Activated B cells express receptors for, and proliferate in response to, pure interleukin 2.

In this study we investigated whether interleukin 2 (IL-2) acts on B cell proliferation and whether activated B cells express IL-2 receptors. First, the functional activity of immunoaffinity-purified or recombinant human IL-2 was studied in a B blast assay using positively selected murine surface Ig-positive cells that had been activated by lipopolysaccharide (LPS) plus anti-Ig antibodies (anti-Ig). In this assay, T cells were not detected by fluorescence-activated cell sorter analysis. It was found that both IL-2 preparations led to optimal B cell proliferation compared with supernatants obtained from murine or human spleen cells or murine cloned T helper cells. Second, we observed that the IL-2 requirement in this assay was about the same as in a proliferation assay using lectin-activated polyclonal murine Lyt-2- positive T cells. Third, analysis of the binding of radiolabeled immunoaffinity-purified IL-2 to B cells indicated that LPS plus anti-Ig- activated B cells expressed a mean of 3,500 IL-2 receptors per cell with an apparent dissociation constant of 150 pM. However, neither nonactivated B cells nor B cells activated by LPS alone exhibited significant specific IL-2 binding. The functional and the receptor data are consistent with the conclusion that IL-2 is a growth factor not only for T cells but also for B cells.

alone had no significant effect, B cells activated by LPS and anti-Ig together proliferated 10-fold better in response to T cell SN than B cells activated by LPS alone (25). Those results suggest that some previously reported B cell growth factors (BCGF) could have (directly or via other cells) participated in B cell activation but may be different from the essential growth factors acting on activated B cells.
In this study it was investigated whether IL-2 acts on B cell proliferation and whether activated B cells express IL-2 receptors. The results demonstrated (a) that either recombinant or immunoaffinity-purified human IL-2 stimulated the proliferation of LPS plus anti-Ig-activated purified murine B cells to the same extent as various T cell and spleen cell SN, (b) that very similar IL-2 concentrations were required for B vs. T cell proliferation, and (c) that LPS plus anti-Igactivated B cells expressed IL-2 receptors.
Culture Media, General Culture Methods. Cell cultures were performed in Dulbecco's modified Eagle's medium (DME) supplemented with additional amino acids and including 10 mM Hepes, penicillin-streptomycin, and 5 x 10 -5 M 2-mercaptoethanol (26). Preparation and Immunohistochemical Analysis of B Cells. Surface Ig (sIg)-positive cells were obtained from C57BL/6 spleen cells by the panning method of Mage et al. (27). The efficiency of this method was checked by the quantitation of sIg + and Thy-1 ÷ cells on acetone-fixed cytocentrifuge smears using biotinylated horse anti-mouse Ig antibody (Vector Laboratories, Inc., Burlingame, CA) or biotinylated monoclonal anti-Thy-1 antibody (AT-15, provided by Dr. F. Fitch, University of Chicago) together with avidinperoxidase conjugate (Vector Laboratories, Inc.) and hematoxylin counterstaining. 3,000 cells were scored. Freshly prepared B cells contained >97% sIg + cells and <0.3% Thy-1 ÷ cells. Control cells (Con A-stimulated thymocytes) were >98% Thy-1 +. In addition, it was found that B cells cultured during 4 d in the presence of LPS, anti-Ig, and EL-4 SN (see below) contained >99% Ig-positive cells and <0.2% Thy-1 + cells.
Murine B Blast Assay for B Cell Growth Factor. To obtain activated B cells, 3 x 10 ~ sIg + cells were cultured during 3 d in 1-ml cultures in the presence of 5 #g/ml of LPS, 10 #g/ ml of Sepharose-anti-mouse Ig, and 10% FCS, followed by removal of anti-Ig beads from the culture suspension by sedimentation (10 min at 1 g), centrifugation of the cells, and resuspension in fresh DME. Assay cultures (150 /zl, triplicates) were set up with 104 activated B cells, 10% FCS, and dilutions of tested supernatants. Thymidine incorporation was measured 3 d later (by standard procedure, using an automated cell harvester), after a 6-h pulse with 1 #Ci of [3H]thymidine (Radiochemical Centre, Amersham, England) added in 50 ~1 of DME.
