For this purpose seven anti-CD45 mAb produced in our laboratory and studied in the International Workshops on Human Leukocyte Differentiation Antigens were used. By competitive binding analysis, we have shown that these mAb recognized three different epitopes (Fig. 1). From all of them only the one recognized by the 136-4B5 mAb was neuraminidase sensitive. In functional assays we have observed that all these anti-CD45 mAb were able to induce Tcell proliferation on anti-CD3-Sepharose-activated cells highly depleted of monocytes (MHDC) (data not shown) as already described for anti-CD45 mAb [ ll] . Moreover, all these anti-CD45 mAb, except 136-4B5 mAb, also inhibited the anti-Iginduced B cell proliferation (Fig. 3) as already reported for the anti-CD45 mAb [14, 151. Surprisingly, 136-4B5 rnAb not only did not inhibit anti-Ig-induced B cell proliferation but greatly enhanced it (Fig. 3). Until now, 136-4B5mAb is
kDa 200-
11592-
62-
3.5 136-4B5 mAb recognizes only the CD45 antigen on B cells 45-
Taking into account that the effect of 136-4B5 on anti-Ig stimulated B cell proliferation is contrary to the effect of all the other anti-CD45 antibodies tested, we were interested in verifying that 136-4B5 does not recognize any other molecule than CD45 on the surface of B cells. In order to test this point 136-4B5, 72-5D3 and 135-4H9, that each react with different epitopes on CD45, were subjected to a Western blot analysis. Dense tonsillar B cell lysates were run on an 8% SDS-PAGE gel under nonreducing conditions. As shown in Fig. 4, all the mAb recognized the four-band pattern characteristic of the CD45 antigen. In
Figure 4. Western blot analysis of 136-4BS mAb from tonsillar B cell lysate. Samples of tonsil B cell lysates were run on an 8%
SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose paper. Each strip was incubated with the appropriately diluted purified mAh listed in the figure for 1 h. Peroxidase-labeled rabbit anti-mouse Ig were used as a second antibody.
2804
Eur. J. Immunol. 1990. 20: 2801-2804
E. Alsinet, J. InglCs-Esteve, R.Vilella et al.
the only anti-CD45 mAb known to induce an increase in the proliferative response of B cells stimulated by anti-Ig. Due to this special feature of 136-4B5mAb, as opposed to all the anti-CD45 mAb tested by different authors [14, 151, it was important to assess whether 136-4B5 mAb really belonged to the antLCD45 mAb family. That this was the case was confirmed in four different ways. First, the cellular and tissue distribution data obtained by us (data not shown) together with that reported in the Fourth International Workshop on Human Leukocyte Differentiation Antigens confirm that 136-4B5 mAb belongs to the anti-CD45 mAb family [21]. Furthermore, the results obtained from the proliferative studies on MHDC (data not shown) agree with the ones described for anti-CD45 mAb [ll].Finally, the Western blot analysis of B cell lysates (Fig. 4) and sequential immunoprecipitation (data not shown) confirms that 136-4B5 recognizes only the CD45 antigen on B cells. The studies reported in the literature on the role of anti-CD45 mAb on the proliferative response are conflicting. In some cases they inhibit Tand B cell proliferation [12, 14, 151. In other situations, anti-CD45 mAb enhanceTcell proliferation [ l l ] . The peculiar behaviour of 136-4B5 m Ab described in this report could be considered as a new conflicting element to the ones already existing in this field. However, it should be taken into account that, in contrast to the results published in which different experimental systems are used, our results were obtained using the same cells and the same stimulations. The only difference in our system was the anti-CD45 mAb used recognized different epitopes.
It is also very surprising that 136-4B5 mAb has an opposite effect on B cells to that of the rest of the anti-CD45 mAb used in this study and to that of the ones described in the literature, although it has the same effect as the rest of anti-CD45 mAb on T cells. Since mAb against different epitopes can mimic the physiological ligands of cell receptors, the results obtained in this study raise again the question of the natural ligands of the CD45 molecule. The extracellular diversity of this molecule on different cell types allows us to assume that they are able to bind different ligands. Moreover, the fact that CD45 is a highly glycosylated molecule the glycosylation pattern of which changes according to the cell lineages and the activation state of the cell induces us to postulate that the carbohydrate structures play a role in the function of the CD45 molecule. The recently described inhibition of binding between NK cells and their targets by anti-CD45 mAb recognizing the carbohydrate residues of the CD45 molecule [27] supports the hypothesis that they are involved in the cell to cell interaction as has been recently suggested [28].The peculiar effect of the 136-4B5mAb on B cells described in our study also favors this suggestion. Taking into account that CD45 has a tyrosine phosphatase activity and the recent direct involvement of the CD45 molecule in T cell proliferation [28], it could be assumed that the effects of the different anti-CD45 mAb on B cell proliferation are related in some way with its phosphatase activity. The fact that in T cells, CD45 phosphotyrosine phosphatase acts on the T cell tyrosine protein kinase pp56ICk[7], which is not present on B cells, allows us to assume that CD45 phosphatase activity on B cells should act on other tvrosine Drotein kinase. In anv case. it can not be ruled out' that [he mechanisms thaf inhibit B cell
proliferation could be different from the ones inducing an increase of the proliferation. We thank Dr. 0.Viiias and J. J. Barcelo for their advice and practical help on flow cytometric analysis. Received May 7, 1990; in revised form July 16, 1990.
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