BRIEF COMMUNICATION
Immunology 1991 74 561-563
Soluble CD23 displays T-cell growth enhancing activity J.-C. LECRON, F. MOREL, J. TANZER, J. GOMBERT & P. GOUBE DE LAFOREST URA C.N.R.S. 1338, Hopital La Miletrie, Poitiers, France Acceptedfor publication 8 July 1991
SUMMARY Soluble CD23 (sCD23) enhances, in a dose-dependent manner, the number of secondary T-cell colonies generated by peripheral blood-derived agar T-colony cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2). This effect is not affected by IL-I or IL-4 but is abolished by an anti-CD23 monoclonal antibody (mAb) or by IgE. No colonies were observed when sCD23 was added to PHA- or IL-2-free cultures. sCD23 also enhanced the cloning frequency of primary T-colony cells in a limiting dilution assay. These data provide the first direct evidence that sCD23 recruits T-cell clones in peripheral blood-born T cells and may be involved indirectly in the regulation of IgE response.
The 45,000 MW CD23 surface antigen was first described as a B-cell restricted activation antigen found at high levels on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and at a low density on normal B cells.' Later on, CD23 was identified as the low affinity receptor for IgE (FcERII).2 A 25,000-27,000 MW soluble form of the CD23 molecule is spontaneously shed from the surface of lymphoblastoid or activated normal B cells.3'34 sCD23 is analogous to Bcell-derived IgE binding factor, and still reacts with CD23
mAb.5 Interestingly, an expanding range of biological activities has been ascribed to sCD23, such as the control of human IgE synthesis,3'6 the regulation of B-lymphocyte differentiation and proliferation,7 and the enhancement of tonsil but not peripheral blood T-cell proliferation.8 On the other hand, we have reported previously that culture supernatants (SN) from B lympho-
cytes9"10 can support in vitro differentiation of bone marrowderived CD7+, 2- 3-, 4-, 6-, 8- precursors. This activity is therefore referred to as prothymocyte differentiating activity (PTDA)."12 PTDA-containing SN were also shown to enhance the growth of secondary T-cell colonies from peripheral bloodborn primary colony cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2).'2"13 Subsequent observations from our laboratory revealed striking biochemical and cell-source homologies between B-cellderived PTDA'2 and sCD23, leading to the hypothesis of their identity. In support of this view, we were able to show that sCD23 can promote thymocyte maturation provided IL-I is added to the cultures.'4 In the present investigation, we have Correspondence: Dr J.-C. Lecron, Centre de Recherche sur les Maladies du Sang, URA CNRS 1338, H6p. La Miltrie, 86021 Poitiers, Cedex, France.
561
attempted to determine whether sCD23 displays a T-cell enhancing activity in the presence of PHA and IL-2. Ficoll-isolated peripheral blood mononuclear cells (PBMC) were obtained from normal human volunteers after informed consent. Mass cultures of PHA-induced (PHA-M; Difco Laboratories, Detroit, MI) primary agar T-cell colonies were grown from PBMC, as described previously.'3"5 Seven days later, colonies growing at the surface of the agar were harvested, pooled and resuspended in RPMI-1640 with 15% autologous plasma and 0-3% agar. Fifty microlitres of this suspension (5 x 104 cells) were layered onto 200 MI underlayers containing cytokines at various dilutions, 20 pg PHA and 0-5% agar, in 24-well microplates (Costar, Cambridge, MA). Colonies of more than 50 cells were scored after 7 days of incubation. Results were compared using the Wilcoxon test for paired data. Recombinant sCD23, a generous gift from Dr Hofstetter (Ciba-Geigy, Basel, Switzerland), was