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explain figure 2 please on the third page

The Jounal of Immunol nes of the National Academy of Sciences USA 89. 6550-6554. 01992, by permision of Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6550-63554, July 1992 Immunolog A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells RANDOLPH J. NOELLE时, MEENAKSHI ROY.. DAVID M. SHEPHERD., IVAN STAMENKOVICt JEFFREY A. LEDBETTER, AND ALEJANDRO ARUFFo Departmend Microbiology. Dartmouth Medical School One Medical Center Dme, Leba NH 037% fann í Myers Se co hrasa Insotute, Seattle, WA 98121: and 'Department of Pathology, Masuacbsvetts Generl Hospital, Harvard Medical School, Boston, MA 02114 Communicated by Leon E. Rosenbers,April 22, 1992 (received for reviee February 5, 1992) ABSTRACT CD40 is n B-cell surface molecule that has been shown to induce B-cel growth upon lization with mone M clonal antibodles. This report shows that triggering via CD40 MATERIALS AND METHODS entlal for the activation of resting B celh by belper T cellswere used for the preparation of filler cells to support the T). A soluble fusion protein of CD40 and human mmano- srowth of T, clones and in the preparation of resting B cells Mice. Female DBA/2J mice (The Jackson Laboratory) T Clones. D1.6, an I-A -restricted, rabbit Ig-specific T.1 Worcester). In this paper, Di.6 will be re prolferation, and differentlation by activated T,l and T2. clone, and CDC3S, an I-Ad-restricted, rabbit Is-specific T2 The ligand for CD40 was identified as a 39-kDa membrane clone, were obtained from David Parker (University of lobulin, CD40-Ig, inhibited the Induction of B-cell cycle monoclonal antibody specific for the 39-kDa proteln Inhiblted ferred to as T,l and CDC35 as T2 (7) CD40-lg binding and also lahibited the activation of B celu by irat oof T, by Ant-CD3. Tal or Ti2 were cultured proteln that was selectively expressed on activated T. Aes. aD (40 ag/4 ml of phosphate-buffered saline per well Preparation of T, Plasma Membranes. Plasma membranes were prepared by discontinuous sucrose gradient sedimen- T, These data indlcate that the 39-kDa membrane protein 10 per wel)incluster wells (six-well, Corning) coated with for 16 hr, as described (5) pressed on activated T, s a binding protela for CD functions to transdace the signal for T dependent B-eel activation. Studies by Mitchison, Benacerraf, and Raff first suggested tation (5). that physical interactions between belper T cells (T and B Preparation of Resting B Cella. Resting splenic B cells were ated from be70-75% a nay, ien were unresponsive to Con A. cells were essential in the development of humoral immune prepared by sedimentation on discontinuous Percoll gradi- ent, cal responses. Later studiesdocumented that Ts formed physical conjugates with class II major histocompatibility complex- g/ml) Percoll interface were typically >95% membrane la te, had a unitorm, low degree of near forward light compatible, antigen-presenting B cells (1) and that it was the B cells within these conjugates that responded to T.(2). With the al Antibodies (mAbe). Tbe following mAbs were that Ti derived lymphokines exerted polenpurified by ion exchange HPLC from ascites grown in mice on B cells, it was proposed that had been irradiated and bone marrow-reconsttuted factors) released in proximity by activated Ts anti-CD3, 145-2C1l 9): anti-a T-cell antigen receptor ctivation of the interacting B cells. However. (TCR). H57-97 (10); anti-CD4, GK1.5 (11): anti-ICAM-1 cloned lymphokines, alone or in YN/1.7.4 (12): anti-LFA-1, FD441.8 (13): and anti-rat combination, manifested the ability to induce B-cell cycle entry. Unlike soluble factors, plasma membrane fractions hamster I from activated T, induced B-cell cycle entry (3-5). Studies K chain, RG-7 (14) using purified plasma membranes from activated T, (PMA) activated Th cells was responsible Preparation of the CD40 Recombinant Globulia (CD40-I. A plasmid containing a cDNA encoding the CD40 antigen (15) was digested with the restriction enzymes Pst I and Sau3A1 The Pst 1-Sau3Al fragment was subcloned into the same plasmid digested with Pst I and BamHI·This allowed the preparation of a DNA fragment