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Isolation of antigen-binding virgin and memory B cells
Journal of Immunological Methods, 92 (1986) 45-57
45
Elsevier JIM 04013
Isolation of antigen-binding virgin and memory B cells * C h r i s t o p h e r D. Myers, Virginia M. Sanders a n d Ellen S. Vitetta ** Department of Microbiology, Universityof Texas Health Science Center, Southwestern Medical School, Dallas, TX 75235, U.S.A,
(Received10 March 1986, accepted 31 March 1986)
One major obstacle in studying the activation of antigen-specific B cells is the small number of B cells reactive with a particular antigenic epitope. In this report, we describe a method by which large numbers of highly purified antigen-binding cells can be obtained. We have shown that by varying the haptenation level of the erythrocytes used for rosetting, we can purify antigen-binding B cells which have different affinities for the antigenic epitope. Thus, memory cells (which have receptors of higher affinity) can be prepared and these cells are essentially free of contaminating virgin cells. The effects of varying the haptenation levels on the red cells used for purifying the B cells can, in turn, be related to the precursor frequency of secreting cells following their activation with T cells and antigen. Key words: B cells, antigen-specific;Memorycell; Lymphocyte
Introduction It is estimated that 1 / 1 0 6 B cells express immunoglobulin (Ig) receptors specific for any given
* This work is supported by NIH Grants AI-11851 and AI21229. ** Address for correspondence: Dr. E.S. Vitetta, Department of Microbiology,Universityof Texas Health ScienceCenter at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235, U.S.A. Abbreviations: ABC, antigen-binding cells (virgin); ACT, hypotonic ammonium chloride; BSA, bovine serum albumin; BSS, balanced salt solution; BSS/5% FCS, balanced salt solution supplemented with 5% fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; DNP, dinitrophenyl; FCS, fetal calf serum; GAMIg, goat anti-mouse immunoglobulin; Gly-Gly, glycyl glycine buffer; HBSS, Hanks' balanced salt solution; HRBC, horse erythrocytes;MABC, memory antigen-binding cell; PBS, phosphate-bufferedsaline; PFC, plaque-forming cells; RFC, rosette-formingcells; IliA, radioimmunoassay; TD, thymus-dependent; Trt, helper T lymphocytes; TNBS, trinitrobenzene sulfonic acid; TNP, trinitrophenyl; TNP-HRBC,TNP-haptenatedhorse erythrocytes; TNPx-KLH,TNP-haptenated keyholelimpet hemocyaninwith X moleculesof TNP per 100000 Mr of KLH.
antigenic epitope. Following interaction of these antigen-specific B cells with antigen and carrierspecific T cells, the B cells expand and secrete antibody specific for this antigen. Proliferation and secretion are driven by a series of soluble cytokines (Howard et al., 1984; Vitetta et al., 1984). Because of their small number, antigenspecific B cells are difficult to isolate and study. As a result, the majority of B cell activation studies are performed with antigen-primed cells (only a small fraction of which are antigen-specific) or polyclonal activators such as anti-Ig or mitogens which activate a fraction of the B cells regardless of their antigen-binding specificity (Defranco et al., 1982). Nevertheless, a number of approaches have been developed for obtaining populations of B cells enriched for antigen-specific cells where activation by antigen, rather than mitogens or anti-Ig can be studied. Three major approaches have been utilized to enrich antigen-binding cells (ABC): (a) soluble fluoresceinated antigen can be bound to cells and the ABC can be separated from the non-ABC by
0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
46
flow cytometry (Julius and Herzenberg, 1974); (b) antigen can be immobilized on solid matrices, e.g., glass beads, plastic, gelatin, and ABC can be absorbed and eluted (Truffa-Bachi and Wofsy, 1970; Choi et al., 1974; Haas and Layton, 1975); and (c) for hapten-binding B cells, haptenated red cells can be bound to the B cells and the rosetted ABC can be purified by velocity and/or density sedimentation techniques (Elliot, 1979; Snow et al., 1983). Each of the above techniques has its advantages and drawbacks. We have utilized the third approach since relatively large numbers of resting ABC can be isolated within a 2 day period. In this report we describe a modification of the rosetting method, originally described by Snow et al. (1983) and Yefenof et al. (1985), which allows us to produce up to 2.5 x 107 B cells, 50-90% of which are antigen-specific. This technique can be applied to both memory and virgin B cells and is readily performed by a single person. Finally, by varying the haptenation density of the red cells used for rosetting, it is possible to isolate B cells bearing receptors of different affinities (Yefenof et al., 1985). This allows for the separation of memory cells (which bear receptors of high avidity) from virgin cells (Yefenof et al., 1985).
at 37 °C for 30 min with occasional gentle mixing. The reaction was stopped by diluting the cell suspension to 30 ml with cold BSS supplemented with 5% fetal calf serum (FCS) (BSS/5% FCS) followed by centrifugation at 4 ° C. Cells were then washed once in 30 ml glycyl glycine buffer (GlyGly) and three times in 30 ml BSS/5% FCS to obtain a cell pellet. Gly-Gly was prepared by dissolving 0.6% (w/v) glycyl glycine in a veronal buffer containing 1.82 mM sodium barbital, 3.11 mM barbital, 0.15 mM calcium chloride, 0.23 mM magnesium chloride and 145 mM sodium chloride.