expressed, as described elsewhere,5 in Chinese hamster ovary (CHO) cells after stable transformation with cDNA coding for the FcERII of the human B-lymphoblastoid cell line RPMI-8866 cloned into a mammalian expression vector. The rsCD23 was purified by affinity chromatography from CHO-transformed culture SN. It had a molecular weight of 25,000 and an amino acid sequence identical to native sCD23. SN from CHO cells transfected with a plasmid deleted from CD23 active domain (amino acid 1-198) was used as negative control. Human rIL-2 (specific activity 3 x 106 U/mg) was kindly provided by Eurocetus (Amsterdam, The Netherlands), HrIL-l (1-3 x 107 U/mg) by Dr Shaw (Biogen, Geneva, Switzerland) and HrIL-4 (5-6 x 106 U/ml) by Drs Banchereau and de Vries (Unicet, Dardilly, France). IgE was obtained by (NH4)2SO4 precipitation of the serum from a patient with IgE myeloma. The CD23 monoclonal antibody (mAb) IOB8 (IgGI K) (Immunotech, Marseille, France) and, as
562 160 -
J.-C. Lecron et al. (0)
C1 In
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100 80 sCD23 ->v 60 50 A 40 4-f 30 c
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IgE:1/27
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sCD23+IgE: 1/22
25 o
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40
20
30
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control, an anti-prostate specific antigen mAb, ER-PR8 (IgG IK), a gift of Dako (Versailles, France), were used in this study. Primary colony cells were also cultured at limiting dilution in RPMI-1640 supplemented with 20% autologous serum, 100 pg/ ml PHA, 20 U/ml IL-2 with or without 15 ng/ml sCD23. Onehundred microlitre aliquots containing 1-32 cells were transferred to 96-well culture plates containing 2 x 103 irradiated (40 Gy) autologous colony cells. Estimated frequency was obtained by fitting experimental data of positive (growing) and negative (non-growing) wells. Phenotypic analysis of primary and secondary T-colony cells was performed using the following mAb: T4 (CD4), T8 (CD8), B4 (CD 19), B6 (CD23), purchased from Coulter (Coulter Electronics, Hialeh, FL), R8 II (CD25) from Dako and WT31 (TcR ax) from Becton-Dickinson (Grenoble, France). These mAb were conjugated with FITC or phycoerythrine for double fluorescence analysis, using a Coulter EPICS ELIT flow
18737575
.lj
150
sCD23 (ng/ml)
Cells cultured /well
Figure 1. sCD23 enhances the cloning capacity of MNC-derived T-colony cells. (a) Plated in agar medium in the presence of PHA 1% and 2 U/ml IL-2 with (-) or without (0) 75 ng/ml sCD23 (-: mean + SEM of seven experiments). (b) Plated at limiting dilution in the presence of PHA 1% and 20 U/ml IL-2 with or without 15 ng/ml sCD23 and/or 2500 U/ml IgE.
n
-
0 05
2 8 32 128 IL-2 (U/ml)
Figure 2. The effect of sCD23 on MNC-derived T-colony cells is dosedependent and distinct from IL-2. MNC-derived T-colony cells were plated in agar medium. (a) In the presence of various concentrations of sCD23 with (-) or without (0) 2 U/ml IL-2. (b) In the presence of various concentrations of IL-2 with (-) or without (0) 75 ng/ml sCD23 (data are the mean + SD of one of four similar experiments).
a
Control sCD23 Anti-CD23 sCD23 + onti -CD23 sCD23+IgE 250 U/ml sCD23 + IgE 2500 U/ml i sCD23 +IgE 25,000 U/ml IgE 250 U/ml IgE 2500 U/ml
H
lI
IgE 25,000 U/ml 0
20
40 60 80 100 120 140 160 180 Colonies / 50,000 cel Is plated Figure 3. IgE or CD23 mAb inhibits enhancing action of sCD23. MNCderived T-colony cells were plated in agar medium with PHA 1%, IL-2 (2 U/ml) with or without sCD23 (15 ng/ml), anti-CD23 (2 pg/ml) and IgE (250-25,000 U/ml) (data are the means + SD of one of four similar
experiments).
cytometer.