Analysis and Separation of Cells with the Fluorescence-activated Cell Sorter (FACS). A FACS II (Beckton, Dickinson & Co., Mountain View, CA) was used in conjunction with monoclonal anti-Thy-I and anti-Lyt-2 antibodies coupled to biotin, with fluoresceinated avidin as a second reagent as described (31,32).
IL-2 Assay Using CTLL Cells. IL-2 biologic activity was determined by the IL-2dependent stimulation of proliferation of the CTLL-2 murine cytotoxic T lymphocyte line (8). CTLL cells (4 x 10S/culture of 200 #1) were cultured during 24 h, in the presence of DME-5% FCS and serial dilutions of tested SN. During the last 5 h, the cultures were pulsed with 1 #Ci of [SH]thymidine. The IL-2 concentration leading to 50% maximal thymidine incorporation was assigned a value of 1 U/ml. Thus a sample containing 100 U IL-2/ml would give 50% maximal stimulation at a dilution of 1:100 in the assay.
Immunoaffinity-purif~ed Human IL-2. IL-2-containing SN were obtained from a subclone (6.8 b) of the IL-2 high-producer Jurkat 6.8 human T leukemia cell line in the absence of serum and the IL-2 purified on an immunosorbent column as described (8). Affi-gel 10 beads coupled with DMS-3 monocional anti-IL-2 antibodies were provided by Dr. K. A. Smith, Dartmouth, NH. The specific activity of the affinity-purified IL-2 was 1 U (see CTLL assay) per 0.3 ng protein. A single stained band of 15,000 tool wt was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Recombinant human IL-2. The construction, cloning, and screening of a human IL-2 cDNA plasmid made from polyadenylated RNA isolated from PHA-stimulated human spleen cells and its expression in E. eoli has been described (11). E. coli-derived recombinant IL-2 was purified to apparent homogeneity according to SDS-PAGE and freed of endotoxin (methods from Biogen SA, Geneva, Switzerland). The specific activity of the recombinant IL-2 was also 1 U/0.3 ng protein.
[SH]IL-2-binding Assay. Biosynthetically radiolabeled IL-2 was prepared (5). Briefly, Jurkat cells were cultured in the presence of DME with 40 #M leucine and lysine (i.e., 5% of the levels in regular DME) and 10 mCi of both [SH]leucine (160 Ci/mM; Amersham Corp., Arlington Heights, IL) and [nH]iysine (85 Ci/mM; Amersham Corp.). ~ The radiolabeled IL-2 was immunoaffinity purified (8), tested for biological activity, and adjusted to 500 cpm/U IL-2 (25,000 cpm/pmol). The cell-binding assay was performed as described by Robbet al. (5). The LPS plus anti-Ig-activated B cells to be tested were obtained as follows. Cultures (250 #1) were set up with 3 x 104 C57BL/6 sIg + cells, 50 #g/ml LPS, 10 #g/ml Sepharose-anti-Ig, and 20% EL-4 SN (see below) in DME-10% FCS (using four complete 96-well plates). After 4 d, the cells were harvested and the anti-Ig beads removed, first by sedimentation (10 min at 1 g), and the remaining beads (~5%) by centrifugation (10 rain at 850 g) of the cells (15 x 106 cells in 5 ml DME) on top of Ficoll (3 ml, density 1.077; Seromed) in 10-ml tubes. By microscopic counting it was found that all beads and also the dead cells sedimented to the bottom of the tubes whereas the activated B cells remained at the interface. The B cells had multiplied 10 times during the 4-d culture period and 75% of the viable cells were recovered after the sedimentation and centrifugation on Ficoll. LPS-activated B cells were obtained in the same way, i.e., the cultures also included 20% EL-4 SN. Con A-activated T cells were obtained by culturing C57BL/ 6 spleen cells during 4 d in the presence of 1 •g/ml Con A and 20% EL-4 SN in DME-5% FCS. Before the binding assay, all cells were washed three times in DME and incubated for 2 h at 37°C to promote the removal of cell-bound IL-2. Aliquots of 5 x 106 cells (in 200 #1 DME) were incubated in the presence of [SH]IL-2 for 20 rain at 37°C, after which time 800 #1 of cold DME was added. Free and cell-bound [SH]IL-2 was determined as described (5).