encoding a CD40 protein that from the transmembrane domain used to investigate the nature of this Was truncated upstream effector function (4, 5). PMA from activated Th, but not The fragment encoding the truncated CD40 was then sub- and BamHI digest. The CD40-ig fusion protein was produced by lack of antigen specificity and class II restrictions, it transient transfection in COS cells and purified on a protein from resting T (PM) excloned into the Ig fusion plasmid (16) by using an Mlu class II-unrestricted manner. Because nonpolymorphic membrane proteins) on-Lynphokines. Recombinant mouse interleukin4 ILA activity expressed by PM required 4-6 hr of generously provided by C. Maliszewski and K. Grabstein was proposed that membrane the activation of in acuvation and de novo RNA synthesis and was protein in chased from R&D Research (Sarrento, CA) (Immunex, Seattle). Recombinant mouse Iし5 was pur- nature (6). Here we show that activated T, express a 39-kDa I prote that bds CD40 Blocking the binding oftsliga d (3x10") were cultured toCD40 inhibited , dependent B-cell activation Th seda a suggest that the binding of the 39-kDa protein on activated T to CD40 on B cells initiates th Induction of B-Cell RNA Synthesis by PMAd. Resting B cells 50μ1 of complete RPMI medium (RPMI i640 plus į0% fetal bovine se una d50A 2 er captoethanol) in A/2 microtiter wells (Costar). To these Abbreviations: T belper T cella); PMAet and PMRe, purified The publication costs of this article were defrayed in part by pag car payment. This article must therefore be bereby marked "advertisement"leukin: mAb, monoclonal antibody n accordance with 18 U.S.C. 11734 soiely to indicate this fact plasma membranes from activated and resting Tb. respectively TCR, T-cell antigen receptor: LPS, lipopolysaccharide: IL, inter To whom reprint requests should be addressed. 6550 PILLARS OF IMMUNOLO ed from Proccedings of the National Academy of Sciences, U.S.A. 89: 6550-6554. 01992, by permission of the authors. 6551 Proc. Natl. Acad. Sel. USA 89 (1992) PMAct İnduced B cell RNA wells, 0.5 Ag of T,l or T,2 membrane protein was added. brane proteins were added to cultures of PMAct and B cells As previously published (5) Forty-tw hours later 2.5pC(92.5 kBq) ofPHJuridine(New England Nuclear) was added to each well. After 6 hr the cells and the radioactivity was determined by spectrometry. Results were expressed as synthesis &-fold over that observed with PMRet (Fig. 1A). The addition of anti-LFA-1, anti-CD4, or anti-ICAM-1 alone, or in combination, did not inhibit induction of B-cell RNA by Induction of B-Cell Ig Secretion by PMA and Lympbokines. Resting B cells were cultured as described above. To each CD40-Iz Inhibits T-Induced B-Cell Cycle Entry, Differen- latlon, and Proliferation. In the human system, it had been of Tl membrane protein, IL (10 shown that anti-CD40 mAb induced B-cell proliferation (18) culture well. ng/m), and IL ( ne/ml) were added. On day 3 of culture, thereby implicating CD40 as an important tnggering an additional 50 pl of complete RPMI was added. On day 6 for B cells. To determine whether CD40 was involved in the of culture, supernatants from individual wells were harvested Induction of B-Cell Proliferation by Activated Tand IL-4 Resting B cells (4 x 10") were cultured in 50 of complete RPMI in A/2 microtiter wells (Costar). To each well, IL4(10 protein of the extracellular domains of human CD40 and the Fe domain of human IgGI (CD40-1g) was added to cultures of PMA and B cells. PMAt derived from hl and T2 were ng/ml) and 10 resting or activated irradiated (500 rads; I rad prepared and used to stimulate B-cell RNA sy 0.1 Gy) Til were added. On day 3 of culture, cells were addition of CD40-1g to culture caused a inhibition of B-cell RNA synthesis that was induced by PMAt from Thl and Th2 (Fig. 18). Half-maximal inhibition of B-cell Production of mAbs Specific for Membrane Proteins In- RNA synthesis induced by PMct from Thl and T2 was duced oa Activated TI. Hamsters were immunized intraperi. achieved with about 5 ㎍ of CD4-Ig per ml. A CDIE-lg incubated with 1 Ci of PHjthymidine, and incorporation toncally with 5-10 x 10 activated Til (D1.6) at weekly intervals for 