Animals
BALB/c female mice, aged 8-16 weeks, were obtained from Cumberland Farms, and were maintained in our own animal facilities. For the preparation of memory antigen-binding cells, mice were primed with a single dose of 100 /tg of TNP-KLH in complete Freund's adjuvant i.p. 10-15 weeks prior to sacrifice. Age-matched untreated mice were used as controls in these experiments.
Preparation of splenocytes Materials and methods
Haptenated horse erythrocyte (HRBC) preparation 5 ml HRBC in Alsever's solution (Colorado Serum Co., Denver, CO) were layered onto 5 ml of Ficoll-metrizoate, density 1.09 (10.08 g Ficoll 400 + 30 ml sodium metrizoate in 102 ml) and centrifuged at 2000 X g for 20 rain at 4°C. Cells at the interface were discarded and the cells in the pellet were washed twice at 4°C in 30 ml of balanced salt solution (BSS) by centrifugation for 5 rain at 1200 × g. This produced a pellet of 1.0 ml packed cells. An alternative to the Ficoll separation was to wash the cells three times in BSS discarding the leukocyte-rich buffy coat after each wash. Trinitrobenzene sulfonic acid (TNBS) (usually 4 or 20 mg for memory or virgin cells respectively) was dissolved in 10 ml cacodylate buffer, 0.28 M, pH 7.4, filter sterilized and added directly to the cell pellet. Cells were resuspended and incubated
All procedures were carried out at 4°C unless otherwise stated. Spleens from 2 to 20 mice were removed and a single cell suspension was prepared in BSS. The suspension was allowed to settle for 10 min on ice and the pellet was discarded. The remaining cells were centrifuged for 10 min at 300 X g and the cells in the pellet were resuspended in 0.5-3 ml (depending on number of spleens) hypotonic ammonium chloride (ACT) (0.8% ammonium chloride, 0.1% KH2PO4) and incubated at 37°C for 4 min to lyse red ceils. After incubation, the cells were immediately diluted with cold BSS and centrifuged. The cells in the pellet were then resuspended in 10 ml BSS/5% FCS per mouse spleen, passed through a 22 gauge needle to prepare a single cell suspension, and layered onto Ficoll-metrizoate (density 1.09 g/ml) using 20-25 ml of cells per 8 ml Ficoll in clear 50 ml tubes (Corning, cat. no. 25339). Tubes were centrifuged for 20 rain at 4°C at 2000 x g. Cells at the interface were ~ollected and washed (2-3 interfaces/50 ml wash) in 50 ml BSS/5% FCS. Cells
47 were pooled and washed at 4 ° C in 50 ml BSS/5% FCS. The first centrifugation after Ficoll was carried out at 660 x g and the second centrifugation was carried out at 300 x g. The A C T and Ficoll steps removed the majority of the mouse erythrocytes. The Ficoll step also removed non-viable cells and the majority of granulocytes.
Purification of ABC Splenocytes prepared as described above were resuspended in BSS/5% FCS at 2.25 X 107cells/ml and trinitrophenyl-haptenated horse erythrocytes ( T N P - H R B C ) were resuspended at a final concentration of 1.5% in BSS/5% FCS. All procedures were carried out at 4 ° C to prevent cell activation (Pike et al., 1983). Percoll gradients were prepared as described in Table I and contained 10 ml of 75% Percoll overlayered with 10 ml of 30% Percoll in alcohol-sterilized, 50 ml round bottomed tubes (Nalgene, cat. no. 3117-0500). 0.5 ml of the T N P - H R B C suspension was added to each ml of splenocytes and 10-20 ml of this mixture was immediately layered onto each gradient. The tubes were covered with foil and centrifuged at 4 ° C for 15 min at 300 x g, accelerating and decelerating gently. Pellets from these tubes were collected by careful aspiration of the supernatant and the cells in the pellet were resuspended immediately in 15 ml 1.09 Percoll per pellet and layered onto 15 ml, 75% Percoll in the same type of tube. These gradients were overlayed with 5 ml, 1.07 Percol~, the tubes were covered with foil and centrifuged at 120 x g for 35 min, again accelerating and decelerating gently. The
cell pellets were collected by aspiration, the cells were resuspended in 15 ml BSS/5% FCS and an aliquot was removed for rosette counting prior to centrifuging at 300 x g for 10 min at 4°C. Cells were then washed once in serum-free RPMI-1640 and were resuspended in 10 ml of serum-free RPMI-1640 containing 25 m M Hepes (Whittaker MA Bioproducts, cat. no. 12-115) with 1.5 m g / m l Trypsin (Sigma, cat. no. T-0134) and 1.5 m g / m l protease (Sigma, cat. no. P-5147). This mixture was incubated for 30 rain at 37°C with vigorous resuspension after 15 and 30 rain. The enzymatic reaction was 'stopped' by the addition of 2 ml of FCS and the cells were pelleted by centrifugation at 300 X g for 10 min at 4°C. Cells were then resuspended in 1.5 ml BSS/5% FCS per original Percoll tube and layered onto Ficoll, using 2 ml Ficoll and 3 ml cell suspension in sterile 12 x 75 polystyrene tubes. Gradients were centrifuged at 2000 x g for 20 min at room temperature and the cells at the interface were collected using a glass pasteur pipette which had been prewashed with FCS to reduce cell adherence. The cells were then washed twice in BSS/5% FCS by centrifugation at 4°C, first at 6 6 0 X g for 10 min and then at 300 × g for 10 min. All procedures between the admixtures of splenocytes with the T N P - H R B C and the enzyme treatment were carried out as rapidly as possible at 4 ° C to avoid cell activation. Finally, cells were resuspended in complete medium (RPMI-1640 plus 10% FCS, 2 m M glutamine, 5 x 1 0 -5 M 2-mercaptoethanol, 50 U / m l streptomycin and 50 /~g/ml penicillin) at 5 x 105/ml and cultured overnight in an atmo-
TABLE I PERCOLL PREPARATIONS a Percoll
75% 1.09 1.07 30%
Density
Volumes (ml)
Calculated (g/l)
Measured (g/l)
Percoll
H20
10 × HBSS
1.098 1.083 1.065 1.039
1.0975 1.0825 1.0650 1.0375
37.5 19.17 5.0 6.0
7.0 7.53 3.9 11.8
5.0 3.0 1.0 2.0
1.0 M Hepes pH 6.8 0.5 0.3 0.1 0.2
a Names and preparations of Percoll mixtures used in gradients. Names and percentages of Percoll are those used by Snow et al. (personal communication). Calculated densities are based on a 1.0 density for reagents other than Percoll and a Percoll density of 1.130. Densities were measured with a narrow range hydrometer and are accurate to +0.0025 g/l. The amounts described produce enough Percoll for two gradients.
