Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3831-3835, June 1987 Immunology Assessment of -specific receptor function of surface immunoglobulin M and D with identical specificity (antigen-binding capacity//gene transfer/relative fluorescence index) ROLAND TISCH*, MOTOO WATANABE*t, MICHELLE LETARTEt, AND NOBUMICHI HozuMI* *Mount Sinai Hospital Research Institute, Department of Medical Genetics, University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada; and tResearch Institute, Hospital for Sick Children, Department of Immunology, University of Toronto, 555 University Avenue, Ontario, M5G 1X5, Canada Communicated by Allan M. Campbell, February 9, 1987

ABSTRACT Monoclonal B-cell lines expressing antigen- determinants. However, whether this apparent difference is specific surface IgM or IgD were established by transferring the attributable to specific properties associated with the binding genes encoding immunoglobulin heavy (,u or 6) and light (K) ofantigen by sIgM or sIgD is not clear. There is also evidence chains specific for the hapten 2,4,6-trinitrophenyl (TNP) into a that suggests sIgM and sIgD differ in signal transduction B-cell lymphoma line. Two IgMTNp and two IgDTNp transform- following antigen binding (19, 20). Recently, however, Chen ants were selected on the basis of similar levels of anti-TNP et al. (21) have demonstrated that crosslinking of sIgM or expression by flow microfluorimetric analysis. The sIgD expressed on normal B cells leads to equivalent levels IgMTw and IgDTNp transformants were compared in quanti- of protein kinase C translocation. Whether these two sIg tative assays for their ability to bind TNP-carrier and present isotypes transduce different signals upon antigen binding still TNP-carrier to carrier-specific T cells. Our results indicate has to be ascertained. that IgMTNp and IgDTNp transformants have an equal capacity The ability of a to interact with Th cells to generate to bind and present specific antigen. Thus surface IgM and an depends on the capacity ofthat B cell to IgD, when present in equivalent amounts, function similarly as bind, process, and present antigen. Therefore it is of great antigen receptors. interest to determine whether sIgM and sIgD function dif- ferently as antigen-specific receptors in these processes. One The primary role ofan immunoglobulin (Ig) molecule expressed problem in trying to assess the specific roles ofsIgM and sIgD on the surface ofa B is to act as an antigen receptor. as antigen receptors is the lack of monoclonal antigen- The binding of antigen by surface Ig (sIg) leads to at least two specific B cells, which has, in general, greatly hindered sIg events associated with the generation of a B-cell immune and specific antigen-binding studies. Without such B cells, response. (i) Bound antigen is endocytosed, processed, and investigations of the specific function of sIgM and sIgD have returned to the surface in association with class II major primarily depended on isolated, small resting B cells (7, 19). histocompatibility complex (MHC) molecules (1-5). This pre- These B cells are not clonal and express variable amounts of sentation enables the B cell to interact with helper T (Th) cells sIgM and sIgD (22), making quantitative studies-in specific, that recognize the processed antigen and that provide signals for antigen receptor function studies-extremely difficult. The further B-cell proliferation and differentiation (6). It is believed study of clonal B cells expressing equal amounts of either that sIgs aid the antigen presentation process by efficiently antigen-specific sIgM or sIgD would permit direct quantita- concentrating antigen on the B-cell surface as well as subse- tive comparison between these two sIg classes