As shown in Fig. 1, sCD23 enhanced secondary T-cell colony growth. Pooled cells from PBMC-born primary T-cell colonies were replated in agar onto underlayers supplemented with PHA, IL-2 (2 U/ml) and sCD23 (75 ng/ml); sCD23-free cultures served as controls. A significant (P < 0-025) increase in the number of secondary colonies obtained was observed in sCD23-supplemented cultures (Fig. Ia). In the limiting dilution assay (Fig. I b) sCD23, when added to PHA- and IL-2containing cultures, increased from 1/28 to 1/14 of the cloning frequency of primary T-colony cells. In contrast, sCD23 did not affect primary T-cell colony formation from PBMC (data not shown), in agreement with a previous report.8 This failure could be due to endogenous release of sCD23 by PHA-stimulated B cells upon PBMC plating in primary cultures, which may mask the effect of exogenous sCD23. In support of this possibility, although adding CD23 mAb to primary cultures failed to depress primary colony formation by PBMC, we found that the SN from PHA-stimulated B cells, but not T cells, yielded significant amounts of sCD23. Furthermore, exogenous sCD23 induced a 30-40% increase in the number of colonies grown from PHA and IL-2 stimulated T cells (E-rosette positive) (data not shown). The effect of sCD23 on secondary colony formation is dosedependent. Exogenous sCD23 increased the number of colonies obtained at concentrations between 5 and 20 ng/ml (Fig. 2a)
similarly to those reported for its differentiating activity on T-cell precursor.' Moreover, sCD23 and IL-2 exerted distinct effects on colony formation. While the number of secondary colonies in PHAand IL-2-containing cultures reached a plateau around 32 U/ml IL-2, sCD23 (75 ng/ml) still increased the number of colonies above this plateau (Fig. 2b). In contrast, sCD23 did not elicit any significant colony growth in PHA- and/or IL-2-free cultures.
The specificity of the effect exerted by sCD23 is documented in Fig. 3, which shows that a CD23 mAb (but not an isotyperelated control mAb, ER-PR8) abolishes the rise in colony number induced by sCD23 while not affecting the number of colonies grown in the presence of PHA and IL-2 alone. Interestingly, IgE could also inhibit, in a dose-dependent manner, the enhancing effect of sCD23 (Fig. 3), without affecting colony growth in control sCD23-free cultures. In the limiting dilution assay, sCD23 growth enhancing activity was in the most part reversed by IgE (Fig. I b). Because of previous studies suggesting that the capacity of sCD23 to induce human thymocyte differentiation depends on the presence of IL-1,'4 we tested the effect of IL-1 in the secondary colony assay. IL- 1 alone increased the plating efficiency of primary T-colony cells stimulated with PHA and
T-cell growth enhancement IL-2. However, when added to sCD23-supplemented cultures, IL-I either enhanced, depressed or failed to affect secondary colony formation (data not shown). On the other hand, IL-4, which is known to induce both expression and production of sCD23, IgE synthesis and T-cell proliferation,34 did not modulate the action of sCD23 on T-cell colonies. While IL-4 increased the number of colonies obtained with IL-2 and PHA, this effect was not reversed by CD23 mAb, nor did sCD23 display any synergistic effect in association with IL-4 (data not shown). So far, we were unable to detect any significant difference in the phenotype of T-colony cells upon secondary culture with or without sCD23, respectively, as judged in five different experiments, by the following cell markers: CD4 (62 + 4% versus 63 +4%), CD8 (43+3 versus 41 +2), CD4-CD8 (9±+1 versus 11 + 1), TcRafl (85 + 4 versus 87 + 3), CD25 (67 + 7 versus 72 + 5), CDl9 ( < 1 versus < 1). However, in two out of the five experiments, an increase (two- to four-fold) in CD4-CD23 coexpression was observed on cells stimulated with sCD23 (data not shown). These results are in keeping with controversial data reported previously.3 8.16 Mitogens were found to induce CD23 expression on tonsil but not peripheral blood T cells,8 while CD4-CD23 co-expression on peripheral lymphocytes induced by antigens that cause an IgE response has been documented.'6 Taken together, our results suggest that sCD23 recruits T cells to become activated and proliferate in the presence of PHA and IL-2. This mechanism may indirectly contribute to the regulation of the IgE response, given that sCD23 seems to be also directly involved in B-cell differentiation.367
ACKNOWLEDGMENTS This work was supported by 'Fondation contre la leucemie' (grant no. 871878), 'Fondation pour la Recherche Medicale' and 'Association pour la Recherche contre le Cancer' (ARC) (grant no. 6535). The technical assistance of Miss D. Pelletier and the secretarial assistance of Miss M. T. Peguin were appreciated. We also thank Dr A. Brizard for help in FACS analysis.