48 sphere of 10% CO2, 7% O2 and 83% N 2 at 1 ml/well in 24-well cluster plates (Costar) to allow re-expression of surface Ig (Snow et al., 1983).
Percoll gradients The constituents of these gradients were based on those described by Snow et al. (1983). The major modifications were in the buffering system. Snow et al. (1983) used 10 x Dulbecco's modified Eagle's medium (DMEM) to prepare the isoosmotic Percoll with bicarbonate buffer. 10 x DMEM is no longer available commercially and the powder is technically difficult to dissolve at 10 x . Thus, we now use 10 x Hanks' balanced salt solution (HBSS). Bicarbonate buffering is intended for use in high CO 2 environments and, in Percoll gradients, often requires pH adjustment with HC1. To avoid problems with pH fluctuations, Hepes buffer, pH 6.8, is now used. Detailed procedures for the preparation of the gradients are described in Table I.
Rosetting techniques The enumeration of rosetted cells has been used as the major criterion for purity throughout this work since the number of rosettes has previously been shown to correlate with presence of ABC (Snow et al., 1983). 100 ffl samples for initial rosette counting were collected from the suspension layered onto the first Percoll gradients; these samples were centrifuged at 70 × g for 10 min at 4°C in 12 × 75 round bottomed tubes and the pellet was incubated at 4°C for 10 min to ensure rosette formation prior to resuspending and counting. Samples taken throughout the preparation were counted directly after collection without further washing. Rerosetting at later times was performed in a manner analogous to the initial rosetting procedure using at least 10 freshly prepared TNP-HRBC per leukocyte. All counting of rosettes was performed in the presence of crystal violet to aid in the enumeration of unrosetted cells. The results represent the mean of 4 counts of each sample.
Radioirnmunoassay (RIA) Direct measurement of the level of hapten expressed on the surface of TNP-HRBC was performed by RIA. Briefly, TNP-HRBC were sus-
pended at 2 x 109 cells per ml in 1% bovine serum albumin/phosphate-buffered saline (BSA)/ (PBS)-azide and 10/~1 of the cells were added per V-bottom ELISA microwell, precoated with 1% BSA/PBS azide. 10 #l/well monoclonal anti-dinitrophenyl (DNP) antibody (29.13 (Scott and Fleischman, 1982)) at 1.5 /~g/ml were added and cells were incubated for 1 h at room temperature before washing them three times in BSS containing 1% BSA. Plates were developed with 125I-goat anti-mouse immunoglobulin (GAMIg) (2× 105 cpm/well), incubated for 1 h at room temperature, washed four times and counted.
Indirect assay for haptenation The total amount of TNBS bound to the HRBC was assessed indirectly by measuring the amount of unreacted TNBS remaining after haptenation. Varying concentrations of TNBS were dissolved in cacodylate buffer, pH 7.4. 0.5 ml samples were removed and added to 2 ml Gly-Gly. After haptenation, cells were pelleted by centrifugation (without dilution) and 0.5 ml of the supernatant was removed and added to 2 ml Gly-Gly. Cells were then washed and utilized as normal. The absorbance at 340 nm (A340) of solutions in GlyGly was then measured and the amount of TNBS remaining was estimated from a standard curve.
Proliferation assays Proliferation assays were carried out as described by Snow et al. (1983). Freshly prepared TNP-ABC or splenic B cells were cultured for 16-20 h at 5 × 10 4 per microwell in 100 /~1 of complete medium. Various reagents, as listed in the Results section, were added to give a final volume of 200/~1. Cells were incubated for 3 days and then pulsed for 16 h with 1 /~Ci per well of [3H]thymidine (ICN Radiochemicals, Irvine, CA) and harvested on a MASH II automated cell harvester. The uptake of [3H]thymidine was assessed by scintillation counting.
Aspirin Acetyl salicylic acid (J.T. Baker, Philadelphia, PA) was dissolved in absolute alcohol at 167 mg/ml immediately prior to use. This stock solution was added, with shaking, to all reagents used between mixing the TNP-HRBC with the spleno-
49 cytes and the enzyme treatment, at a final concentration of 0.4 mg/ml (Snow et al., 1983). The Hepes buffering of Percoll gradients minimized the pH effects of aspirin and no further adjustments were made; however, BSS and BSS/5% FCS were neutralized after addition of aspirin using 1 M NaOH.
