Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining

Journal oflmmunologicalMethods, 109 (1988) 235-244 Elsevier

235

JIM04741

Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining * Alan R. Brown 1 and Eric Claassen 2 ~ Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38101, U.S.A., and 2 Department of lmmunology, TNO, Med. Biol. Lab., 2288 GJ Rijswijk, The Netherlands

(Received 6 November 1987, accepted 11 December 1987)

Methods are described for the iinmunocytochemical staining of cryostat sections of lymphoid tissue with enzyme conjugates of antigen, idiotype (Id) and anti-idiotype. Results established this as a useful approach, for simultaneously detecting Id and anti-Id antibody forming cells (AFC) in situ. As a model, the 5AF6 Id family associated with the BALB/c mouse antibody response against the p-azophenylarsonate (Ar) epitope was examined by two-color immunocytochemical staining, allowing the simultaneous detection of both Id ÷ and Id- anti-Ar AFC. Spleens from mice secondarily immunized with Ar antigen but not normal mice contained anti-Id AFC stained with the 5AF6 Id but not with another immunoglobulin of the same isotype. A sequential staining method was developed which allowed the detection of both Id and anti-Id AFC in the same tissue, thus providing a means of examining Id and anti-Id antibody networks in intact lymphoid tissues. Key words: Idiotype; Arsonate; Immunocytochemistry; Anti-idiotype; Antibody-forming cell; (Mouse)

Introduction

The study of idiotype (Id) and anti-idiotype (anti-Id) antibody formation during normal immune responses is complicated by their inherent capability to bind to each other. As a conse-

Correspondence to: A.R. Brown, Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38101, U.S.A. * This research was supported in part by CA 34105 from the National Institute of Health and by American Lebanese Syrian Associated Charities (ALSAC). Abbreviations: AFC, antibody forming cell; anti-Id, anti-idiotype; AP, alkaline phosphatase; Ar, azophenylarsonate; BGG, bovine gamma globulin; BSA, bovine serum albumin; HRP, horseradish peroxidase; Id, idiotype; Ig, immunoglobulin; i.p., intraperitoneal; i.e., intravenous; KLH, keyhole limpet hemocyanin; PBS, phosphate-buffer saline.

quence, the assessment of the potential influence of Id and anti-Id antibodies as regulating components in an idiotype network (Jerne, 1974) has been difficult (Rajewsky and Takemori, 1983). Innovative use of immunocytochemical staining has been of considerable utility in studying antibody forming cells (AFC) in situ (Van Rooijen and Claassen, 1986; Van Rooijen et al., 1986). Table I lists some of the advantages of this approach in studying antibody responses. In the context of Id and anti-Id AFC, application of antigen, idiotype and anti-idiotype reagents conjugated with enzymes and applied in appropriate staining procedures would have the following advantages. (1) In situ staining would allow the examination of Id and anti-Id AFC in the context of normal tissue morphology. (2) The number of Id or anti-Id AFC and their clone sizes, could be estimated by counting clustered cells. (3) Detec-

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

236 TABLE I ADVANTAGES OF IMMUNOCYTOCHEMICAL DETECTION OF ANTIBODY FORMING CELLS (AFC) (1) Positivedetection of rare AFC (1 in 106-107) (2) Detection of AFC having low or blocked secretion (3) Intracytoplasmicantibody detection before or after active secretion (4) Detection of membrane antibody positive cells (5) Possibilityof detecting multiple antibody specificitiesin a preparation (6) Determinationof tissue compartments, distances between cells, and clone size of AFC (7) Sequential application of cross-reactiveagents is possible (8) Use of frozen tissue is convenientfor storage and experimental design (9) Mounted slides provide a permanent record of results (10) Capacity to combine irnmunocytochemicalstaining with in situ hybridization (11) Potential for quantitation of AFC

