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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6550-6554, July 1992 Immunology A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells RANDOLPH J. NOELLE*t, MEENAKSHI RoY*, DAVID M. SHEPHERD*, IVAN STAMENKOVICO, JEFFREY A. LEDBETTER§, AND ALEJANDRO ARUFFO§ *Department of Microbiology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756; *Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121; and *Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 Communicated by Leon E. Rosenberg, April 22, 1992 (receivedfor review February 5, 1992) ABSTRACT CD40 is a B-cell surface molecule that has MATERIALS AND METHODS been shown to induce B-cell growth upon ligation with mono- clonal antibodies. This report shows that triggering via CD40 Mice. Female DBA/2J mice (The Jackson Laboratory) is essential for the activation of resting B cells by helper T cells were used for the preparation of filler cells to support the (Th). A soluble fusion protein of CD40 and human immuno- growth of Th clones and in the preparation of resting B cells. globulin, CD40-Ig, inhibited the induction ofB-cell cycle entry, Th Clones. D1.6, an I-Ad-restricted, rabbit Ig-specific ThO proliferation, and differentiation by activated Thi and Tb2. clone, and CDC35, an I-Ad-restricted, rabbit Ig-specific Th2 The ligand for CD40 was identified as a 39-kDa membrane clone, were obtained from David Parker (University of protein that was selectively expressed on activated Tb. A Massachusetts, Worcester). In this paper, D1.6 will be re- monoclonal antibody specific for the 39-kDa protein inhibited ferred to as Thl and CDC35 as Tb2 (7). CD40-Ig binding and also inhibited the activation of B cells by Activation of Th by Anti-CD3. Thl or Th2 were cultured (8 Th. These data indicate that the 39-kDa membrane protein x 106 per well) in cluster wells (six-well, Coming) coated with expressed on activated Tb is a binding protein for CD40 and anti-CD3 (40 pug/4 ml of phosphate-buffered saline per well) functions to transduce the signal for Tb-dependent B-cell for 16 hr, as described (5). activation. Preparation of Tb Plasma Membranes. Plasma membranes were prepared by discontinuous sucrose gradient sedimen- tation (5). Studies by Mitchison, Benacerraf, and Raff first suggested Preparation of Resting B Cells. Resting splenic B cells were that physical interactions between helper T cells (Th) and B prepared by sedimentation on discontinuous Percoll gradi- cells were essential in the development of humoral immune ents (8). Cells isolated from the 70-75% (density, 1.087-1.097 responses. Later studies documented that Th formed physical g/ml) Percoll interface were typically >95% membrane Ig- conjugates with class II major histocompatibility complex- positive, had a uniform, low degree of near forward light compatible, antigen-presenting B cells (1) and that it was the scatter, and were unresponsive to Con A. B cells within these conjugates that responded to Th (2). With Monoclonal Antibodies (mAbs). The following mAbs were the discovery that Th-derived lymphokines exerted potent purified by ion-exchange HPLC from ascites grown in mice growth and differentiative effects on B cells, it was proposed that had been irradiated and bone marrow-reconstituted: that soluble factor(s) released in proximity by activated Th anti-CD3, 145-2C11 (9); anti-ap3 T-cell antigen receptor mediated the activation of the interacting B cells. However, (TCR), H57-597 (10); anti-CD4, GK1.5 (11); anti-ICAM-1, none of the molecularly cloned lymphokines, alone or in YN1/1.7.4 (12); anti-LFA-1, FD441.8 (13); and anti-rat/ combination, manifested the ability to induce B-cell cycle hamster Ig K chain, RG-7 (14). entry. Unlike soluble factors, plasma membrane fractions Preparation of the CD40 Recombinant Globulin (CD401g). from activated Th induced B-cell cycle entry (3-5). Studies A plasmid containing a cDNA encoding the CD40 antigen (15) using purified plasma membranes from activated Th (PMAct) was digested with the restriction enzymes Pst I and Sau3AL. suggested that a protein expressed on the membrane of The Pst I-Sau3Al fragment was subcloned into the same activated Th cells was responsible for initiating humoral plasmid digested with Pst I and BamHI. This allowed the immunity (5, 6). preparation of a DNA fragment encoding a CD40 protein that PMAct have been used to investigate the nature of this was truncated upstream from the transmembrane domain. effector function (4, 5). PMAct from activated Th, but not The fragment encoding the truncated CD40 was then sub- purified plasma membranes from resting Th (PMRest), ex- cloned into the Ig fusion plasmid (16) by