Limiting dilution assay Limiting dilution assays of TNP-ABC were performed as described by Yefenof et al. (1985). Briefly, after overnight incubation, ABC were harvested and their viability was assessed by trypan blue exclusion. Between 10 and 1000 viable cells were cultured with 5 x 104 irradiated, KLHprimed T cells and 0.4/tg/ml TNP33-KLH in 10 /zl per Terasaki well. After 5 days, cultured cells were washed and transferred to 96-well microtiter plates with TNP-sheep erythrocytes (SRBC) and guinea pig complement (absorbed with SRBC) in 75 /~1 BSS/5% FCS. Plates were centrifuged to form a cell lawn and incubated at 37°C for 1 h before scoring for the presence of PFC on a Zeiss inverted microscope at 40 x magnification. Sixty replicates were scored per condition. Precursor frequencies were assessed using the Poisson distribution as described by Lefkovitz and Waldmann (1979).
Results
Enrichment of TNP-ABC In evaluating this modified procedure for the purification of TNP-ABC, we have assessed total cell yield and the percentage of rosetting cells. Table II shows these values for several such cell preparations. These values are representative of our experience with a large number of similar purifications. In this procedure, 1 ml packed cell aliquots of HRBC were haptenated with either 10, 20, 40 or 120 mg of TNBS. BALB/c splenocytes from 12 spleens were collected and the erythrocytes were lysed by ACT treatment. Remaining red cells, dead cells and granulocytes were removed on a Ficoll gradient to give a total cell yield of 1.16 x 109cells which was subsequently divided into four
aliquots. The cell yield is subject to large variations. Spleens from 7-12-week-old BALB/c mice yield 9.2 x 107-t- 20% cells per spleen after lysis and Ficoll separation. Omission of the Ficoll step at this stage has no deleterious effect on the preparation but increases the total numbers of cells being processed. Another alternative is to utilize a Percoll gradient to purify high density resting cells (Snow et al., 1983) for which we now use a modification of the method of Layton et al. (1985). As shown in Table II, the yield of rosettes in the first centrifugation step is quantitative, i.e., the number of rosettes recovered in the pellet was close to the number originally layered onto the gradient. This is one major modification in the procedure described by Snow et al. (1983). This single step replaces the repeated rosetting and short centrifugation steps, but the cell yields and purity are similar. The second Percoll gradient is not significantly different from that used by Snow et al. (1983) and the resulting cell population is similar. There is a consistent small loss of rosettes in this step as can be seen in Table II; however, the purity of the rosetting cells recovered was t'outinely in the range of 50-90% Disruption of the rosettes and removal of contaminating TNP-HRBC remains a problem. We have been unable to improve upon the method of Snow et al. (1983) using proteolytic enzymes followed by Ficoll-metrizoate separation. This gives a significant cell loss (Table II) but a variety of other methods have shown no improvement (see below). Cells are routinely incubated overnight at 5 x 105/ml in complete medium, in Costar 24-well clusters at 1 ml/well or microwells at 100/d/well. After overnight culture, the majority of the cells plated are recovered and these cells are 80-95% viable.
Enrichment of TNP-(memory)ABC (MABC) Few modifications of the above methodology are required for purification of memory cells. Mice are primed 10-15 weeks prior to use with 100/~1 TNP-KLH at 1 mg/ml in complete Freund's adjuvant and the TNP-HRBC are haptenated at the
50 TABLE II PURIFICATION OF TNP-ABC a Haptenation (mg/ml packed cells)
Virgin cells 10
Memory cells 20
40
120
4
Number of cells (after Ficoll) ( x 10- 8)
2.9
2.9
2.9
2.9
11.0
Initial rosettes (%)
1.25
2.28
5.64
6.45
1.1
Rosettes after 1st Percoll step Yield (rosettes) ( x 10 -6) Purity (%)
3.7 45.6
6.1 40.7
13.0 51.2
14.8 51.6
8.1 12.1
Rosettes after 2nd Percoll step Yield (rosettes) ( × 10-6) Purity (%)
2.9 67.0
4.1 73.0
10.1 73.2
13.4 74.2
7.0 54.0
1.3
2.8
3.7
7.5
7.1
0.8 84
1.6 80
3.1 85
7.5 83
5.3 84
56
52
51
49
30
YieM After Ficoll step ( X 10 -6) Yield after overnight incubation Viable cells recovered ( x 10 -6) Viability (%) Rerosetting percentage
a Four virgin cell purifications were performed in parallel using the same initial splenocyte preparation. The purification of MABC was carried out with splenocytes from immunized mice at a separate time. Aliquots of cells were removed at all stages of the purification and their content assessed. The above data are from representative experiments. In a series of 10 consecutive purifications of virgin ABC, we recovered 5.0 × 105 (+_ 2.9 × 105) cells after Ficoll for each 108 cells initially rosetted with a mean purity of 70.8% (+8.3%). After overnight incubation, 3.4 × 105 (+1.9 x l0 s) cells at 54.4% (+9.6%) purity was recovered. In a similar series of 10 purifications of memory ABC, the cell yield was 5.1 x 103 (+ 3.2 x 105) per 108 cells rosetted with a mean purity of 59.6% (+ 6.7%). Overnight viability counts and rerosetting percentages were not routinely measured for MABC.