Murine B Blast Assay for B Cell Growth
Factor. In a recent report (25) by one of us it was shown that LPS and polyvalent rabbit anti-mouse Ig antibodies coupled to Sepharose beads (anti-Ig) together induced optimal growth factor responsiveness of murine B cells in a T cell-depleted polyclonal B cell system. Based on these results, a quantitative B blast assay for BCGF has now been developed. To obtain activated B cells, positively selected sIg + cells were cultured (3 × 105/ml) during 3 d in the presence of anti-Ig (10 #g/ml) and LPS, followed by one cell wash and depletion of anti-Ig beads by sedimentation. A concentration of 5 #g/ml of LPS was found to be optimal with regard to the subsequent detection of BCGF. On day 3, the B cells had multiplied by a mean 2.5 times and become >90% large ceils according to FACS analysis. To further establish the optimal assay conditions, cultures (150 #1) were set up with different numbers of activated B cells suspended in fresh medium-10% FCS-10% EL-4 SN as a source of T cell factors (or control cultures without SN). 3 d later, thymidine incorporation was measured, after a 6-h pulse (see Fig. 1A). The highest stimulation index (cpm per test culture/cpm per control culture) was obtained in cultures with 104 activated B cells. This cell number was thus routinely used. It was also found in cultures with 104 B cells that the stimulation index was lower when thymidine incorporation was measured either on day 2 (because of a higher control response) or on day 4 (because of overgrowth of the cultures, data not shown). In confirmation of the immunohistochemical analysis of B cells (see Materials and Methods), T cells, i.e., Thy-1 + cells, were not detectable by the FACS in either the starting or the LPS/anti-Ig-activated B cell populations (not shown) or in the cell population recovered after the 3-d cultures performed in the presence of EL-4 SN (see Fig. 1 B).
Effects of lL-2 and Various T Cell SN in the B Blast Assay. Dose-response curves for the effects of IL-2 and different sources of T cell factors on thymidine incorporation in the B blast assay are shown in Fig. 2. Human IL-2, purified to apparent homogeneity according to SDS-PAGE analysis, was obtained either by immunoaffinity purification of IL-2 generated by Jurkat cells or by biochemical purification of recombinant IL-2 generated by E. coli (see Materials and Meth- ods). It was found that either affinity-purified or recombinant IL-2 supported B cell proliferation as well as (or better than) lectin-stimulated murine or human total spleen cell SN, murine MLC SN, or SN obtained from murine cloned T helper cells (TH-HY6) (25) and T cell hybridomas (FS6 and AOFS) (13,14,23,35). TH-HY6 provide themselves, in conjunction with anti-Ig, all signals required by B cells for a PFC response (25). Moreover, as can be seen in Fig. 2, there was a striking correlation between the IL-2 activity of different T cell SN and their activity in supporting B cell proliferation. Only some batches of EL SN (e.g., batch 22) but not others (e.g., batch 25) led to a two-to threefold higher maximum proliferative response than IL-2. It is not known whether this variation was due to the presence of other growth factors (19) or phorbol ester in certain batches of EL-4 SN. Finally, the SN from the monocytic cell line P388D1, which had a high IL-1 activity in a thymocyte proliferation assay (not shown) and was previously found (24) to strongly enhance T cell-dependent B cell activation, had absolutely no effect in the B blast assay when tested either alone or in conjunction with IL-2.