tion of intracellular antibody by immunocytochemical staining would not be subject to potential blocks in the secretion of these cells. (4) Sequential staining as described in this report would allow the detection of both Id and anti-Id A F C in the same section providing the opportunity to examine the tissue microenvironment and relative position of cells making complementary antibodies. (5) It should be possible by densitometry analyses to determine the antibody concentrations within individual Id and anti-Id AFC. (6) Analyses of Id + AFC that are specific for antigen, as well as, parallel set (Goldberg et al., 1983) AFC that are Id ÷ but have undefined or unrelated antigen specificity could provide information about the involvement of parallel set cells in network regulation. Considering these attractive features we have examined the feasibility of utilizing immunocytochemical staining as a means of analyzing B cells making Id and anti-Id antibodies. The idiotype model examined was the 5AF6 idiotype family associated with the B A L B / c mouse antibody response against the p-azophenylarsonate (Ar) hapten (Brown, 1983). This family represents about 30% of the serum anti-Ar in B A L B / c mice immunized with keyhole limpet hemocyanin coupled with Ar (KLH-Ar) (Brown, 1984a) and is related idiotypically and structurally to the 36-60 anti-Ar

family of A strain mice (Brown, 1984b; Brown and Fried, 1987).

Materials

and methods

Animals and immunizations B A L B / c mice (5-6 weeks old) were obtained from Charles Rivers (Wilmington, MA). To study primary immune responses, mice received a single intravenous (i.v.) injection of 0.2 ml of saline containing 10-50 #g of keyhole limpet hemocyanin (KLH) conjugated with the diazonium salt of p-arsanilic acid (KLH-Ar) (Nisonoff, 1967). To examine secondary responses, mice received a primary intraperitoneal (i.p.) immunization of 100 /~g of K L H - A r in 200 #1 of 1 : 1 complete Freund's adjuvant: 30-34 days after priming, mice received a secondary i.v. challenge with 50 ~g of K L H - A r in saline. Mice were bled retroorbitally and spleens were removed and flash frozen by immersion in liquid nitrogen. Frozen tissue was stored at - 20 ° C until sectioning. Reagents The monoclonal anti-Ar antibody, 5AF6, an IgG1 B A L B / c anti-Ar antibody, was produced and specifically purified from tumor ascites fluids by affinity chromatography on bovine gamma globulin-Ar (BGG-Ar)-Sepharose and hapten elution, as previously described (Brown, 1983). Alternatively, 5AF6 was partially purified from ascitic fluids on protein A-Sepharose (Pharmacia, Upsala) by application of the ascites to the adsorbent in a 3.0 M NaC1, 1.5 M glycine, p H 8.9 buffer and subsequent washing with that buffer, followed by elution with 0.1 M citrate buffer pH 6.0. An irrelevant B A L B / c IgG1 hybridoma antibody 32A3 (Melvin et al., 1984) was similarly purified from ascitic fluids. IgG1 monoclonal anti-Id 6BA1 was purified by immunoadsorption to 5AF6-Sepharose, followed by elution by Ar hapten as previously described (Brown and Sealy, 1986a). Purified 5AF6 (Id) and 6BA1 (anti-Id), and irrelevant IgG1 32A3 were conjugated with alkaline phosphatase (AP) (no. P-6772, Sigma Chemical Co., St. Louis, MO) with glutaraldehyde using the method of Boorsma (1984). To prepare an Ar containing enzyme con-