lower rate of 4 m g / m l p a c k e d cell v o l u m e (Yefenof et al., 1985). A l l steps used in the p u r i f i c a t i o n are i d e n t i c a l a l t h o u g h these rosettes are t r e a t e d even m o r e gently w h e n r e s u s p e n d e d b e t w e e n gradients. A s can b e seen in T a b l e II, the level of purific a t i o n achieved with the first Percoll step is lower with m e m o r y R F C t h a n virgin R F C and, a l t h o u g h the second Percoll g r a d i e n t i m p r o v e s this yield, the final p u r i t y of M A B C is i n v a r i a b l y lower t h a n t h a t of virgin A B C . F o l l o w i n g overnight culture, viability of M A B C is good, b u t the recovery of rosettes tends to b e lower t h a n t h a t o b t a i n e d with virgin ABC. Finally, in M A B C purifications, the n u m b e r of T N P - H R B C in the rosette-rich fraction is always higher t h a n in c o r r e s p o n d i n g fractions f r o m virgin A B C , b u t this is rectified b y the F i c o l l
step. The m e m o r y cells purified in this w a y have b e e n extensively c h a r a c t e r i z e d b y Y e f e n o f et al. (1985, 1986). Analysis o f the parameters of the different procedures used T N P - H R B C to splenocyte ratio Snow et al. (1983) d e s c r i b e d rosette f o r m a t i o n using T N P - H R B C at 0.5% p a c k e d r e d cells a n d leukocytes at 15 x 1 0 6 / m l . This yields a T N P H R B C : s p l e n o c y t e ratio of b e t w e e n 6 a n d 7 to 1. Since c o n t a m i n a t i o n with T N P - H R B C at the end of the p r o c e d u r e c a n b e a p r o b l e m , we e v a l u a t e d p r o c e d u r e s using fewer r e d cells in the p r e p a r a t i o n of the T N P - A B C . A s c a n b e seen in T a b l e III,
51 T A B L E III R E D CELL R E Q U I R E M E N T S a
%
TNP-HRBC per white cell
Final cell yield ( X 106 )
Rosettes
% of rerosettes (after cell culture)
6.7 5 4 3 2 1
2.03 2.11 1.84 1.03 0.50 0.30
70 63 66 53 70 50
70 74 63 68 50 58
a Cell preparations were performed and contained identical numbers of splenocytes with varying numbers of T N P - H R B C at 10 m g T N B S / m l of cells. The yield of rosette-forming cells, purity and rerosetting efficiency were measured. Similar results were obtained in a second experiment.
below a ratio of 5 : 1 for T N P - H R B C : splenocytes, the absolute cell yield began to fall although purity was not affected down to a 2 : 1 ratio. From these data we have chosen to continue using a 0.5% packed cell volume of TNP-HRBC.
Percoll gradients (a) Medium and buffering system.
As stated above, 10 x D M E M is no longer available and cannot be prepared reliably from powder. Therefore, we have examined other combinations based on HBSS, RPMI-1640 and 10 × PBS buffered either with bicarbonate or Hepes. Hepes buffer consistently gave better buffering than bicarbonate and, when prepared at p H 6.8, overcame the basic nature of the Percoll. HBSS was chosen since it consistently gave results equivalent to the b i c a r b o n a t e / D M E M mixture. RPMI containing high concentrations of glucose was not used because of cell clumping with Hepes buffer and Percoll (data not shown). (b) Inclusion of FCS in Percoll. It was thought that the long period during which the cells were maintained in serum-free conditions would be deleterious both to cell viability and purity of the preparation due to non-specific aggregation of cells. Consequently, a series of purifications was carried out using 5% FCS in all Percoll layers. In six experiments, the final cell yield was increased by 17.5% + 11.3% without any significant effect on either final purity or the % of rerosetting cells after overnight incubation. However, there was a significant increase in red cell
contamination after the Ficoll step. Therefore, the use of FCS was not adopted. (c) Percoll densities. The bottom layer of Percoil provides the major purification step. Therefore, we tested the effects of changing the density of this layer. Increasing the density of the bottom layer to 78% Percoll caused a decrease in cell yield, whereas 81% Percoll completely prevented rosettes from reaching the pellet, even when additional centrifugations were ~erformed (data not shown). Using the procedures described in Table I, no significant variations in yield were obtained using 4 different batches of commercially available Percoll (data not shown). (d) Centrifugation time. In calibration experiments, almost all rosettes reach the cell pellet within 15 min on the first Percoll gradient and 25 min on the second gradient. However, on a routine basis, we have found it necessary to centrifuge the second gradient for 30-35 min to ensure consistently good cell recoveries. Since both gradients are separating cells based on both bouyant density and sedimentation velocity, it is important not to centrifuge the cells too long since the number of unrosetted leukocytes and erythrocytes in the pellet increases with time. (e) Cell loading. The majority of data presented are based on purifications in which splenocytes were resuspended at 1.5 × 107cells/ml and 10-12 ml were loaded on each gradient. However, recent data show that up to 25 ml of cells at the same concentration can be loaded onto each gradient without significant effects on the yield or
52
purity of the cells recovered. Thus, a purification based on 12-15 spleens can be performed using only four gradients in each stage.