using an Mlu I and pressed an activity that induced B-cell cycle entry in an BamHI digest. The CD40-Ig fusion protein was produced by antigen-nonspecific, class II-unrestricted manner. Because transient transfection in COS cells and purified on a protein of the lack of antigen specificity and class II restrictions, it A column (16). was proposed that nonpolymorphic membrane protein(s) on Lymphokines. Recombinant mouse interleukin 4 (IL-4) was activated Th mediated the activation ofinteracting B cells. In generously provided by C. Maliszewski and K. Grabstein addition, the activity expressed by PMAct required 4-6 hr of (Immunex, Seattle). Recombinant mouse IL-5 was pur- activation and de novo RNA synthesis and was protein in chased from R&D Research (Sarrento, CA). nature (6). Here we show that activated Th express a 39-kDa Induction ofB-Cell RNA Synthesis by PMAd1. Resting B cells protein that binds CD40. Blocking the binding of this ligand (3 x 104) were cultured in 50 yA of complete RPMI medium to CD40 inhibited Th-dependent B-cell activation. These data (RPMI 1640 plus 10%o fetal bovine serum and 50 ,uM 2-mer- suggest that the binding ofthe 39-kDa protein on activated Th in microtiter wells (Costar). To these to CD40 on B cells initiates thymus-dependent humoral captoethanol) A/2 immune responses. Abbreviations: Th, helper T cell(s); PMACt and PMReSt, purified plasma membranes from activated and resting Th, respectively; The publication costs ofthis article were defrayed in part by page charge TCR, T-cell antigen receptor; LPS, lipopolysaccharide; IL, inter- payment. This article must therefore be hereby marked "advertisement" leukin; mAb, monoclonal antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6550 Downloaded by guest on September 30, 2021 Immunology: Noelle et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6551 wells, 0.5 gg of Thl or Th2 membrane protein was added. brane proteins were added to cultures of PMACt and B cells. Forty-two hours later 2.5 1Ci (92.5 kBq) of [3H]uridine (New As previously published (5), PMACt induced B-cell RNA England Nuclear) was added to each well. After 6 hr the cells synthesis 8-fold over that observed with PMReSt (Fig. 1A). were harvested, and the radioactivity was determined by The addition ofanti-LFA-1, anti-CD4, or anti-ICAM-1 alone, liquid scintillation spectrometry. Results were expressed as or in combination, did not inhibit induction of B-cell RNA cpm per culture (mean + SD). synthesis by PMACt. Induction ofB-Cell Ig Secretion by PMAd and Lymphokines. CD4O-Ig Inhibits Tb-Induced B-Cell Cyde Entry, Differen- Resting B cells were cultured as described above. To each tiation, and Proliferation. In the human system, it had been culture well, 0.5 pug of Thl membrane protein, IL-4 (10 shown that anti-CD40 mAb induced B-cell proliferation (18), ng/ml), and IL-5 (5 ng/ml) were added. On day 3 of culture, thereby implicating CD40 as an important triggering molecule an additional 50 1d of complete RPMI was added. On day 6 for B cells. To determine whether CD40 was involved in the ofculture, supernatants from individual wells were harvested induction of B-cell RNA synthesis by PMACt, a soluble fusion and quantitated for IgM and IgG1 (5). the Fc Induction of B-Cell Proliferation by Activated Th and IL-4. protein ofthe extracellular domains ofhuman CD40and Resting B cells (4 x 104) were cultured in 50 1.l of complete domain of human IgG1 (CD40-Ig) was added to cultures of RPMI in A/2 microtiter wells (Costar). To each well, IL4 (10 pMACt and B cells. PMACt derived from Thl and Th2 were ng/ml) and 104 resting or activated irradiated (500 rads; 1 rad prepared and used to stimulate B-cell RNA synthesis. The = 0.1 Gy) Thl were added. On day 3 of culture, cells were addition of CD40-Ig to culture caused a dose-dependent incubated with 1 ,.Ci of PH~thymidine, and incorporation inhibition of B-cell RNA synthesis that was induced by PMAC, was determined as described (5). from ThO and Th2 (Fig. 1B). Half-maximal inhibition of B-cell Production of mAbs Specific for Membrane Proteins In- RNA synthesis induced by PMACt from Thl and Th2 was duced on Activated Tbl. Hamsters were immunized intraperi- achieved with about 5 gg of CD40-Ig per ml. A CD7E-Ig toneally with 5-10 x 106 activated ThO (D1.6) at weekly intervals for 6 weeks. When the serum titer against murine 70. A Thi was >1:10,000, the hamster splenocytes and NS1 mouse PMRest PMAct myeloma cells were fused in the presence of polyethylene 60- T glycol. Supernatants from wells containing growing hybrid- omas were screened by flow cytometry for reactivity with 50- T resting and activated Thl.

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