237 jugate for staining anti-Ar forming B cells, horseradish peroxidase (HRP) (no. P-8375, Sigma, RZ3) was conjugated to bovine serum albumin (BSA) using the periodate method of Wilson and Nakane (1978). HRP-BSA was then conjugated with Ar using the diazonium salt of p-arsanilic acid (Nisonoff, 1967). Alternatively, BSA-Ar was conjugated directly to HRP by the same method. Purified monoclonal anti-Ar, 5AF6, was also conjugated to HRP by the above method. Following conjugations all enzyme-linked reagents were dialyzed against phosphate-buffered saline, 0.15 M NaC1/10 mM phosphate buffer, pH 7.4 (PBS), filter sterilized, and stored at 4°C. Inhibitors utilized in immunocytochemical staining reactions were: the IgG1 myeloma protein MOPC 21 in the form of a globulin fraction obtained by 18% Na2SO 4 w / v precipitation of ascites fluids obtained from BALB/c mice injected with MOPC 21 tumor cells obtained from Hazelton Laboratories America (Vienna, VA) through NCI contract N01-CB-71085, 5AF6 Id ÷ hybridoma anti-Ar in both purified and ascitic fluid forms and the IgG2a anti-Ar hybridoma 3C6 idiotopically unrelated to 5AF6. Other inhibitors were BSA-Ar and parsanilic acid (Kodak, Rochester, NY) which was twice recrystallized from 50% ethanol in water.

Immunocytochemical staining of spleen sections Cross-sections (8 /~m) were cut from frozen spleen tissue using a Hacker-Bright Model 2212 Cryostat (Hacker Instruments, Fairfield, N J). Generally, sections from both normal spleens and spleens of KLH-Ar immunized mice were picked up on a single slide to allow comparable staining of all sections. Slides were kept overnight in a humid environment and used immediately or stored at - 2 0 ° C in foil wrapped boxes until stained. Enzyme-linked reagents were diluted in 1.0% BSA in PBS (BSA-PBS); appropriate dilutions for staining were determined by titrating the reagents on cytocentrifuge preparations of hybridoma cells producing antibodies reactive with the reagents. Hybridoma ceils making Id ÷ anti-Ar 5AF6 were used to test for staining by BSA-ArHRP and anti-Id 6BA1-AP. Hybridoma cells making anti-Id 6BA1 were used to test the binding of 5AF6-AP. Staining experiments included the appropriate cell type as a positive control and a

cytocentrifuge prepared slide of cells from the Abelson virus induced immunoglobulin (Ig) negative murine leukemia cells line ABE 19 (obtained from Dr. Marshall Sklar) as a negative control. Methods for staining were those described by Claassen et al. (1986a). Slides were fixed in acetone (analytical grade) containing 0.02% hydrogen peroxide (to destroy any endogenous tissue peroxidase activity) for 10 min. Slides were moistened in PBS and incubated with 0.5 ml (0.25 ml for cytocentrifuge preparations) of appropriately diluted enzyme conjugate in BSA-PBS. Slides were incubated under high humidity for 4 h at 37 o C or overnight at 4°C. The most consistent results were obtained by overnight incubations. Inhibitors were included in some staining procedures; best inhibition was generally obtained when slides were preincubated 4-6 h with the inhibitor; the inhibitor was recovered from the slide, mixed with the enzyme conjugate, and reapplied to the slide for 4 h or overnight incubation. Slides were then washed three times in PBS and stained for their appropriate enzymes. Cells incubated with AP conjugates were stained blue by the method of Burstone (1958), using Fast Blue BB (no. F-0125, Sigma) or stained red with fast red TR salt (No. F-1500, Sigma) as indicator dyes with naphthol AS-MX phosphate (no. N-4875, Sigma) as the AP substrate. Levamisole (no. L-9756, Sigma) was added to the staining reaction at 0.25 m g/ m l to inhibit endogenous AP activity in the tissue. Sequential staining using alkaline phosphataseconjugated anti-Id 6BA1-AP and Id 5AF6-AP were performed by a 4-5 h room temperature incubation with 6BA1-AP which included 0.5 mg MOPC 21. Slides were washed and then overstained with Fast Blue BB to give blue Id + AFC. The slides were then washed three times, preincubated 2-3 h with 1 mg of MOPC 21, and then 5AF6-AP was added for overnight incubation at 4 ° C. Slides were stained with Fast Red to visualize anti-Id AFC. Cells incubated with BSA-ArHRP were stained red for 7 rain with a solution of 4.0 mg of 3-amino-9-ethylcarbazole (no. A-5754, Sigma) dissolved in 250/~1 of dimethylformamide and then added to 9.75 ml of 0.05 M acetate buffer pH 5.0 containing 0.06% hydrogen peroxide (Graham et al., 1965). Slides were washed three times in PBS, once in water, and then counter-