Effects of aspirin Aspirin (Acetylsalicylic acid) was originally included in all purification steps where TNP-HRBC and splenocytes were together to avoid activation of the B cells (Snow et al., 1983). Since that time we have made further changes in the purification procedure and the effects of aspirin have been re-examined. In our current experiences aspirin has no effect on purification (Table IV), proliferation of TNP-ABC or anti-TNP PFC responses (data not shown). In addition, the presence or absence of aspirin has no effect on phospholipid metabolism induced by antigen in these cells (Myers et al., manuscript in preparation). Since Snow et al. (1983) found that the addition of aspirin was essential in avoiding cell activation during the isolation procedures, we can only conclude that the lower hapten density and shorter period of B cell contact with hapten avoids the problem of activation seen previously. Thus, all preparations are currently performed in the absence of aspirin.
Disruption of rosettes Snow et al. (1983) describe disruption of rosettes by treating the rosettes with trypsin and protease. We have titrated the enzyme concentration and confirmed that 1.5 m g / m l of each enzyme is opti-
T A B L E IV E F F E C T OF ASPIRIN ON T H E P U R I F I C A T I O N OF TNPABC a Parameter
With aspirin
Without aspirin
T N P - A B C Yield (n = 5) % Purity (n = 5) % Rerosetting (n = 2)
1.05 × 107 65.8 55.0
0.97 × 107 65.2 59.7
a Results of cell preparations carried out in parallel with and without aspirin. Data represent mean values for the number of experiments listed. Standard deviations for the % purity were less than 6% and a close correlation was seen in cell yields of all experiments.
TABLE V TITRATION OF PROTEOLYTIC ENZYMES a Enzyme concentration (mg/ml)
Rosettes remaining (%)
Recovery of cells after culture ( × 10- s )
Rerosetting cells (%)
3.0 1.5 0.75 0.38 0.19
0.39 0.60 2.50 2.62 2.32
8.85 8.50 9.02 8.20 9.10
59.5 64.5 56.0 56.8 53.6
a T N P - A B C were taken immediately after the final Percoll step and incubated for 30 min at 3 7 ° C with various concentrations of trypsin and protease. The rosettes remaining immediately after enzyme treatment were counted and the cells were Ficolled and cultured. Viable cell yields and rerosettes were measured 18 h later. Similar results were obtained in a second experiment. Significant cytotoxicity was seen at 4.5 m g / m l of trypsin and protease.
mal for disruption of rosettes (Table V) with 750 ~tg/ml yielding a percentage of unbroken rosettes and 4.5 m g / m l causing significant cytotoxicity to the lymphocytes. There are clearly a number of different effects occurring to induce this disruption since the enzymes alone would not be expected to remove all the surface Ig (Calvert et al., 1981). To further examine the effects of enzyme treatment on the cells, TNP-ABC were prepared and incubated overnight as usual. 74% of these cells rerosetted. Aliquots of these rosettes incubated at 37°C for 30 min in BSS contained 50% rosettes after treatment, but when incubated at 37°C for 30 min in RPMI (which is isotonic for human cells and hypotonic for murine cells), the level of rosettes dropped to 24%. Similarly, an aliquot of TNP-ABC was incubated in enzymes in the usual manner and then rosetted with fresh TNP-HRBC producing 11% rosettes. 15% rosettes were present when fresh TNP-ABC were rosetted with enzyme-treated TNP-HRBC. This suggests that the enzyme treatment removes some surface Ig from the TNP-ABC and some TNP from the TNP-HRBC, but at least part of the effect is due to low tonicity of the incubation medium. Enzyme treatment has been shown to activate some cells (Vischer, 1974, 1984) and to change the activation responses of B cells (Kern, 1985; Ramanadham et al., 1985). Therefore, other proce-
53 dures for disrupting rosettes were examined. Osmotic shock with A C T was found unsuitable in two experiments due to lowered viability after overnight incubation. Monomeric antigen in the form of TNP-lysine was shown to be effective at disrupting rosettes prepared with H R B C haptenated with 12 mg TNBS per ml packed cell volume (Fig. 1). Disruption was time and dose-related with an optimal concentration of 1 m g / m l in complete medium (Fig. 1). When utilized in BSS, this concentration caused cytolysis, probably due to lack of adequate buffering (data not shown). However, in agreement with previous data (Wigzell and Maleka, 1970), this method failed to disrupt rosettes prepared with red cells haptenated with higher concentrations of TNBS (20 m g / m l , Fig. 1). The monomeric antigen probably remained on the cell surface since the percentage of rerosettes formed by cells disrupted with TNP-lysine followed by overnight incubation was only 10% compared with 65% for the rosettes disrupted with proteolytic enzymes.