in their role as quently internalizing it (2, 5, 7). (ii) Antigen binding also results antigen receptors. Our laboratory has recently established in the crosslinking of sIg, which, in turn, triggers activational techniques for transferring Ig chain genes (prNP, KTNp) into signals rendering the B cell receptive to stimuli provided, for lymphoid cells enabling us to generate antigen-specific sIgMTNp instance, by Th cells (8, 9). monoclonal B cells (5). We now report on the establishment of Almost all B cells ofperipheral lymphoid organs carry two antigen-specific B-cell lines expressing sIgD following the classes of slg: IgM, expressed early during B-cell ontogeny, transfer of 8 and K Ig chain genes specific for the hapten TNP. and IgD, appearing later in development at the immunocom- We have quantitated the ability of transformants expressing petent mature B-cell stage (10, 11). The reasons for the either sIgMTNp or sIgDNp to present TNP-carrier to carrier- expression of two types of sIg antigen receptors with iden- specific T cells and to bind TNP-key hole limpet hemocyanin tical specificity (12) on an immunocompetent B cell remain an (KLH) with the aim of determining whether sIgM and sIgD enigma. Variations in structural and physical properties function similarly as antigen receptors. between sIgM and sIgD have led to speculation that the function of these two sIg receptors may differ. IgD is more MATERIALS AND METHODS susceptible to proteolysis than IgM (13, 14), and its rapid degradation may facilitate dissociation of bound and Cell Lines. A B-lymphoma line, A20-2J producing sIgG of endocytosed antigen-possibly enhancing antigen presenta- an unknown specificity and surface lad antigen, was obtained tion. IgD heavy chain has an extended hinge region that may from D. McKean (Mayo Clinic, Rochester, MN). An antigen- provide a greater degree of segmental flexibility, allowing specific T-cell hybridoma (CAK1-22) specific for the carrier more efficient crosslinking of polyvalent than that KLH and Iad-restricted was established as previously de- obtained with IgM (15, 16). This notion is supported by the fact that IgD appears to be more effective than IgM in Abbreviations: IL-2, 2; KLH, keyhole limpet hemocya- activating B cells by thymus-dependent antigens (17, 18), a nin; MHC, major histocompatibility complex; Ova, ovalbumin; class of antigens characterized by a low density of antigenic Pi/NaCl, phosphate-buffered saline; slg, surface immunoglobulin; Th cell, helper ; TNP, 2,4,6-trinitrophenyl; RFI, relative fluorescence index. The publication costs of this article were defrayed in part by page charge tPermanent address: Central Research Laboratory, Mitsubishi Pet- payment. This article must therefore be hereby marked "advertisement" rochemical, Co. Ltd., 8-3-1 Chuo, Ami, Inahiki, Ibaraki 300-03, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Japan. Downloaded by guest on September 23, 2021 3831 3832 Immunology: Tisch et al. Proc. Natl. Acad. Sci. USA 84 (1987)

scribed (5). The interleukin 2 (IL-2)-dependent cell line Assay for Antigen Presentation. Antigen presentation was CTL.L, utilized for assessing IL-2 production by the T-cell carried out as previously described (5). In brief, 105 B-cell hybridoma following antigen presentation, was maintained in transformants and 105 T cells were cultured in 96-well plates RPMI 1640 medium containing 10% fetal bovine serum and in 0.2 ml of medium. After 24 hr of culture, the supernatants 10% supernatant from concanavalin A-stimulated rat were collected for assay of IL-2 production. IL-2-dependent cells (23). DNA synthesis was determined by adding 20 ,ul of superna- Vectors and Gene Transfer. The construction ofpR-HLTNP tant to 8 x 103 CTL.L cells and incubating