As shown in Fig. 3, the capacity of either recombinant IL-2, EL-4 SN, or murine MLC SN to support the generation of protein A-PFC in the B blast assay correlated with the capacity to support proliferation; i.e., the relationship between proliferation and differentiation into PFC was the same with different sources of T cell factors in this assay.

Comparison of lL-2 Requirements for the Proliferation of T Cells vs. B Cells. 1 U
of IL-2/ml was defined as the IL-2 concentration leading to 50% maximal thymidine incorporation in the 24-h assay using CTLL-2 cells. However, to compare the IL-2 requirements for the proliferation of normal, polyclonal T vs. B cell populations, a recently developed thymidine incorporation assay was used. 3 Cultures (200 pl) were set up with 2 x 103 FACS-selected Lyt-2 ÷ (murine) splenic T cells, leukoagglutinin, and dilutions of recombinant human IL-2. As shown in Fig. 4, the concentration of IL-2 required in this system for 50% maximal thymidine incorporation on day 3 of culture (6 U/ml) was identical with that required in the B blast assay. Moreover, it appears that an increase from 80 to 160 U IL-2/ml still led to an increased proliferative response when the thymidine incorporation was measured on day 5. A similar requirement for a high IL-2 concentration was found for B cell proliferation in cultures with 104 B cells, LPS, and anti-Ig that lasted 5 d (data not shown).

But these authors did not detect IL-2 receptors on LPS-activated murine B cells. In view of the results shown above, we performed this assay with B cells activated with LPS and anti-Ig for 4 d in the presence of EL-4 SN (LPS plus anti-Igactivated B cells). Nonactivated B cells or B cells cultured during 4 d in the presence of LPS and EL-4 SN but not anti-Ig (LPS-activated B cells) were also tested. Since B cells cultured in the presence of anti-Ig and EL-4 SN alone died rapidly (24), such cells could not be analyzed.
By using immunoaffinity-purified [SH]IL-2 at a concentration that was saturating for T cells, ~ we found (see Table I

) (a) that LPS plus anti-Ig-activated B cells bound [SH]IL-2 in a specific, coid-competable manner, (b) that the amount of [SH]IL-2 specifically bound by such activated B cells at equilibrium was, on a per cell basis, one-third of that bound by Con A-activated T cells, and (c) that nonactivated B cells (i.e., freshly prepared sIg + cells) did not bind [3H]IL-2.
Although anti-Ig beads were not detected by microscopic examination of LPS plus anti-Ig-activated B cells after Ficoll centrifugation, the possible influence of contaminating beads on the binding of [3H]IL-2 was also tested: 18 #1 of packed beads (corresponding to a 50% removal from cultured B blasts) were found to have no effect on the binding of [3H]IL-2 to nonactivated B or T cells (data not shown).
Next, an IL-2-binding curve was obtained by incubating LPS plus anti-Igactivated B cells in the presence of serial dilutions of [SH]IL-2 (Fig. 5A). The Scatchard plot established after correcting for nonspecific binding (Fig. 5B) indicated that thus-activated B cells expressed -3,500 specific binding sites per cell with an apparent dissociation constant of 150 pM. In addition, it was confirmed that B cells activated by LPS alone did not specifically bind IL-2 (see Fig. 5A).