238 s t a i n e d with h e m a t o x y l i n ( R i c h a r d - A l l a n M e d i c a l Industries, R i c h l a n d , M I ) for 5 - 1 0 s. A f t e r further w a s h i n g in water, slides were m o u n t e d in 10% G e l v a t o l ( M o n s a n t o , St. Louis, M O ) a n d 30% glycerol in PBS p H 7.4. Slides s i m u l t a n e o u s l y i n c u b a t e d with H R P a n d A P conjugates were first s t a i n e d with F a s t Blue BB to d e t e c t A P a n d then with 3 - a m i n o - 9 - e t h y l c a r b a z o l e to detect H R P .

10 m g / m l B S A - P B S insured that non-specific b i n d i n g of the conjugates to the tissue sections d i d n o t take place. As expected, spleen sections f r o m K L H - A r i m m u n i z e d mice i n c u b a t e d with u n c o n j u g a t e d H R P p r o d u c e d no staining, i n d i c a t i n g the a b s e n c e of p l a s m a cells m a k i n g a n t i b o d i e s specific for H R P alone. V i r t u a l l y all the cells s t a i n e d with A r h a d large c y t o p l a s m s c h a r a c t e r i s t i c of p l a s m a cells.

Results

Detection of l d + A FC W e also wished to d e t e r m i n e the feasibility of detecting B cells in spleen sections m a k i n g a p a r ticular i d i o t y p e d e f i n e d b y a m o n o c l o n a l a n t i - I d a n t i b o d y . M o n o c l o n a l a n t i - I d 6BA1, an I g G 1 ant i b o d y that was derived f r o m a B A L B / c m o u s e i m m u n i z e d with B A L B / c a n t i - A r 5 A F 6 , reacts with an i d i o t o p e on 5 A F 6 a n d a few o t h e r related m o n o c l o n a l anti-Ar, a n d b i n d s to o n l y a subset of the 5 A F 6 f a m i l y r e p r e s e n t i n g a b o u t 7% of the B A L B / c a n t i - A r r e s p o n s e m e a s u r a b l e in s e r u m (Brown a n d Sealy, 1986b). 6BA1 recognizes an i d i o t o p e requiring b o t h heavy a n d light chains for its expression (Brown a n d Sealy, 1986a). Spleen sections o b t a i n e d f r o m B A L B / c mice d u r i n g a p r i m a r y a n t i - A r response were s t a i n e d with 6BA1A P a n d d e v e l o p e d with F a s t Blue BB i n d i c a t o r dye, p r o d u c i n g a blue staining of cells that b e a r

Detection of anti-Ar A F C Bovine s e r u m a l b u m i n c o n j u g a t e d with A r a n d then c o u p l e d to h o r s e r a d i s h p e r o x i d a s e ( B S A - A r H R P ) was chosen as an e n z y m e - l i n k e d A r p r o t e i n c o n j u g a t e to d e t e c t a n t i - A r p r o d u c i n g cells. Prel i m i n a r y studies e s t a b l i s h e d that H R P c o u l d not r e a d i l y be d i a z o t i z e d directly with A r a n d still retain the c a p a c i t y to detect a n t i - A r A F C . Spleen sections f r o m B A L B / c mice s e c o n d a r i l y imm u n i z e d with K L H - A r were f o u n d to c o n t a i n n u m e r o u s a n t i - A r A F C while n o r m a l spleen was d e v o i d of a n y a n t i - A r A F C . S t a i n i n g c o u l d be c o m p l e t e l y b l o c k e d b y the a d d i t i o n of either BSAA r (5 m g / s l i d e ) o r 10 - 2 M free arsanilic acid h a p t e n to B S A - A r - H R P c o n j u g a t e on tissue slides ( T a b l e II). D i l u t i o n of e n z y m e - l i n k e d reagents in