them for 15-20 hr. and pR-TKgpt has been described previously (24, 25). In brief, DNA synthesis was estimated by incorporation of [methyl- the rearranged /TNP and KTNP chain genes specific for TNP 3H] thymidine (1 ,uCi per well, 25 Ci/mmol, Amersham; 1 Ci were either both inserted into the vector pSV2-neo (pR- = 37 GBq) during an 8-hr period. Data represent averages HLTNP) that confers resistance to the antibiotic G418 (26) or calculated from triplicate cultures. only the KTNP chain gene was inserted into vector pSV2-gpt (pR-TKgpt) which confers resistance to mycophenolic acid RESULTS (26). A germ-line 8 chain gene was obtained from L. Hood Relative Expression of sIgMTNP and sIgDTNP on B-Cell (California Institute of Technology). The complete 8 chain Transformants. The B-lymphoma A20-2J was chosen as the gene and rearranged variable heavy gene segment specific for recipient cell line to establish sIgMTNp and sIgDTNP TNP (VHTNP) were inserted into pSV2-neo. In brief, an transformants. A20-2J cells, which express sIgG ofunknown EcoRI fragment carrying the VHTNp-encoding gene segment specificity and lad antigens, have previously been shown to was ligated to a partial EcoRI-digested fragment carrying all be capable of expressing transferred Ig gene products on the 8 encoding exons. The vector was designated pR-C8TNP. The cell surface and of presenting antigen to T cells (5). The vectors pR-HLTNP, pR-C8TNP, and pR-TKgpt were transfected vector pR-HLTNP, used to establish the sIgMTNP transform- into rK mK Escherichia coli strain K803. A protoplast fusion ants, and vectors pR-CdTNp and pR-TKgpt (Fig. 1), used to technique was used to transform the A20-2J cells with the generate sIgDTNP transformants, were introduced into three vectors (25). Surface IgDTNp-expressing transformants A20-2J cells by a modified protoplast-fusion technique (25). were obtained by serial gene transfers. The vector pR-C8TNP Following the appropriate drug selection, resistant clones was initially introduced into the A20-2J cells, after which were screened for sIgM or sIgD expression by flow G418-resistant clones were pooled and transfected with microfluorimetric analysis using fluorescein-conjugated goat pR-TKgpt. anti-mouse ,u or rabbit anti-mouse 8 . Several Antigens and Antibodies. Hapten (TNP) conjugates ofKLH clones were selected that express varying amounts of sIgM or and ovalbumin (Ova) were prepared as described (27). The sIgD. Fig. 2 illustrates the reactivity of two sIgD clones B-cell hybridoma 34-5-3S, obtained from D. H. Sachs (Na- (ASK4, ASK10) and two sIgM clones (HL, HL8) with anti-8 and tional Institutes of Health, Bethesda, MD), was used as a anti-,u, respectively. Immunoprecipitation of intracellular source of anti-Iad . The B-cell hybridoma anti- [35S]methionine-labeled lysates by class-specific antibodies Sp603, producing antibody (anti-Sp603, IgG1K) against the showed that equivalent amounts of KTNP (24-kDa), /TNP TNP idiotype was a gift from G. Kohler (Max-Planck- (76-kDa), and &rNP (61-kDa) chains were expressed by the Institute, Freiburg, F.R.G.). appropriate transformants and comigrated with Ig chains Flow Microfluorimetric Analysis. (i) Determination of expressed by the Sp603 hybridoma (/TNP, KTNP) (24) and sIgM, sIgD, and Ia levels. The appropriate cells (2 x 105) B188.1 hybridoma X) (29). were incubated for 30 min with fluorescein-conjugated goat (8, anti-mouse ,u antibody (Cappel Laboratories, Cochranville, cb PA) or with fluorescein-conjugated rabbit anti-mouse 8 an- tibody (Nordic Immunological Laboratories, El Toro, CA) or with either biotinylated anti-idiotypic antibody (anti-Sp603) or biotinylated anti-Iad antibody (34-5-3S). After washing in phosphate-buffered saline (Pi/NaCl) containing 0.5% fetal