REFERENCES 1. GORDONJ., FORES-ROMOL.,CAIRNSJ.A.,MILLSUMM.J., LANE P.J., JOHNSON G.D. & MCLENAN I.C.M. (1989) CD23: a multi-functional receptor/lymphokine? Immunol. Today, 10, 153.
563
2. BONNEFOY J.Y., AUBRY J.P., PERONNE C., WIFDENES J. & BANCHEREAU J. (1987) Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD23 is a low affinity receptor for IgE. J. Immunol. 138, 2970. 3. DELESPESSE G., SARFATI M. & HOFSTETTER H. (1989) Human IgEbinding factors. Immunol. Today, 10, 159. 4. DEFRANCE T., AUBRY J.P., ROUSSET F., VANBERVLIET B., BONNEFOY J.Y., ARAI N. et al. (1987) Human recombinant interleukin 4 induces FcE receptors (CD23) on normal human B lymphocytes. J. exp. Med. 165,1459. 5. LUDIN C., HOFSTETTER H., SARFATI M., LEVY C.A., SUTER U., ALAIMO D., KILCHERR G., FROST H. & DELESPESSE G. (1987) Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor. EMBO J. 6, 109. 6. PENE J. (1989) Regulatory role of cytokines and CD23 in the human IgE antibody synthesis. Int. Arch. Allergy appl. Immunol. 90, 32. 7. ARMITAGE R.J. & GOFF L.K. (1988) Functional interaction between B cell populations defined by CD23 expression. Eur. J. Immunol. 18, 1753. 8. ARMITAGE R.J., GOFF L.K. & BEVERLEY P.C.L. (1989) Expression and functional role of CD23 on T cells. Eur. J. Immunol. 19, 31. 9. MOSSALAYI M.D., LECRON J.C., GOUBE DE LAFOREST P., JANOSSY G., DEBRE P. & TANZER J. (1988) Characterization of prothymocytes with cloning capacity in human bone marow. Blood, 71, 1281. 10. LECRON J.C., MOSSALAYI M.D., TANZER J., GOMBERT J. & GOUBE DE LAFOREST P. (1988) Signal requirements for the generation of Tlymphocytes from depleted bone marrow cells: a sequential study. E.xp. Hematol. 17, 785. 11. MOSSALAYI M.D. & GOUBE DE LAFOREST P. (1985) B+null cell derived human prothymocytes differentiating activity. In: Cellular and Molecular Biology of Lymphokines (ed. C. Sorg), p. 509. Academic Press. 12. LECRON J.C., MOSSALAYI M.D., SUTTON L., GOMBERT J., TANZER J. & GOUBE DE LAFOREST P. (1988) B+ null cell-derived prothymocyte differentiating activity (PTDA): biological properties and physicochemical characterization. In: Lymphocyte Activation and Differentiation (eds J. C. Mani and J. Dornand), p.297. Walter de Gruyter & Co., Berlin, New York. 13. MOSSALAYI M.D., GOUBE DE LAFOREST P., GUILHOT F., KALLIL G., NTYANE E., LARROQUE V., FELLOUS M. & TANZER J. (1985) Agar human T cell colony growth promoted by a B+ null cell-derived lymphokine distinct from IL2. J. Immunol. 134, 2400. 14. MOSSALAYI M.D., LECRON J.C., DALOUL A.H., SARFATI M., BERTHO J.M., HOFSTETTER H., DELESPESSE G. & DEBRE P. (1990) Soluble CD23 (FcERII) and interleukin I synergistically induce early human thymocyte maturation. J. erp. Med. 171, 959. 15. GOUBE DE LAFOREST P., THOMAS P., PELLETIER D. & TANZER J. (1979) Studies on cell interactions using a micromethod for growing T-lymphocyte colonies in agar culture. Immunology, 38, 561. 16. PRINZ J.C., BAUR X., MAZUR G. & RIEBER E.P. (1990) Allergendirected expression of Fc receptors for IgE (CD23) on human T lymphocytes is modulated by interleukin 4 and interferon-T. Eur. J. Immunol. 20, 1259.