TABLE II SUMMARY OF RESULTS OBTAINED STAINING SPLEEN SECTIONS WITH ENZYME CONJUGATES OF BSA-Ar ANTIGEN, 6BA1 ANTI-IDIOTYPE AND 5AF6 IDIOTYPE Approximate numbers of stained cells/section: - , no specific staining; _+_+,rare stained cells < 2/section; +, 2-10/section; + +, 10-20/section; + + +, 20-50/section, + + + +, > 50/section; ND, not done. Staining reagent

Inhibitor

Normal BALB/c

KLH-Ar immune BALB/c

BSA-Ar-HRP

10 2 M hapten BSA-Ar MOPC 21 (IgG1) MOPC 21 + Ar hapten MOPC 21 + 3C6 (IgG1, Id-) MOPC 21 + 5AF6 (IgG1, Id ÷) MOPC 21 (IgG1) MOPC 21 (IgG1) + 3C6(IgG2a, Id ) MOPC 21 (IgG1) + 5AF6(IgGI, Id ÷ ) MOPC 21

ND ND _+ + _+ + +_ + ND

+ ± i + + + + + + + + + + -

6BA1-AP (IgG1, anti-Id)

5AF6-AP (IgG1, Id) 32A3-AP (IgG1 irrelevant)

-

+++ +++ +++ + +++ + + +

239 the 6BA1 recognized idiotope, To preclude the possibility of detecting any cells producing autoanti-IgG1 antibodies, slides were preincubated with 1 mg of a globulin fraction of ascites containing the B A L B / c IgG1 myeloma protein, MOPC 21 prior to the addition of the 6BA1-AP conjugate. Table II summarize experiments demonstrating the specificity of this staining. Spleen sections obtained from mice 4 days after KLH-Ar immunization contained substantial numbers of cells bearing the 6BA1 recognized idiotope. Occasional cells were found to be stained with 6BA1-AP in normal B A L B / c spleen sections. Staining of cells with 6BA1-AP was inhibited approximately 50% by the addition of 10 -2 M arsanilate hapten. This is contrasted with the almost complete inhibition of 6BA1 binding to 5AF6 in a radioimmunoassay (Brown and Sealy, 1986a). Of interest was the finding that hapten was a poor inhibitor of 6BA1-AP binding in normal mouse spleen. Preincubating the 6BA1-AP anti-Id with an excess of 5AF6 inhibited staining about 70%, but similar treatment with another IgG1 B A L B / c anti-Ar antibody lacking the 5AF6 idiotope, failed to inhibit staining with 6BA1-AP. As a control, sections from spleens of normal mice and from spleens taken at several times after primary and secondary immunization were stained with an irrelevant IgG1 hybridoma antibody enzyme conjugate with and without preincubation with IgG1 MOPC 21. Stained cells were very rare and were virtually nonexistent when preincubations were carried out with MOPC 21. This demonstrated that virtually no auto-anti-IgG1 producing cells could be demonstrated under these conditions. Together these findings establish the binding specificity of enzyme-linked anti-Id 6BA1. Double staining to detect Id + and ld A FC

anti-Ar

We wished to determine whether BSA-Ar-HRP and 6BA1-AP could be used in a double staining procedure like that developed by Van Rooijen and Kors (1985) to allow the simultaneous detection of all anti-Ar AFC and the Id + subset of those cells in situ. Spleen sections from B A L B / c mice immunized with K L H - A r were stained at the same time with BSA-Ar-HRP and 6BA1-AP. Fig. l a shows a photomicrograph of a stained section