EcoRI bovine serum, cells that had been treated with the biotinyl- VHTnp ated antibody were incubated for 30 min with fluorescein- conjugated avidin (Sigma). (ii) Determination of relative binding capacity for TNP- KLH. The relative binding capacity of the sIgMTNp and sIgDTNP transformants for TNP-KLH was carried out by EcoRI incubating 2 x 105 B-cell transformants with varying amounts VKTnp of TNP-KLH in 100 ,ul of Pi/NaCl containing 0.5% fetal bovine serum and 0.2% sodium azide on ice for 60 min. Cells were washed and then incubated on ice for 30 min with saturating amounts of fluorescein-conjugated rabbit anti- KLH (Cappel, Cochranville, PA). Cells were washed and analyzed with an EPICS-C Coulter flow microfluorimeter on a logarithmically amplified scale. The relative fluorescence index (RFI) was used to convert the BamHI log scale to a linear scale in order to compare samples. The RFI was calculated using the following equation (28): FIG. 1. Structure of the transducing vectors pR-C&1Np and pR-TKgpt. The vector pR-C&TNP carries the rearranged &rNP (17 a- b kilobase pairs) gene at the EcoRI site of pSV2-neo. The vector RFI = 2 2S 6 pR-TKgpt carries the rearranged KTNp (9.6 kilobase pairs) at the BamHI site of pSV2-gpt. Black boxes indicate coding segments for where a is the channel number the TNP-specific heavy chain variable region (VHTNp), for the 8 of mean logarithm of inte- heavy chain constant (CS) region, and for the K light chain constant grated green fluorescence of the test sample and b is the (CK) and TNP-specific variable (VKTNp) regions. The direction of channel number of mean logarithm of integrated green transcription of Ig genes and the simian 40 (SV40) early fluorescence of the control sample. promoter are indicated by arrows. Downloaded by guest on September 23, 2021 Immunology: Tisch et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3833

irrelevant antigen specificity but of the same Ig class as I A anti-Sp603 was shown to give no significant binding to any of the four 1gTNP clones (Table 1). This suggests that the a anti-Sp603 binding was specific and was not occurring via recognition. The four sIgTNp transformants were U) 41 stained with a biotinylated anti-Iad antibody (34-5-3S). The amount of Ia expressed was not significantly different be- 0 tween the sIgMTNP, the sIgDTNp transformants and the 0) B parental cell line (Table 1). E Comparison of sIgMTNp and sIgDp Transformants in z k) Specific Antigen Presentation. A previous report demonstrat- ed that a sIgMTNp transformant can efficiently present z~~~~ specific antigen to the appropriate T cells (5). We wanted to determine whether sIgDTNP could also participate in specific antigen presentation and, if so, how it would compare to sIgMTNP. The sIgDTNp and sIgMTNp transformants were Log of Fluorescence Intensity tested for presentation ofTNPrKLH to the KLH-specific Th FIG. 2. Detection of sIgM and sIgD on the transformants by flow cell hybridoma line CAK1-22. The two sIgDTNp transform- microfluorimetric analysis. sIgDTNp transformants ASK4 (broken ants, ASK4 and ASK10 cells, were observed to present line) and ASK10 (solid line) (A) and sIgMTNp transformants HL (solid TNP6-KLH to the CAK1-22 cells as efficiently as the two line) and HL8 (broken line) (B) were stained with fluorescent anti-8 sIgMTNP transformants, HL and HL8 (Fig. 4). Antigen and anti-,a antibodies, respectively. In addition, the parental cell line presentation activity could be initially detected with 40 ng of A20-2J (traces a)'was stained with the Ig class-specific antibodies. TNP6-KLH per ml and reached saturation at 200 ng/ml for both the sIgMTNp and the sIgDTNP transformants. In com- The relative amounts of sIgTNp expressed by sIgMTNp and parison, antigen