Discussion
This report shows that IL-2 is not only a TCGF but also a BCGF, that both activities occur at very similar concentrations of this lymphokine, and that appropriately activated B cells express IL-2 receptors. In particular, it demonstrates that two independently derived human IL-2 preparations, i.e., recombinant IL-2 and immunoaffinity-purified IL-2, of apparent homogeneity and high specific TCGF activity (1 U/per 0.3 ng protein, where 1 U/ml gives 50% maximal thymidine incorporation in the CTLL-2 assay), exhibited identical BCGF activity. It is, therefore, unlikely that contaminating substances caused the effects on B cells. It is also highly unlikely that the effect of IL-2 in the murine B blast assay using LPS plus anti-Ig-activated B cells was due to the IL-2-stimulated generation of other factors by contaminating T cells (37). It was previously shown (25) that LPS plus anti-Ig-dependent B cell activation itself requires neither T helper cells nor macrophages and, in the present study, T cell contamination, not detectable by the FACS, was <0.3% when checked by immunohistochemical analysis. Furthermore, a variety of sources of T cell factors, including total spleen cell SN, cloned T helper cell SN (25), and T hybridoma SN (13,14,23,35), did not lead to stronger B cell proliferation than IL-2 alone. In fact, there appeared a striking correlation between the IL-2 TCGF activity and the BCGF activity of all tested supernatants. P388Dl SN had no effect in this assay.
These results do not exclude the existence of BCGF distinct from IL-2. But, in providing more direct evidence than some previous studies (3,4,(12)(13)(14)23) for the functional role of IL-2 in B cell responses, the data raise questions regarding the conclusions reached by many other authors (15)(16)(17)(18)(19)(20)(21)(22)37), i.e., that IL-2 has no BCGF activity. Several aspects need to be discussed: (a) There exists no discrepancy with regard to the experimental data themselves in that the effects of IL-2 on LPS plus anti-Ig-activated murine B cells were previously not investigated. It is thus possible that some of the previously described factors participate (directly or via other cells) in B cell activation, whereas IL-2 would only act on appropriately activated B cells. Accordingly, it is conceivable that different "activation factors" would be detected in different B cell activation systems (16). (b) In addition, different types of BCGF could theoretically act on different subsets of B cells (16). (c) Certain B cell lines may respond to other factors than activated normal B cells (20,38). However, since most T helper cells are capable of generating IL-2 (39), the demonstrated functional role of IL-2 in B cell proliferation is certainly relevant in the physiologic B cell response.
In addition to the functional demonstration of the BCGF activity of IL-2, this study shows that LPS plus anti-lg-activated B cells express IL-2 receptors; i.e., the receptor assay using radiolabeled IL-2 indicated a mean of 3,500 specific IL-2 binding sites per cell. It is known from limiting dilution analysis that in our system ~ 1 of 8 B cells become responsive to T ceil-derived growth factors in the presence of LPS and anti-Ig (25). We assume that during 4 d of mass culture in the presence of EL-4 SN, these cells outgrow those that do not respond to IL-2, thereby generating a homogeneous population with regard to IL-2 receptor expression. However, this point needs further investigation, e.g., by FACS analysis of the proportion of B cells bearing IL-2 receptors, using anti-IL-2 receptor antibody. In any case, in agreement with previous observations by Robb et al. (5), B cells activated by LPS alone, like nonactivated B cells, did not significantly express IL-2 receptors. This also agrees with our previous finding (25) that anti-Ig is required for the induction of growth factor responsiveness of LPS-activated B cells. Whereas LPS plus anti-Ig-activated B cells expressed only two to three times fewer IL-2 receptors than we and others (5) detected on activated normal murine T cells, the receptor affinity was found to be five-(our unpublished data) to ninefoid (5) lower than that of such T cells. This lower receptor affinity on B cells appears to contrast with the finding that B and T cell proliferation required similar IL-2 concentrations, but two points need to be discussed: (a) T and B cells may differ with regard to the level of IL-2 receptor occupancy required for cell cycle progression, and (b) the apparent receptor affinity measured with binding assays may be influenced by complex variables, including the cell activation stage (i.e., culture conditions), receptor modulation, and possible heterogeneity of the receptors themselves. Until these variables are known, the value of 150 pM should be considered a minimal estimate of the IL-2 receptor affinity on activated B cells.