Haptenation ratio of TNP-HRBC Snow et al. (1983) reported haptenation of horse erythrocytes (HRBC) by the method of Kettman and Dutton (1970) using 120 mg TNBS per packed ml HRBC. As can be seen in Table II, the cell yield is highly dependent on haptenation ratio. We have re-examined this phenomenon by a number of parameters. Table II shows that the rate of initial rosette formation increases rapidly with increasing hap-
tenation levels. We have no explanation for the large differences in levels of initial rosettes described now versus previously. We have found that inclusion of aspirin at the time of rosetting causes a slight decrease in the % of initial rosettes, but this does not explain the observed difference. Examination of the rerosetting potential of the cell preparations described in Table II is presented in Table VI. As can be seen, rerosetting with cells haptenated at the same level as used in the purification were consistently below the levels of purity seen at the time of preparation. Higher levels of haptenation revealed a modest increase in the level of rerosetted cells, while decreased haptenation resulted in a marked decline in the percentage of rosettes. Similar findings have been demonstrated with T N P - M A B C (Yefenof et al., 1985). This finding suggests that TNP-ABC represent a population of cells bearing surface Ig with widely varied affinities for TNP, and that those cells prepared with the lower haptenation ratio represent the higher affinity population. To further address this point, the data shown in Fig. 2 were collected. Here the response of hapten bound to the T N P - H R B C , the hapten available on the surface of the T N P - H R B C and number of rosettes formed with splenocytes from unimmunized mice (initial rosetting level) were titrated versus increasing amounts of TNBS added to the HRBC. It should be noted that even at the lowest haptenation levels, this reaction does not go to completion, so one must take great care with the time and temperature of haptenation, as well as TNBS concentration, in order to obtain consistent
TABLE VI REROSETTING OF TNP-ABC a Haptenation ratio of TNP-HRBC used in preparation (mg TNBS/ml HRBC)
Haptenation ratio of TNP-HRBC used for rerosetting (%) 10
20
40
120
10 20 40 120
55.9 + 5.7 28.9 + 4.2 19.1 ± 5.1 6.9 ± 2.1
70.7 ± 2.1 51.6 ± 4.5 22.8 ± 7.5 13.2 ± 2.8
75.7 + 1.8 62.4 ± 2.2 51.2 ± 5.2 35.0 ± 9.0
76.5 + 5.0 65.0 ± 7.0 57.2 + 3.7 49.2 ± 6.8
a Cells from the preparation described in Table II were incubated overnight and then rerosetted with freshly prepared TNP-HRBC at the four haptenation ratios used in the preparation. Results represent mean + standard deviation from four separate counts on each sample. These results are representative of many similar experiments.
54
results. Secondly, as suggested by the previous data, the percentage of cells which initially form rosettes is related to the level of haptenation and thus markedly effects of final yield. The final test of the purification procedure is to determine the level of precursors responding to antigen and T cells. We examined this response using a limiting dilution assay for plaque-forming cells (PFC) in response to a thymus-dependent antigen, TNP30-KLH and the KLH-primed, irradiated helper cells (TH). As shown in Fig. 3, the precursor frequency in the TNP-ABC population is inversely proportional to the level of haptenation of the TNP-HRBC, suggesting that the cell
70 60 50
yield achieved with the more highly haptenated TNP-HRBC represents cells of lower affinity which do not respond to antigen in a PFC assay. Calculations based on the three experiments used for Fig. 3 show that the total yield of precursors (i.e., overnight viable cell count × precursor frequency) was constant at all haptenation levels at 4.2 × 10 4 (___0.5 × 10 4) with no obvious trends. The largest variation between the lowest and highest yield within a single experiment was twofold. With cells prepared usiJ~g; TNP-HRBC haptenated using 10 mg per ml packed cell volume, all points in the limiting dilution assay fell on a straight line. However, cells prepared with more heavily haptenated TNP-HRBC had a tendency to fall to the right of the line when higher input cell numbers were used. This phenomenon is similar to that described by Aarden et al. (1980) and Lefkovits et al. (1980) suggesting that a significant number of T suppressor cells are present in the populations purified with the more highly haptenated
40 U) LU
F-
50 (I)
0 r~
-
t51
1.5
60
i
~
]~
I
I
-I 6
5
10
' ~ A~L050
20 ~o--o o
1.0 I
I0 0
20
5
-15 t.2 0.3
b
-I 4
I
0
10 20 50 40 50 PERIOD OF INCUBATION (rain)
60
Fig. 1. Disruption of rosettes by TNP-lysine. TNP-ABC were prepared using HRBC haptenated with either 20 or 12 mg of TNBS per ml packed cells. After overnight incubation, cells were rerosetted with similarly prepared TNP-HRBC. At time 0, various concentrations of TNP-lysine were added and the number of rosettes remaining during incubation at 4°C was assessed. Cells were resuspended by brief, genre vortexing prior to removing for sampling. This treatment had little effect on cells lacking TNP-lysine. Experiments were performed in complete medium. Similar results were obtained in two other experiments, zx represents TNP-ABC prepared and rerosetted with TNP-HRBC haptenated with 20 nag TNBS/ml packed cell volume incubated in TNP-lysine at 1.5 mg/ral. The remaining curves were derived from data using TNP-ABC prepared and rerosetted with TNP-HRBC haptenated with 12 mg TNBS/ml packed cell volume and incubated in TNP-lysine at 0.5 mg/ml ( 0 ) , 0.68 mg/ml (O), 0.80 mg/ml ([]), and 1.0 mg/ml (11).
F-
20
t0
5
z5
t2
06o30t5'c
0
[TNBS](mg/ml)
Fig. 2. TNP-HRBC were prepared using various levels of TNBS to prepare the HRBC. The dilutions of TNBS used were also used to set up a standard curve after reaction with Gly-Gly. After haptenation, the supernatants were sampled and the absorbance at 340 nm (A340) of this Gly-Gly reaction (A) was compared with the standard curve (,x) (insert) to ascertain the amount of TNBS bound (n). The level of TNP expressed on the cell surface after haptenation was measured by RIA using monoelonal anti-DNP antibody (29.13) and an 125I-anti-mouse Ig (11). TNP-HRBC produced were then used in rosetting experiments both with freshly prepared splenocytes (initial rosetting) (e) and TNP-ABC prepared the previous day using 20 mg/ml TNBS on the TNP-HRBC (©). Similar results were obtained in two other experiments.