where a group of red staining anti-Ar AFC are intersPersed with several darkly staining Id + antiAr AFC. Close examination of doubly stained cells illustrated in Fig. lb showed they were violet in color due to the deposition of both red and blue dyes. Occasionally cells were observed which appeared to be stained with only blue indicating that some cells made antibodies bearing the 6BA1 idiotype but that were not specific for Ar. Double staining of sections from spleens of normal mice confirmed results of staining with BSA-Ar-HRP and 6BA1-AP alone and indicated that no anti-Ar AFC could be detected but that occasional cells stained with anti-Id alone were evident. Results for both normal and K L H - A r immunized B A L B / c mice indicated their spleens contain B cells producing the 6BA1 idiotype but not having anti-Ar activity. Histological examination of spleen sections from K L H - A r immunized mice showed that both Id + and ld anti-Ar AFC were found mainly in the periphery of the periarteriolar lymphocyte sheaths and in the lymphoid sheaths around the terminal arterioles, as previously described for anti-trinitrophenyl AFC (Claassen et al., 1986b; Van Rooijen et al., 1986). Detection of anti-Id A F C

Studies above have established the feasibility of immunocytochemical staining as an approach for examining antibody and Id production. Idiotype network theory (Jerne, 1974) predicts that anti-Id AFC should also exist and that the number of these cells ought to be increased in antigen-immunized animals producing the Id. In situ immunocytochemical staining would offer a unique and attractive means of detecting anti-Id AFC that would obviate the difficulty of detecting these cells by plaquing methods or detecting their secreted anti-Id products in the face of circulating Id. Monoclonal anti-Ar antibody 5AF6 preparations purified by affinity chromatography on BGG-Ar by hapten elution or on protein A-Sepharose were conjugated with alkaline phosphatase (5AF6-AP) to determine whether immunocytochemical staining with 5AF6 might detect such anti-ld AFC in situ. Spleen sections from normal B A L B / c mice and B A L B / c secondarily immunized with KLH-Ar were stained with 5AF6-AP in the presence of and after preincubation with an

240

241 excess (1 mg/slide) of the IgG1 MOPC 21 to prevent non-specific staining. Results established that virtually no cells in normal spleen were stained by 5AF6-AP, but increased numbers of 5AF6-AP stained cells were detectable in the spleens of the immunized mice. Fig. 2a shows a photomicrograph of a spleen section containing cells stained with 5AF6-AP. While most binding of 5AF6-AP was by solitary cells, some groups of Id binding cells could be found in tight clusters implying that these cells were clonally expanded. 5AF6-AP conjugates prepared with 5AF6 specifically purified on a BGG-Ar or with 5AF6derived by protein A purification from ascitic fluids from 5AF6 tumor-bearing mice, were equally effective in detecting Id binding cells. Use of protein A derived 5AF6 ruled out that staining was due to possible contamination of 5AF6 with BGG-Ar. Leaching of antigens from immunoadsorbents has been described as a problem (Hagen and Strejan, 1987) and it was possible that contaminating BGG-Ar complexes in the antigen purified 5AF6-AP reagent might bind to high affinity anti-Ar cells and account for staining with the 5AF6-AP. This possibility was ruled out. The specificity of 5AF6 staining was established by the capacity of unlabeled 5AF6 to inhibit the staining of 5AF6-AP while an irrelevant anti-Ar failed to inhibit. Failure of unlabeled I d - anti-Ar antibody to inhibit also rules out the unlikely possibility that injected K L H - A r might have accumulated in plasma cells and that 5AF6 staining occurred by virtue of its capacity to bind intracytoplasmic Ar antigen (Table II). As a further test, sections from spleens known to have many 5AF6-AP binding cells incubated with a large amount of the irrele-