presentation by A20-2J cells was first de- sIgDTNP clones were determined with an anti-idiotype anti- tected at 10 Mg ofTNP6-KLH per ml and required 200 ,g/ml body. Anti-Sp603 recognizes a determinant specific for TNP to reach a saturation level similar to that observed for the found in the variable region of the heavy chain (R.T., sIgTNp transformants. Nonspecific presentation of KLH to unpublished data) and therefore should bind equally well to the CAK1-22 cells was not significantly different between the sIgMTNp and sIgDTNP, irrespective of heavy chain class. sIgTNp transformants. Antigen presentation ofTNPrKLH to Biotinylated-IgG-anti-Sp603 was incubated in saturating the CAK1-22 cells by the transformants could be blocked by amounts with the sIgMTNP and sIgDTNP transformants. Rel- a competing antigen. The addition of0.1 Mig ofTNP6-Ova per ative binding of anti-Sp603 was quantitated following the ml inhibited 88% and 92% ofthe ability ofEIL and ASK10 cells, addition of fluorescein-conjugated avidin by flow microfluo- respectively, to present 0.5 Mg of TNP6-KLH per ml. This rimetric analysis and by calculation of the RFI values. Two suggests that the observed efficient presentation activity by sIgMTNP clones (HL, HL8) and two sIgDTNp clones (ASK4, the transformants was due to the transferred sIgTNp. The ASK10) were selected that express similar amounts of the above results suggest that sIgDTNP can participate efficiently TNP-specific idiotype (Fig. 3). The amount of anti-Sp603 in specific antigen presentation and that this activity is bound to the four clones was 43 times that bound by the comparable to that seen with sIgMTNP. parental cell line (Table 1). A biotinylated IgGlK molecule of Relative Binding Capacity of Antigen by sIgMTNp and sIgDTNp Transformants. The sIgMTNp transformants HL, HL8, and sIgDTNp transformants AU4 and ASK10 that ex- A press equal amounts of sIgTNp, wef assessed for their capacity to bind TNPrKLH. The conditions used, Pi/NaCl a containing 0.2% sodium azide and all incubations and washes I at 4°C, should not allow capping and internalization of sIg I I following antigen binding. We determined the binding of .11 TNPrKLH by indirect flow microfluorimetric analysis, .h.. .,k using saturating amounts of fluorescein-conjugated anti- B KLH. An RFI value was calculated for each antigen dose. As a 0) demonstrated in Fig. 5, the relaive antigen-binding capacity 0. of the sIgMTNp and sIgDTNp transformants was not signifi- a) cantly different. Binding could be detected after the addition . of 5 ng of TNPr--KLH and reached a plateau at 100 ng for all z en I~~~ four transformants. In comparison, the parental A20-2J cells cl Table 1. Quantitative expression of Sp603 idiotype, Ia, and sIg on the transformants o RFE IgG1 Control Cell line anti-Sp603 IgG1 Anti-Iad Anti-,u Anti-S HL(,u,K)TNp 43.4 1.1 72.0 31.3 1.0 Log of Fluorescence Intensity HL8(G,K)TNp 43.7 1.3 69.6 29.7 1.0 ASK4(S,K)TNp 43.1 1.0 65.9 1.0 34.2 FIG. 3. Quantitation of Sp603 idiotype on the transformants by ASK10(S,K)TNp 40.8 1.0 66.0 1.0 32.5 flow mirofluorimetric analysis. sIgDTp transformants AUK4 (dotted A20-2J 1.3 1.4 68.0 1.0 1.0 line) and ASK10 (solid line) (A), sIgMTNptransformants HL (solid line) and HL8 (dotted line) (B), and A20-2J parental cell (C) were The sIgTNp transformants and A20-2J cells were stained with the incubated with biotinylated anti-idiotype Sp603 antibody and appropriate antibodies as described. An RFIvalue of 1.0 corresponds fluorescent avidin or with only fluorescent avidin (traces a). to that of the negative control. Downloaded by guest on September 23, 2021 3834 Immunology: Tisch et A Proc. Natl. Acad. Sci. USA 84 (1987) 200 Table 2. Blocking of TNP-KLH binding with competitor TNP-Ova TNP-KLH, TNP-Ova, 0 Transformant ng ng RFI Inhibition, % HL 100 0 11.6 ± 0.5 0 0 10 4.6 ± 0.4 32 a) x 100 50 1.2 ± 0.3 100 *' E ASK10 100 0 12.9 ± 0.6 .0 E0 10 6.7 ± 0.5 56 50 1.0 ± 0.2 100