6 weeks. When the serum titer against murine Tl was >1:10,000, the hamster splenocytes and NS1 mouse myeloma cells were fused in the presence of polyethylene glycol. Supernatants from wells containing growing hybrid- resting and activated Tl. One particular hybridoma, which produced a mAb that selectively recognized activated T was further tested and subcloned to derive MR1. The MR mAb was produced in ascites and purified by ion-exchange of 10 Expressed o T Resting and activated T (16 hr with anti-CD3) were harvested and incubated at 10 cells per 50 l with fusion protein for 20 min at 4'C, followed by fluorescein conjugated goat anti-human IgG (25 ㎍/ml; Southern Bio- technology Associates, Birmingham, AL). Propidium iodide was added (2 μ8/ml) to all samples. Flow cytonuoro metric analysis was performed on a Becton Dickinson FACScan After positive gating of cells by forward vs. side scatter, and None CD4 IFAI ICAMI Mix by red negativity (for propidium iodide exclusion), the green o 2 fluorescence (logarithmic scale) of viable cells was ascer- tained. At least 5000 viable cells were analyzed for the determination of percent positive cells. Staining with MRL 30 employed fluorescein-conjugated RG7, a mouse anti-rat/ 100 and Fluorography. Til were rested or were activated with insolubilized anti-CD3 for 16 hr. Resting and activated Ti (20 x 10 per ml) were labeled with 1 mCi of (NS)methionine/ cysteine for l hr, washed twice in RPMI-1640 with 10% fetal bovine serum, and lysed in extraction buffer (17). Purified antibodies or fusion proteins (1-10 ㎍) were added to 500㎕ of lysate (5 x 10 cell equivalents) at 4'C for 16 hr. At that time, the lysates were transferred to tubes containing S0ul of packed protein A-Sepharose. The pelleted protein A-Sepharose was resuspended and tubes were incubated at 4"C for 1 hr with 10 Jo Fio. 1. Effect of mAbs and CD40-Ig on the induction of B-cell RNA synthesis by PMAct, (A) Resting B cells were cultured with PMRes or PM from T,l. Individual mAb (anti-CD4, anti-LFA-1 or anti-ICAM-1,25 ug/mi) or a combination of all the mAbs (each at 25Ag/ml) (Mix) was added. B-cell RNA synthesis was assessed from Results presented are the arithmetic means - of triplicate cultures and are representative of five such exper. on. The samples were then washed three times with 42 to 48 hr of culture. wash buffer. The pelleted protein A-Sepha S rose was suspended in 30 μ1 of SDS sample buffer and run in 10% polyacrylamide gel. After electropboresis, the proteins imens, un Resting B cells were cultured with PMA" fronta andT2 a. To these cultures, to fusion protein was added or were fixed in the gel and fluorography was performed CD40Ig(A, a) or CDTE-la (o) was added at 1-25s/ml. B-cell RNA synthesis was assessed as in A. Results are the arithmetic means t iments. (C) Resting B cells were cultured with LPS (S0 ug/ml) or PMAd. CD40-1g (25 ㎍/m, hatched bar) or CD7E-lg (25 ㎍/m2 Effect of mAbs on the Induction of B-Cell RNA Synthesis by stppled bar) wa PMA. To define the cell surface molecules that mediate the Results are the induction of B-cell cycle entry by PMA, mAbs to Th mem- epresentative of three such experiments stippled bar) was added. B-cell RNA syathesis was assessed as A arithmetic means SD of triplicate cultures and are fl ner.U.SA 89 6550-6554. С 1992, by permission of the authors ader fron Pro erdings of the National Proc. Natl. Acad Ser USA 89 (1992) 52Immunology: Noelle et al. and ILS S e/mil Ether at the initiation ef culture, or eo day 1. 2, or 3 after initiation of culture. CD40-1g or CDTE-Is (23 Hg/ml) was added. On day 6 of culture, supernatants trom individual wells were harvested and quanticated for IsM () and IgG1 () by anti-isotype-specific ELISA 3) In the presence of PMA, 114, and IL-5 Gn the absence of added CDO-la. IgM and IgG1 were 4.6 ㎍/ml and i 26 ng/m, respectively. Cultures thal received CD18-lg (25 Pg/ml) on day 0 produced 2.4 ㎍/ml and 89 ng/ml of IgM and igG1, respectively. The control of 100% is based on the response in the presence of CD7E-ls la the abseace of IL4 and IL-5, no lgM or IG1 was detected, Results are representative of three chexperiments un Tal were rested or activated with ant-CD) for