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Affinity-Purified Interleukin 2 Induces Proliferation of Large but Not Small B Cells (Lymphoklnes/B-Cefl Activation/Size-Fractionated B Cells) JAMES J

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Affinity-Purified Interleukin 2 Induces Proliferation of Large but Not Small B Cells (Lymphoklnes/B-Cefl Activation/Size-Fractionated B Cells) JAMES J Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1518-1521, March 1985 Immunology Affinity-purified interleukin 2 induces proliferation of large but not small B cells (lymphoklnes/B-cefl activation/size-fractionated B cells) JAMES J. MOND*, CRAIG THOMPSONt, FRED D. FINKELMAN*, JOHN FARRARt, MARY SCHAEFER* AND RICHARD J. ROBB§ *Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814; tNaval Medical Research Institute, Bethesda, MD 20814; *Department of Immunopharmacology, Hoffmann-La Roche, Nutley, NJ 07100; and §Central Research and Development, E. I. Du Pont de Nemours & Company, Glenolden Laboratory, Glenolden, PA 19036 Communicated by Michael Heidelberger, October 29, 1984 ABSTRACT Immunoaffinity-purified interleuWn 2 (IL2) er subclone (J6.8.9.15.32) of the human T-celi line Jurkat stimulated proliferation of large but not smal B cells. Stimula- (11). The cells (4 x 106 per ml) were stimulated in serum-free tion was observed even when B cells were cultured at very low medium with phytohemagglutinin (PHA; 1.5 .g/ml; HA-16, cell densities (3 x 104 per microwell containing 0.2 ml of medi- Wellcome Reagents) and phorbol 12-myristate 13-acetate um). Addition of small numbers of purified splenic T cells did (PMA; 50 ng/ml; Consolidated Midland, Brewster, NY). not enhance the 1L2-induced B-cell proliferative response. The supernatant was harvested after 15 hr at 370C and stored These results suggest that IL2 was not operating through con- at 40C. IL2 was purified by passing the supernatant over a taminating T cells. B cells cultured with anti-Ig antibody in column of CNBr-activated Sepharose 4B coupled to a mu- vitro showed enhanced proliferation when cultured 'with ETA rine monoclonal antibody (8 mg per ml) reactive with human thymoma-derived B-cell growth factor but not when cultured IL2 (11, 12), Bound IL2 was recovered by elution with 1.5% with HL2. A direct role for 1L2 in B-cell activation is discussed. acetic acid. The eluted material migrated as a single spot on two-dimensional gel electrophoresis (silver stain detection) Although interleukin 2 (IL2) has been shown to influence B- at pI =8 and apparent Mr 15,500 and as a single peak on cell activation, it is not known whether its effect is exerted reversed-phase HPLC, had a single NH2-terminal amino directly on the B cell or indirectly by induction of other T- acid sequence (11) which matched that predicted from cell-derived lymphokines (1-6). We and others have shown cDNA data (13), and was free ofPHA and PMA asjudged by that T-cell-derived lymphokines that are enriched for IL2 ac- gel electrophoresis and radiolabeled PMA tracer analysis tivity enhance antigen-specific as well as polyclonpl B-cell (11). The specific activity of the IL2 was 315,000 ± 20,000 activation and that this stimulatory activity is diminished units of bioactivity/mg of protein. when IL2 is removed by adsorption onto an IL2-dependent IL2 Bloactivity. 1L2 activity was measured by proliferation T-cell line (1, 3, 5). Other investigators, however, have been ([3H]thymidine uptake) of an IL2-dependent, cloned murine unable to demonstrate a direct influence of IL2 on B-cell cytotoxic T-cell line [CTLL-2, subclone 1SH (14)]. Serial di- differentiation (7-9) or to demonstrate unequivocally specif- lutions of IL2 were incubated with 4000 CTLL cells in a vol- ic binding of radiolabeled IL2 onto resting or activated B ume of 200W4 for 16 hr, followed by a 4-hr pulse of0.5 pCi of cells. Because of the prior unavailability of homogeneous [3H]thymidine (1 Ci - 37 GBq). The reciprocal of the dilu- preparations of IL2, it was uncertain whether or not the B- tion of the sample yielding half-maximal thymidine uptake cell-enhancing effects ofpreparations enriched for IL2 could was divided by that of an arbitrary standard to give the activ- have been due to other B-cell-stimulatory lymphokines con- ity in units per ml. The standard (1 unit/ml) gave half-maxi- tained in these fractions. To study this more carefully, we mal [3H]thymidine incorporation at about 1:10 [dilution be- examined the ability of immunoaffinity-purifiqd IL2 to influ- fore addition of an equal volume (100 p.1) of CTLL cells]. ence B-cell activation directly. Since large B cells are more BCGF. BCGF was prepared by chromatography on phe- responsive than small B cells to the stimulatory effects of B- nyl-Sepharose (15). cell growth factor (BCGF) (unpublished