vant B A L B / c IgG1 conjugate 32A3-AP in the absence of any MOPC 21 IgG1 absorbent produced virtually no staining. This again substantiated the Id specific staining by 5AF6-AP. Detection of Id and anti-Id A F C in the same tissue It has been established that both Id and anti-Id producing cells can be detected in situ by immunocytochemical means. It would be very attractive to detect both of these cell types simultaneously in the same tissue. This could afford the opportunity to examine the spacial relationships between A F C with complementary specificities. Since the Id and anti-Id enzyme conjugates bind to each other, a sequential staining method was developed in which tissue was stained first with anti-Id (6BA1-AP) and developed with blue dye and subsequently stained with Id (5AF6-AP) and developed with a red dye. Staining of Id A F C blue and anti-Id A F C red was chosen to allow unambiguous identification of anti-Id forming cells as red cells. Sequential staining caused Id + A F C cells to stain violet in part because overstaining with blue may not have exhausted all the enzyme activity of 6BA1-AP, and because 6BA1 binding of 5AF6-AP could cause additional enzyme binding necessary for the deposition of red dye. Similar sequential staining was also possible using an H R P conjugate of 5AF6. Fig. 2b is a photomicrograph of a sequential staining of a B A L B / c spleen section from a mouse 4 days following a secondary immunization with KLH-Ar. It shows an anti-Id A F C cell in close proximity to several Id A F C and illustrates that Id and anti-Id A F C were frequently found close to each other in tissue. Sequential staining of this type was not inhibited

Fig. 1. a: Spleen section of KLH-Ar immunized mouse, double stained with BSA-Ar-HRP to stain for anti-Ar AFC (red), and 6BA1-AP to stain for Id ÷ anti-Ar AFC (violet). Bar represent 22 /~m. b: Spleen section stained like a, showing deposition of both red and blue dye in a double stained (violet) cell, which is therefore, an Id + anti-Ar antibody forming cell. Red cell is not Id +. Bar represents 4/~m. Fig. 2. a: Spleen section from a KLH-Ar immunized mouse, incubated with 5AF6-AP,showinggroups of Id binding cells (blue). Bar represents 44 p.m. b: Spleen section from a 4-day secondarily immunized BALB/c mouse incubated sequentially with 6BA1-APand developed with a blue dye resulting in blue Id + AFC and then stained with 5AF6-APand developed with a red dye staining anti-ld AFC red. Both antibody enzymeconjugates were incubated in the presence of excess IgG1. Bar represents 5.0 #m.

242 by preincubation with an irrelevant anti-Ar further confirming the Id and anti-Id binding specificities of these stained cells.

Discussion In this report we have described the application and established the feasibility of using double and sequential immunocytochemical staining procedures in the identification and the analysis of antibody, Id, and anti-Id AFC in lymphoid tissues. A sequential staining method was demonstrated which allowed the simultaneous detection of both Id and anti-ld AFC in the same tissue section. This should be a powerful approach for examining the occurrence of Id and anti-Id AFC during normal immune responses. In situ detection of the T15 anti-phosphorylcholine idiotype using rabbit anti-Id and an enzyme-linked anti-rabbit second antibody has been described (Razzeca et al., 1986). Our approach differs in that purified monoclonal enzyme-linked anti-Id was used to detect Id + A F C simultaneously with a second enzyme conjugate to detect all cells making anti-antigen antibodies. Studies reported here also describe the detection of anti-ld AFC and concurrent detection of Id and anti-ld AFC in the same tissue. Most of the cells examined in this study had extensively stained cytoplasms indicating they were filled with antibody. Consequently, they were considered terminally differentiated B plasma cells with relatively short lifespans. In a previous study it has been demonstrated that quiescent B cells bearing only membrane Ig can also be detected by in situ immunocytochemical staining (Claassen et al,, 1986a). It remains to be established whether committed but unstimulated B cells with Id and anti-ld specificities can be practically detected by this approach. It is important to point out that secreted extracellular antibody is not detected by this staining methodology. The thinness of the sections and the fixation and washing steps insure that free antibody will be washed away, leaving only membrane-bound, intracytoplasmic, or immune complexed antibody to be stained (Van Rooijen and Claassen, 1986).