ishr, imdialed, and cultured (10, per well) with resting B cells (4 10) in the presence of TC4 (10 ng mn CD#4g (A) or CD7E-1 .) was added at 0-25 ㎍/mL From 66 to 72 hr of culture, cells were incubated with 1 Cof lH)thy midime and then harvested. Brokeo line, response of B cells to resting To without the addition of fusion protein. Results / Po. 2. cD0-Is inhabits B-ell fferestiuation and proliíteration ()Resting B cells were cultured with PM, arr the arithmetic means ± SD of triplicate altures and are representative er two such apenmenis. protein (19) was without effect even when used at 25s also involved in the activation of B cells by intact, viabl B cells by T-cell-independent activators, B cells were cul cultured with B Salmonella typhosa) and CD40-Is. On day 2,. RNA synthesis enous source of activated TTl were activated for 16 hr with insolubilized To investigate whether CD40-lg inhibited the activation of anti-CD3, harvested, and irradiated. The irradiated Thl were cells in the presence of IL4 and B-cell on was determined on day 3 of culture. An exog was assessed (Fis. 1C). CD40-g eration because Thl do not produce IL4 (20). CD40-lg response of B inhibited the induction of B-cell proliferation by irradiated, activated T in a dose-dependent manner, similar to that of PMA IL-4, and IL, B cells poly. observed with PMA (Fig. 2B). The negative control CDTE- Ig, exerted no eff clonally differentiated to produce Ig (4, 5). To evaluate the requirements for CD40 signaling in this process, CD40-1s was added at the initiation of culture or on subsequent days of culture. The addition of CD40-1g (Fig. 2A) at the initiation expressed a binding protein for CD40, resting and activated (16 of culture inhibited >99% of the polyclonal IgM and IgG1 hr) Ta were stained with CD40-lg or CDIE-lg, followed by production compared with control levels in its absence. In fluorescein-conjugated anti-human IgG. Binding of CD40-1g contrast, the addition of CD40-1g on day 1 or 2 of culture was assessed by flow cytometry (Fig. 3). Activated Thl, but showed little if any inhibitory effect on IgM and IgG1 production. These dataindisated that after via CD40 was no lonser essentiakforth a Molecule Expressed on Activated T, But Not on Resting T. To investigate whether activated T.l not resting Tal, stained 56% positive with CD40-Ig, but not 24he signaling with the control CD7E-lg. To identify the CD40-1g-binding protein, Thl proteins were biosynthetically labeled with of B ed CD4Oin the activation of B cells by PMa, Studies were performed to assure that CD40 was SJmethionine/cysteine and proteins were tated with CDW-Ig or CDE-lg·The immunoprecipitated proteins were resolved by Sps/PAGE and fuorography (Fig. 10 10 10 10 10 010 10 logigreen proteins for 20 min at 4°C, followed by by the analysis of at least 5000 cells per sample. experiments. On resting Th, staining on activated Tabu·thot resting Ta-Resting U) or activated un Th were incubaled with thia ample. The thresbold for positive cells was set at channel 35. Results are representative of six sach with CD40-1g and staining with CDTE-g are completely overlapping and identical in distribution

Answer 1

In figure 2.A - shown that CD inhibit T-cell independent (without T cell), B cell activation. when B cell are culture with (PM^act,IL-4 and IL-5) CD40-Ig at initial period, their is approximately 95 % inhibition is occur in IgG1 and IgM synthesis or poly clonal Ab production , but if CD40-Ig applied in 2 or 3 day of B cell culture then their is very less or no inhibitor effect is seen in both antibody production. While in control solution were CD40-Ig is absent their is normal production of poly clonal Ab from B cell through independent manner, due to the presence of PM^actand (IL-4 and IL-5).

In figure 2B.Indicate CD-40 help B cell to active from fully intact T cell , IL-4 and IL-5 not from the radiation influence T cell. In control conditions were CD-40 Ig is absent and other are same conditions but their is decrease B cell proliferation is seen (negative control) because of distract T cell interaction.

So the Role of CD-40 Ig is to insure B cell activate through intact T cell only (T dependent activation only).