observations) and B Antibodies. Preparation of affinity-purified anti-IgM and cells activated by bacterial lipopolysaccharide express re- anti-IgD as well as their fluorescent conjugates was as de- ceptors for IL2 (10), we studied the effects of affinity-puri- scribed (16). fied IL2 not only on small resting B cells but also on large B B-Cell Purification. Splenic cells were enriched for B cells cells in later stages ofthe cycle of cell activation. Our results by a modification of the method of Liebson et al. (1). Mice indicate that (i) large but not small B cells can be stimulated were injected intraperitoneally with 0.04 ml of anti-thymo- to proliferate by IL2, (it) this stimulatory effect does not ap- cyte serum (M. A. Bioproducts, Walkersville, MD). Two pear to be mediated through T cells, and (iii) B cells that days later spleen cells were harvested and treated at 370C for have been activated by soluble anti-Ig antibody in vitro to 45 min with a mixture containing a mouse monoclonal anti- show an increase in cell size cannot be stimulated to prolifer- Thyl.2 and a noncytotoxic rat monoclonal anti-Lytl anti- ate by IL2. body (15D6) [American Type Culture Collection (ATCC), Bethesda, MD]. This was followed by treatment for 45 min at 370C with mouse monoclonal anti-rat Ig (MAR 18.5, MATERIALS AND METHODS ATCC) and newborn rabbit complement (1:5 dilution). Fluo- Mice. CBA/J mice (8-12 weeks old; The Jackson Labora- rescence-activated cell sorter (FACS) analysis of B cells ob- tory) were used in all experiments. tained by this method showed no fluorescent Thyl.2' cells Preparation of IL2. IL2 was prepared from a high-produc- above background staining. B cells prepared in this manner failed to proliferate in response to stimulation with the T-cell mitogen concanavalin A (Con A) after 2, 3, or 4 days of cul- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: IL2, interleukin 2; BCGF, B-cell growth factor. 1518 Downloaded by guest on October 1, 2021 Immunology: Mond et aL Proc. Natl. Acad Sci. USA 82 (1985) 1519 Table 1. IL2 induces B-cell proliferation in large but not small B lymphocytes [3H]Thymidine incorporation,* cpm B cells used Control IL2 Con A Unfractionated 24,197 ± 510 38,359 ± 2306 18,691 ± 782 Fraction 1 (small) 13,452 ± 2214 14,994 ± 1276 8,389 ± 175 Fraction 5 (large) 23,603 ± 1686 71,653 ± 2227 23,133 ± 1178 B cells were prepared as described in Materials and Methods and cultured at 2.5 x 105 per microwell with Con A (1 pg/ml) or with IL2 (10 units/ml). [3H]Thymidine was added at 48 hr and the cultures were harvested 18 hr later. *Values in all tables represent the arithmetic mean ± SEM for triplicate culture wells. ture. The number of surface Ig-bearing (sIg+) cells in the which the cells in fraction 1 had a mean volume of 115.8 ± experiments described was 93-95% of those analyzed. The 3.7 ,um3 (mean ± SD) and the cells in each successive frac- remaining 5-7% of the cells may have included B cells ex- tion had a progressively larger mean volume. The largest pressing sIg at low density as well as cells that were of the cells (fraction 5) had a mean volume of 168.0 ± 6.0 um . phenotype Thyl.2-sIg-. Cell Culture and Proliferation. Cells were cultured at vari- T-Cell Separation. For preparation of splenic T cells, ous densities in modified Mishell-Dutton medium that con- spleen cells were incubated for 1 hr in Petri dishes coated tained 10% endotoxin-free (<0.1 ng/ml) fetal bovine serum with an affinity-purified goat anti-mouse Ig K chain antibody (HyClone, Logan, UT) and 50 ,uM 2-mercaptoethanol in flat- and nonadherent cells were recovered and incubated for an bottomed Microtiter plates (Costar, Cambridge, MA) (0.2 ml additional hour on anti-IgM antibody-coated Petri dishes. of medium per well). Plates were incubated for up to 4 days Nonadherent cells thus obtained contained <1% sIg+ cells in a humidified 5% CO2 atmosphere at 37°C. Cultures (tripli- and >95% Thyl.2+ cells as determined by FACS analysis of cate) in medium containing IL2, BCGF, or anti-Ig antibodies cells stained with fluorescence-labeled anti-8 or anti-Thyl.2 were incubated for 16 hr with 1.0 ,uCi of [methyl-3H]thymi- antibodies. dine ([3H]thymidine) and then harvested. Results are ex- B-Cell Subpopulations. The enriched B-cell population was pressed as the arithmetic mean ± SEM of cpm incorporated separated into five size-dependent subpopulations with a per culture. counterflow centrifuge (elutriator) as previously worked out for murine spleen cells (17). Counterflow centrifugation op- RESULTS poses an outwardly directed centrifugal force with an in- wardly directed force generated by the flow of fluid through 1L2 Induces B-Cell Proliferation in Large but Not Small B the centrifuge's separation chamber.
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