C,,I /2' /^ ,O,,, ~~~~HLand ASK10 cells (2 x 101) were incubated with TNP6-KLH in /s,',,'the presence or absence of competitor TNP5-Ova. Binding of TNP6-KLH was measured by flow microfluorimetric analysis using )I7|~Iww'' |a fluorescent anti-KLH antibody. RFI values are an average of 0 0.001 0.01 0.1 1 10 100 triplicate samples. An RFI value of 1.0 corresponds to that of the Antigen, jig/ml negative control. FIG. 4. Antigen presentation to KLH-specific Th cells. CAK1-22 cells (10) were cultured with either the sIgDTNp transformants (10-) DISCUSSION ASK4 (U) and ASK10 (i), or the sIgMTNp transformants (105) HL (o) Differences in the structural and physical properties of IgM and HL8 (e), or A20-2J cells (10') (A) with TNP-KLH (solid line) or and IgD have prompted investigators to propose that these KLH (broken line). CTL.L cells (8 x 103) were used for assay of two Ig classes should have different antigen receptor func- IL-2-dependent [PHithymidine incorporation. Each data point rep- tions (30). We have established a system in which direct and resents an average of cultures. triplicate quantitative comparisons can be made between hapten- specific sIgM and sIgD in their role(s) as antigen-specific exhibited no significant binding of TNP6-KLH at all doses receptors. tested. In addition, no significant binding of KLH by the In order to make quantitative comparisons between the sIgTNp transformants was observed (Fig. 5). The binding of sIgMTNp and sIgDTNp transformants in antigen presentation TNP6-KLH by the sIgTNp transformants could be blocked by and antigen binding, clones were selected on the basis of the addition of competing TNP6-Ova (Table 2), on an equivalent levels of sIgTNp and surface, Ia expression, and equimolar basis: 440 ng of TNP6-Ova and 1.0 jig of comparable nonspecific antigen-presentation activity. There TNP6-KLH are equivalent in TNP concentration. This fur- is evidence suggesting that the efficiency of antigen presen- ther suggests that sIgMTNp and sIgDTNp are essential for the tation is proportional to the quantity of sIg expressed (R.T., observed antigen binding. unpublished data) and to the amount of Ia found on antigen- We determined whether the density of the antigens presenting cells (31-33). As demonstrated in Table 1, equiv- TNP1-KLH versus TNP12-KLH altered the binding ability of alent levels of both sIgTNp and surface Ia were expressed by the sIgMTNp and sIgDTNP transformants. As demonstrated in the transformants. The presentation of KLH by the clones Fig. 6, no difference was observed between the HL and ASK10 was considered to be a measure of the efficiency of internal cells in binding either the low or high epitope density antigen processing, which could potentially alter the mea- TNP-KLH antigens. However, both transformants bound surements of specific antigen presentation. As demonstrated greater amounts of TNI12-KLH than of TNP1-KLH, indi- in Fig. 4, the transformants and parental cell line exhibited cating that the binding to either sIgM or sIgD receptor depended on the density of TNP . 16 -

14 14 -

12- a)x 12- X) a) *' 10- 0 10- a) C 0 )8 0 C Co II . 8- 0 ,,I d) .1 I, 'I 0 I .3 8I6- U) 6 ,,,,S co (D cu 4 4 -

(D

2 2-

A t~n,1~:J!.------=-= ------_-- v 0 50 100 150 200 50 100 Antigen, ng Antigen, ng