Immunology 1991 74 561-563
Soluble CD23 displays T-cell growth enhancing activity J.-C. LECRON, F. MOREL, J. TANZER, J. GOMBERT & P. GOUBE DE LAFOREST URA C.N.R.S. 1338, Hopital La Miletrie, Poitiers, France Acceptedfor publication 8 July 1991
SUMMARY Soluble CD23 (sCD23) enhances, in a dose-dependent manner, the number of secondary T-cell colonies generated by peripheral blood-derived agar T-colony cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2). This effect is not affected by IL-I or IL-4 but is abolished by an anti-CD23 monoclonal antibody (mAb) or by IgE. No colonies were observed when sCD23 was added to PHA- or IL-2-free cultures. sCD23 also enhanced the cloning frequency of primary T-colony cells in a limiting dilution assay. These data provide the first direct evidence that sCD23 recruits T-cell clones in peripheral blood-born T cells and may be involved indirectly in the regulation of IgE response.
The 45,000 MW CD23 surface antigen was first described as a B-cell restricted activation antigen found at high levels on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and at a low density on normal B cells.' Later on, CD23 was identified as the low affinity receptor for IgE (FcERII).2 A 25,000-27,000 MW soluble form of the CD23 molecule is spontaneously shed from the surface of lymphoblastoid or activated normal B cells.3'34 sCD23 is analogous to Bcell-derived IgE binding factor, and still reacts with CD23
mAb.5 Interestingly, an expanding range of biological activities has been ascribed to sCD23, such as the control of human IgE synthesis,3'6 the regulation of B-lymphocyte differentiation and proliferation,7 and the enhancement of tonsil but not peripheral blood T-cell proliferation.8 On the other hand, we have reported previously that culture supernatants (SN) from B lympho-
cytes9"10 can support in vitro differentiation of bone marrowderived CD7+, 2- 3-, 4-, 6-, 8- precursors. This activity is therefore referred to as prothymocyte differentiating activity (PTDA)."12 PTDA-containing SN were also shown to enhance the growth of secondary T-cell colonies from peripheral bloodborn primary colony cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2).'2"13 Subsequent observations from our laboratory revealed striking biochemical and cell-source homologies between B-cellderived PTDA'2 and sCD23, leading to the hypothesis of their identity. In support of this view, we were able to show that sCD23 can promote thymocyte maturation provided IL-I is added to the cultures.'4 In the present investigation, we have Correspondence: Dr J.-C. Lecron, Centre de Recherche sur les Maladies du Sang, URA CNRS 1338, H6p. La Miltrie, 86021 Poitiers, Cedex, France.
561
attempted to determine whether sCD23 displays a T-cell enhancing activity in the presence of PHA and IL-2. Ficoll-isolated peripheral blood mononuclear cells (PBMC) were obtained from normal human volunteers after informed consent. Mass cultures of PHA-induced (PHA-M; Difco Laboratories, Detroit, MI) primary agar T-cell colonies were grown from PBMC, as described previously.'3"5 Seven days later, colonies growing at the surface of the agar were harvested, pooled and resuspended in RPMI-1640 with 15% autologous plasma and 0-3% agar. Fifty microlitres of this suspension (5 x 104 cells) were layered onto 200 MI underlayers containing cytokines at various dilutions, 20 pg PHA and 0-5% agar, in 24-well microplates (Costar, Cambridge, MA). Colonies of more than 50 cells were scored after 7 days of incubation. Results were compared using the Wilcoxon test for paired data. Recombinant sCD23, a generous gift from Dr Hofstetter (Ciba-Geigy, Basel, Switzerland), was expressed, as described elsewhere,5 in Chinese hamster ovary (CHO) cells after stable transformation with cDNA coding for the FcERII of the human B-lymphoblastoid cell line RPMI-8866 cloned into a mammalian expression vector. The rsCD23 was purified by affinity chromatography from CHO-transformed culture SN. It had a molecular weight of 25,000 and an amino acid sequence identical to native sCD23. SN from CHO cells transfected with a plasmid deleted from CD23 active domain (amino acid 1-198) was used as negative control. Human rIL-2 (specific activity 3 x 106 U/mg) was kindly provided by Eurocetus (Amsterdam, The Netherlands), HrIL-l (1-3 x 107 U/mg) by Dr Shaw (Biogen, Geneva, Switzerland) and HrIL-4 (5-6 x 106 U/ml) by Drs Banchereau and de Vries (Unicet, Dardilly, France). IgE was obtained by (NH4)2SO4 precipitation of the serum from a patient with IgE myeloma. The CD23 monoclonal antibody (mAb) IOB8 (IgGI K) (Immunotech, Marseille, France) and, as
562 160 -
J.-C. Lecron et al. (0)
C1 In
20 H
ur 80
100 80 sCD23 ->v 60 50 A 40 4-f 30 c
a} 40
lo
0
Ctrolol
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IgE:1/27
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control, an anti-prostate specific antigen mAb, ER-PR8 (IgG IK), a gift of Dako (Versailles, France), were used in this study. Primary colony cells were also cultured at limiting dilution in RPMI-1640 supplemented with 20% autologous serum, 100 pg/ ml PHA, 20 U/ml IL-2 with or without 15 ng/ml sCD23. Onehundred microlitre aliquots containing 1-32 cells were transferred to 96-well culture plates containing 2 x 103 irradiated (40 Gy) autologous colony cells. Estimated frequency was obtained by fitting experimental data of positive (growing) and negative (non-growing) wells. Phenotypic analysis of primary and secondary T-colony cells was performed using the following mAb: T4 (CD4), T8 (CD8), B4 (CD 19), B6 (CD23), purchased from Coulter (Coulter Electronics, Hialeh, FL), R8 II (CD25) from Dako and WT31 (TcR ax) from Becton-Dickinson (Grenoble, France). These mAb were conjugated with FITC or phycoerythrine for double fluorescence analysis, using a Coulter EPICS ELIT flow
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sCD23 (ng/ml)
Cells cultured /well
Figure 1. sCD23 enhances the cloning capacity of MNC-derived T-colony cells. (a) Plated in agar medium in the presence of PHA 1% and 2 U/ml IL-2 with (-) or without (0) 75 ng/ml sCD23 (-: mean + SEM of seven experiments). (b) Plated at limiting dilution in the presence of PHA 1% and 20 U/ml IL-2 with or without 15 ng/ml sCD23 and/or 2500 U/ml IgE.
n
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0 05
2 8 32 128 IL-2 (U/ml)
Figure 2. The effect of sCD23 on MNC-derived T-colony cells is dosedependent and distinct from IL-2. MNC-derived T-colony cells were plated in agar medium. (a) In the presence of various concentrations of sCD23 with (-) or without (0) 2 U/ml IL-2. (b) In the presence of various concentrations of IL-2 with (-) or without (0) 75 ng/ml sCD23 (data are the mean + SD of one of four similar experiments).
a
Control sCD23 Anti-CD23 sCD23 + onti -CD23 sCD23+IgE 250 U/ml sCD23 + IgE 2500 U/ml i sCD23 +IgE 25,000 U/ml IgE 250 U/ml IgE 2500 U/ml
H
lI
IgE 25,000 U/ml 0
20
40 60 80 100 120 140 160 180 Colonies / 50,000 cel Is plated Figure 3. IgE or CD23 mAb inhibits enhancing action of sCD23. MNCderived T-colony cells were plated in agar medium with PHA 1%, IL-2 (2 U/ml) with or without sCD23 (15 ng/ml), anti-CD23 (2 pg/ml) and IgE (250-25,000 U/ml) (data are the means + SD of one of four similar
experiments).
cytometer.