The double staining procedure used to simultaneously detect Id ~ and anti-At AFC in these studies differs mechanistically from most double staining methods in that the Ar and anti-ld compete for overlapping epitopes in the combining sites of these antibodies. It is probable that the affinity of the anti-Id for its idiotope relative to the affinity of the cell's antibody for Ar influences the balance of double staining, Staining intensity is also influenced by the quantity of intracellular antibody in individual cells, as well as, the length of enzyme substrate exposure, Consequently, an appropriate balance of anti-ld and antigen conjugates is required to produce doubly stained ld + violet cells. The finding of some ld + cells in normal mice lacking anti-Ar production, as well as, occasional Id + cells not binding BSA-Ar in Ar immunized mice indicated that Id ÷ AFC having another antigen binding specificity might exist. This would be similar to parallel sets described in the A strain anti-Ar C R I a (Hornbeck and Lewis, 1985) and other idiotype systems (Goldberg et al., 1983: Marion et al., 1984). Further specificity studies and quantitative analyses are being performed to confirm a parallel set(s) in this model. Immunocytochemical staining could offer an attractive method for examining parallel set expression during normal immune responses. Monoclonal I g G l anti-Ar 5AF6, prototype member of the 5AF6 anti-Ar family, was shown to be bound intracytoplasmically in spleen cells from mice immunized with KLH-Ar. The number of stained cells were much higher than the rare stained cell observed in normal mouse spleen. Some clusters of Id binding cells were found, implying that clonal expansion had probably occurred from a single precursor. Increased numbers of ld binding cells in mice secondarily immunized with K L H - A r is consistent with an antigen-driven expansion of ld ~ cells, followed by expansion of anti-ld AFC. Sequential immunocytochemical staining for Id and anti-Id AFC established that cells producing complementary antibodies can be detected and may occur in close proximity to each other, Analyses of the spacial relationships and distances between ld + AFC (both antigen-specific and antigen-non-specific parallel sets) and anti-ld AF("

243 m a y help to establish if these cells influence each other at a systemic level through their secreted p r o d u c t s or m i c r o e n v i r o n m e n t a l l y as suggested b y Pollock a n d K e a r n e y (1984). T h e p r o d u c t i o n of Id a n d a n t i - I d d u r i n g imm u n e responses has b e e n detected b y other approaches. Circulating a n t i - I d a n t i b o d i e s a n d antiId p l a q u e - f o r m i n g cells have b e e n observed in other idiotype systems d u r i n g n o r m a l i m m u n e responses (Cosenza, 1976; B i n i o n a n d Rodkey, 1982). A n t i - I d a n t i b o d i e s eluted from the surfaces of Id ÷ p l a q u e - f o r m i n g cells, based o n h a p t e n reversible i n h i b i t i o n of p l a q u e f o r m a t i o n by A F C , have b e e n i m p l i c a t e d in i n h i b i t i n g the secretion of Id + a n t i b o d i e s from p l a s m a cells (Goidl et al., 1984; Wieties a n d Moticka, 1986). T h e significance of these a n t i - I d to the course of a n t i b o d y responses has b e e n difficult to assess. I n vivo analysis of secreted a n t i - I d is difficult due to the f o r m a t i o n of rapidly cleared I d - a n t i - I d complexes. Analyses of a n t i - I d p l a q u e - f o r m i n g cells suffer from the technical difficulties of c o m p l e m e n t m e d i a t e d lysis of I d - c o n j u g a t e d erythrocytes (Cosenza, 1976). Plaque analysis for b o t h Id a n d a n t i - I d secreting cells m a y u n d e r e s t i m a t e A F C if they are n o t actively secreting or if their secretion is b e i n g blocked by the b i n d i n g of c o m p l e m e n t a r y antibodies. The i m m u n o c y t o c h e m i c a l s t a i n i n g m e t h o d s described in this report avoid m a n y of the difficulties associated with these alternative m e t h o d s a n d will allow the detailed study in intact tissues of the frequencies a n d kinetics of Id a n d a n t i - I d A F C p r o d u c t i o n in the B A L B / c antia r s o n a t e a n d other idiotype models.

Acknowledgement T h e authors would like to t h a n k R.E. Sealy for excellent technical assistance.

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