FIG. 5. Binding capacity ofTNP6-KLH by sIgTNp transformants. FIG. 6. Binding capacity of sIgTNP transformants for TNP,-KLH TNP6--KLH (solid line) or KLH (broken line) was incubated with the versus TNP,2-KLH. TNP1-KLH (broken line) or TNP,2-KLH sIgDTNp tratisformants (2 x 10-) ASK4 (n) and ASK10 (0), the sIgMTNp (solid line) was incubated with the sIgDTNp transformant (2 x 10') transformants (2 x 105) HL (o) and HL8 (e), and A20-2J cells (A). ASK10 (o), and the sIgMTNp transformant (2 x 105) HL (o). Antigen Antigen binding was measured by flow microfluorimetric analysis binding was measured by flow microfluorimetric analysis with a with a fluorescent anti-KLH antibody. Each data point represents an fluorescent anti-KLH antibody. Each data point represents an average of duplicate samples. average of duplicate samples. Downloaded by guest on September 23, 2021 Immunology: Tisch et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3835 comparable nonspecific antigen-processing activities. The sIgD antigen-specific-induced signal transduction, required selected clones were thus very similar with respect to the for proliferation and differentiation. three criteria of selection, ensuring that any difference observed between the transformants should be due to their We thank J. Pawling and M. Wong for expert technical assistance. expression of a distinct heavy chain, p, or S. This work was supported by the National Cancer Institute and The sIgMTNp and sIgDTNP transformants showed equiva- Medical Research Council. R.T. holds a Medical Research Council lent TNP-KLH binding capacity that depended on the TNP Studentship. M.L. is a Terry Fox Research Associate ofthe National epitope density. These results suggest that, despite structural Cancer Institute. differences, sIgD does not bind and crosslink antigen more 1. Chestnut, R. W. & Grey, H. M. (1981) J. Immunol. 126, efficiently than does sIgM. The apparent higher intrinsic 1075-1079. ability of sIgD to bind antigens of low epitope density, such 2. Rock, K. L., Benacerraf, B. & Abbas, A. K. (1984) J. Exp. as thymus-dependent antigens, leading to B-cell activation is Med. 160, 1102-1113. probably due to higher amounts ofthis receptor expressed on 3. Abbas, A. K., Haber, S. & Rock, K. L. (1985) J. Immunol. the surface. Havran et al. (34) have demonstrated that most 135, 1661-1667. splenic B cells express 410-fold more sIgD than sIgM. 4. Lanzavecchia, A. (1985) (London) 314, 537-539. Experimental systems that have shown a differential require- 5. Watanabe, M., Wegmann, D. R., Ochi, A. & Hozumi, N. ment over in (1986) Proc. Natl. Acad. Sci. USA 83, 5247-5251. for sIgD sIgM thymus-dependent antigen-B-cell 6. Singer, A. & Hodes, R. J. (1983) Annu. Rev. Immunol. 1, responses generally involved the removal or blocking of 211-241. either sIgD or sIgM (17, 18, 35). The removal of sIgM (on 7. Tony, H. P., Phillips, N. E. & Parker, D. C. (1985) J. Exp. average <10% ofthe total slg) would be less inhibitory ofthe Med. 162, 1695-1708. ability of a B cell to bind antigen and transduce activating 8. Monroe, J. G. & Cambier, J. C. (1983) J. Exp. Med. 158, signals than would the removal of the more abundant sIgD. 1589-1599. The ability ofthe sIgDTNp transformants to present TNP6- 9. Kakiuchi, T., Chesnut, R. W. & Grey, H. M. (1983) J. Immu- KLH to the KLH-reactive Th hybridoma CAK1-22 was nol; 131, 109-114. demonstrated. This observation agrees with results obtained 10. Sher, I. S., Sharrbw, S. O., Wistar, R., Asofsky, R. & Paul, B W. E. (1976) J. Exp. Med. 144, 494-502. by Tony et al. (7), who demonstrated that normal cells 11. Goding, J. W. & Layton, J. E. (1977) J. Immunol. Rev. 37, could present rabbit anti-mouse 8 antibodies to Th cell lines 152-173. reactive with rabbit Ig. Further, we have demonstrated that 12. Goding, J. W. & Layton, J. E. (1976) J. Exp. Med. 144, the specific antigen presentation activities ofthe sIgDTNp and 852-861. sIgMTNP transformants are equal. The ability of the sIgMTNP 13. Bourgois, A., Abney, E. R. & Parkhouse, R. M. (1977) Eur. J. and sIgDTNP transformants to present small amounts of Immunol. 