As shown in Fig. 1, sCD23 enhanced secondary T-cell colony growth. Pooled cells from PBMC-born primary T-cell colonies were replated in agar onto underlayers supplemented with PHA, IL-2 (2 U/ml) and sCD23 (75 ng/ml); sCD23-free cultures served as controls. A significant (P < 0-025) increase in the number of secondary colonies obtained was observed in sCD23-supplemented cultures (Fig. Ia). In the limiting dilution assay (Fig. I b) sCD23, when added to PHA- and IL-2containing cultures, increased from 1/28 to 1/14 of the cloning frequency of primary T-colony cells. In contrast, sCD23 did not affect primary T-cell colony formation from PBMC (data not shown), in agreement with a previous report.8 This failure could be due to endogenous release of sCD23 by PHA-stimulated B cells upon PBMC plating in primary cultures, which may mask the effect of exogenous sCD23. In support of this possibility, although adding CD23 mAb to primary cultures failed to depress primary colony formation by PBMC, we found that the SN from PHA-stimulated B cells, but not T cells, yielded significant amounts of sCD23. Furthermore, exogenous sCD23 induced a 30-40% increase in the number of colonies grown from PHA and IL-2 stimulated T cells (E-rosette positive) (data not shown). The effect of sCD23 on secondary colony formation is dosedependent. Exogenous sCD23 increased the number of colonies obtained at concentrations between 5 and 20 ng/ml (Fig. 2a)
similarly to those reported for its differentiating activity on T-cell precursor.' Moreover, sCD23 and IL-2 exerted distinct effects on colony formation. While the number of secondary colonies in PHAand IL-2-containing cultures reached a plateau around 32 U/ml IL-2, sCD23 (75 ng/ml) still increased the number of colonies above this plateau (Fig. 2b). In contrast, sCD23 did not elicit any significant colony growth in PHA- and/or IL-2-free cultures.
The specificity of the effect exerted by sCD23 is documented in Fig. 3, which shows that a CD23 mAb (but not an isotyperelated control mAb, ER-PR8) abolishes the rise in colony number induced by sCD23 while not affecting the number of colonies grown in the presence of PHA and IL-2 alone. Interestingly, IgE could also inhibit, in a dose-dependent manner, the enhancing effect of sCD23 (Fig. 3), without affecting colony growth in control sCD23-free cultures. In the limiting dilution assay, sCD23 growth enhancing activity was in the most part reversed by IgE (Fig. I b). Because of previous studies suggesting that the capacity of sCD23 to induce human thymocyte differentiation depends on the presence of IL-1,'4 we tested the effect of IL-1 in the secondary colony assay. IL- 1 alone increased the plating efficiency of primary T-colony cells stimulated with PHA and
T-cell growth enhancement IL-2. However, when added to sCD23-supplemented cultures, IL-I either enhanced, depressed or failed to affect secondary colony formation (data not shown). On the other hand, IL-4, which is known to induce both expression and production of sCD23, IgE synthesis and T-cell proliferation,34 did not modulate the action of sCD23 on T-cell colonies. While IL-4 increased the number of colonies obtained with IL-2 and PHA, this effect was not reversed by CD23 mAb, nor did sCD23 display any synergistic effect in association with IL-4 (data not shown). So far, we were unable to detect any significant difference in the phenotype of T-colony cells upon secondary culture with or without sCD23, respectively, as judged in five different experiments, by the following cell markers: CD4 (62 + 4% versus 63 +4%), CD8 (43+3 versus 41 +2), CD4-CD8 (9±+1 versus 11 + 1), TcRafl (85 + 4 versus 87 + 3), CD25 (67 + 7 versus 72 + 5), CDl9 ( < 1 versus < 1). However, in two out of the five experiments, an increase (two- to four-fold) in CD4-CD23 coexpression was observed on cells stimulated with sCD23 (data not shown). These results are in keeping with controversial data reported previously.3 8.16 Mitogens were found to induce CD23 expression on tonsil but not peripheral blood T cells,8 while CD4-CD23 co-expression on peripheral lymphocytes induced by antigens that cause an IgE response has been documented.'6 Taken together, our results suggest that sCD23 recruits T cells to become activated and proliferate in the presence of PHA and IL-2. This mechanism may indirectly contribute to the regulation of the IgE response, given that sCD23 seems to be also directly involved in B-cell differentiation.367
ACKNOWLEDGMENTS This work was supported by 'Fondation contre la leucemie' (grant no. 871878), 'Fondation pour la Recherche Medicale' and 'Association pour la Recherche contre le Cancer' (ARC) (grant no. 6535). The technical assistance of Miss D. Pelletier and the secretarial assistance of Miss M. T. Peguin were appreciated. We also thank Dr A. Brizard for help in FACS analysis.
REFERENCES 1. GORDONJ., FORES-ROMOL.,CAIRNSJ.A.,MILLSUMM.J., LANE P.J., JOHNSON G.D. & MCLENAN I.C.M. (1989) CD23: a multi-functional receptor/lymphokine? Immunol. Today, 10, 153.
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