A monoclonal antibody directed against the murine T cell IL-2 receptor was previously found by Malek et al. (7) to react, although weakly, with LPS-activated B cells. Our own studies in progress show that another monoclonal antibody against the murine T cell IL-2 receptor (obtained by Dr. M. Nabhoiz, Epalinges) inhibits B cell proliferation in an IL-2 dose-dependent way and that the radiolabeled antibody binds to LPS plus anti-Ig-activated B cells to almost the same extent as to Con A-activated normal T cells (unpublished data).
We are also currently investigating the effect of IL-2 on human B cell proliferation. But, in confirmation of previous studies (14,(24)(25)(26)(40)(41)(42) showing that murine or human B cell activation normally requires a direct T-B cell interaction (alone or in conjunction with T-dependent antigens or anti-Ig), and because LPS does not bypass this T helper signal in human B cell systems, we have thus far not obtained a T cell-free human BCGF assay. Nonetheless, in an assay using irradiated mutant EL-4 thymoma cells, which provide an activation signal(s) to murine and human B cells, IL-2 optimally stimulated murine and human B cell proliferation (R. Zubler, C. Mingari, L. Moretta, and H. R. MacDonald, unpublished data). Thus, the previous observation that a monoclonal antibody reacting with the human T cell IL-2 receptor (6) did not react with human B cells (43) could have been related to a suboptimal B cell activation. In fact, it has been more recently (44) demonstrated that this antibody (anti-Tac) recognizes on certain human B tumor cells a molecule of the size of the IL-2 receptor (44).
Finally, we have observed (see Fig. 3) that IL°2 led to the generation of PFC in the murine B blast assay. However, the questions concerning the nature of the B cell differentiation signals in this assay and whether, in fact, differentiation signals are different from activation/proliferation signals in the B cell response (45), were not addressed in this study. The Ig class/subclass distribution of the PFC responses (46) remains to be studied in our systems.

Summary
In this study we investigated whether interleukin 2 (IL-2) acts on B cell proliferation and whether activated B cells express IL-2 receptors. First, the functional activity of immunoaffinity-purified or recombinant human IL-2 was studied in a B blast assay using positively selected murine surface Ig-positive cells that had been activated by lipopolysaccharide (LPS) plus anti-Ig antibodies (anti-Ig). In this assay, T cells were not detected by fluorescence-activated cell sorter analysis. It was found that both IL-2 preparations led to optimal B cell proliferation compared with supernatants obtained from murine or human spleen cells or murine cloned T helper cells. Second, we observed that the IL-2 requirement in this assay was about the same as in a proliferation assay using lectin-activated polyclonal murine Lyt-2-positive T cells. Third, analysis of the binding of radiolabeled imunoaffinity-purified IL-2 to B cells indicated that LPS plus anti-Ig-activated B cells expressed a mean of 3,500 IL-2 receptors per cell with an apparent dissociation constant of 150 pM. However, neither nonactivated B cells nor B cells activated by LPS alone exhibited significant specific IL-2 binding. The functional and the receptor data are consistent with the conclusion that 1L-2 is a growth factor not only for T cells but also for B cells.
We wish to thank Dr.J.C. Cerottini and Dr. M. Nabholz for helpful discussions; Marianne Van Laer, Patricia Corth6sy, and Rose Lees for expert technical help; M. Schreyer for performing the immunohistochemical analyses of B cells; Dr. K. A. Smith for kindly providing DMS-3 monoclonal antibody-coupled Affi-gel 10 beads; and Biogen SA, Geneva, for the gift of purified recombinant IL-2. Note added in proof'. Additional studies using very high [3H]IL-2 concentrations indicate that activated B and T cells express both high and low affinity IL-2 receptors. The corresponding mean apparent dissociation constants (~30 pM and ~300 pM, respectively) do not differ significantly for B and T cells.