7, 210-213. antigen (40 ng/ml) is comparable to that seen with enriched 14. Vitetta, E. S. & Uhr, J. W. (1976) J. Immunol. 117, 1579-1583. normal TNP-specific B cells (2, 3). In addition, the sIgMTNP 15. Vitetta, E. S. & Uhr, J. W. (1977) Immunol. Rev. 37, 50-88. and sIgDTNp transformants presented TNP-Ova to the Th 16. Tucker, P. W., Chih-Ping, L., Mushinskit 1. F. & Blattner, National Jewish F. R. (1980) Science 209, 1353-1358. hybridoma 3DO-54.8 (P. Marrack, Hospital 17. Marshall-Clarke, S., Keeler, K. D. & Parkhouse, R. M. (1983) and Research Center, Denver), specific for Ova, with equiv- Immunology 48, 393-400. alent efficiency (data not shown). This suggests that the 18. Cambier, J. C., Ligler, F. S., Uhr, J. W., Kettman, J. R. & presentation activities demonstrated by the sIgTNp transform- Vitetta, E. S. (1978) Proc. Natl. Acad. Sci. USA 75, 432-436. ants are not restricted to the carrier nor the responding Th cell 19. Cambier, J. C. & Monroe, J. G. (1984) J. Immunol. 133, line. Efficient antigen presentation by the B-cell transform- 576-581. ants to the Th hybridoma requires antigen processing. The 20. Vitetta, E. S., Cambier, J. C., Ligler, F. S., Kettman, J. k. & sIgMTNP transformant HL, when fixed with glutaraldehyde Uhr, J. W. (1977) J. Exp. Med. 146, 1804-1811. prior to antigen pulsing, was unable to present antigen to the 21. Chen, Z. Z.,. Coggeshall, K. M. & Cambier, J. C. (1986) J. cells (M.W. and N.H., unpublished work). Immunol. 136, 2300-2304. Th 22. Scher, I., Titus, J. A. & Finkleman, F. D. (1983) J. Immunol. The classical approach in determining antigen binding to 130, 619-625. the cell surface has been the use of RIA techniques. In this 23. Rock, K. L. & Benacerraf, B. (1983) Immunol. Rev. 76, 29-57. paper we demonstrate that quantitative antigen binding 24. Ochi, A., Hawley, R. G., Hawley, T., Shulman, M. J., measurements can be obtained by flow microfluorimetric Traunecker, A., Kohler, G. & Hozumi, N. (1983) Proc. Nitl. analysis. Recently, a direct comparison between flow micro- Acad. Sci. USA 80, 6351-6355. fluorimetric analysis and cellular RIA in measuring HLA-DR 25. Ochi, A., Hawley, R. G., Shulman, M. J. &Hozumi, N. (1983) antigens on human B-lymphoblastoid cell lines demonstrated Nature (London) 302, 340-342. 26. Southern, P. J. & Berg, P. (1982) J. Mol. App!. Genet. 1, 327-341. that results obtained by the two methods were very similar 27. Good, A. H., Wofsy, L., Henry, C. & Kimura, J. (1980) in (28). Here we demonstrate that flow microfluorimetric anal- Selected Methods in Cellular Immunology, eds. Mischell, ysis is highly sensitive in that <10 ng ofTNP-KLH bound by B. B. & Shiligi, S. M. (Freeman, San Francisco), pp. 343-350. the sIgTNp transformants could be detected, and linearity 28. Letarte, M., Addis, J., Iturbe, S. & Petsche, D. (1985) Blosci. could be demonstrated by proportional increases in REI Rep. 5, 923-931. values with in the 29. Neuberger, M. S. & Rajewsky, K. (1981) Proc. Nail. Acad. increases amounts of antigen added. Sci. USA 78, 1138-1142. It is apparent from our findings that the structural and 30. Blattner, F. R. & Tucker, P. W. (1984) Nature (London) 307, physical properties associated with sIgM and sIgD do not 417-422. confer noticeable differences in their ability to bind, process, 31. Unanue, E. R. (1984) Annu. Rev. Immunol. 2, 395-428. and present antigen. The results presented here diminish the 32. Beller, D. I. (1984) Eur. J. Immunol. 14, 138-143. number ofpotential funtional roles specific for either IgM and 33. Bekkhoucha, F., Naquet, P., Pierres, A., Marchetto, S. & IgD as surface antigen receptors. Iffunctional differences do Pierres, M. 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