55 100 _J ..J
~ w
w Z
o_
5o
I
1-
lo6
100 200
400 600 INPUT CELLS PER WELL
800
1000
Fig. 3. Cells were purified using TNP-HRBC haptenated with 10 (C)), 20 (@), 40 (rq), or 120 (11) mg TNBS/ml packed cell volume (c.f. Table II). After overnight incubation, cells were harvested and plated at between 10 and 1000 viable cells/well in limiting dilution assay (see materials and methods section). Presence of PFC was scored on day 6 and plotted as the log of the proportion of negative wells vs. cells per well. Results represent mean±standard deviation from three separate experiments with 60 wells/point each. Points falling to the right of the least squares line probably represent suppressor effects at high cell concentration (see results section) and least squares lines were calculated using 4 (O), 3 (@,D) or 2 (11) points and the origin to give precursor frequencies of 1/72 with 10 mg TNBS, 1/167 with 20 mg TNBS, 1/418 with 40 mg TNBS and 1/649 with 120 mg TNBS. Correlation coefficients were all better than 0.98.
TNP-HRBC. However, further studies will be required to substantiate this hypothesis.
Discussion
Over the past 20 years, steady progress has been made in the purification of antigen binding B cells. Various approaches have been attempted, each with its own advantages and disadvantages and each producing populations with varied properties. The earliest attempt to purify antigen-binding cells was reported by Wigzel and Andersson (1968). Primed lymph node cells were passed over antigen-coated beads and the adsorbed cells were eluted by mechanical agitation. Significant retention of antigen-specific PFC was achieved, but the eluted fraction showed only 2.5-fold enrichment in PFC and considerably decreased viability. There was also a large amount of non-specific adsorp-
tion of cells to the beads resulting in significant cell loss. Immunoadsorbents based on polyacrylamide (Truffa-Bachi and Wofsy, 1970), agarose (Davie and Paul, 1971) and Sephadex (Schlossman and Hudson, 1973) reduced nonspecific binding and permitted recovery of cells by digestion of the matrix. Other solid-phase systems that were investigated include binding to plastic tubes (Choi et al., 1974) and nylon fibers (Rutishauer et al., 1973). By 1976, a system utilizing anti-fluorescein conjugated to Sephadex G-200 could bind a fluoresceinated protein antigen and act as an immunoadsorbent for antigen-binding B cells. Scott (1976) achieved a 1% yield of input cells containing 30% ABC with this technique. Haas and Layton (1975) described a solid-phase immunoadsorbent system in which ABC were adsorbed to hapten-derivatized gelatin-coated petri dishes. This system had the advantages of low non-specific adherence, simple recovery of bound cells by melting the gel at 37°C and easy regulation of the hapten density. Dependent on the haptenation level, between 0.25% and 2% of input cells were recovered (c.f. Table II, Fig. 2). However, cells released by melting the gelatin apparently retained bound antigen causing specific antigen non-responsiveness. Collagenase treatment reversed this effect and allowed detection of up to 42% RFC in the purified population. Various modifications of this approach have produced increased purities of the recovered populations (Nossal and Pike, 1978; Pike and Nossal, 1984). The method holding the greatest promise for achieving 100% pure populations of ABC is cell sorting. However, starting with less than 1% positive cells, extremely long sorting periods are required. Thus, this method is applicable to some in vivo biological assays (Julius et al., 1972; Julius and Herzenberg, 1974) but is impractical for most in vitro cell culture and biochemical assays. The other major approach to ABC purification, rosetting, was described by Brody (1970) and Brody and Papermaster (1970). Using rate zonal sedimentation in BSA gradients, yields were as low as 0.1-0.01% of input cells with purity usually below 10%. Early experiments with velocity sedimentation produced less than 5% RFC (Osaba, 1970; Wilson, 1973), but by 1979, Elliot had developed a two-step velocity sedimentation tech-
56 nique selecting for large rosettes giving rise to 75-90% pure RFC. However, this technique required specialized equipment, was technically exacting and limited in cell handling capacity. Density gradient centrifugation of rosettes on Ficoll (Sulitzeanu and Axelrad, 1973) or BSA (Tanenbaum and Sulitzeanu, 1975) produced puffties up to 20% and modification of this technique by Kenny et al. (1978) to include one buoyant density and one sedimentation velocity centrifugation on Ficoll produced 50-100% RFC from primed spleens and 20-40% RFC from virgin splenocytes. This approach was further improved by Snow et al. (1983) using Percoll gradients to achieve 60-70% RFC from unprimed spleens. The above methods all suffer to a greater or lesser extent from the same problems, namely non-specific cell contamination, poor overall yields, and difficulties in removing bound antigen. These problems have been identified and discussed by the authors of the papers dealing with each method. The current technique must be compared with other available methods. With regard to purity, Kenny et al. (1978) and Snow et al. (1983) demonstrated significant enrichment of ABC from unimmunized splenocytes by RFC measurement and our results of - 70% RFC equal or surpass their preparations. The present study, along with those of Snow (1983) and Yefenof (1985, 1986) are the only ones in which rerosetting has been reported. We feel that this is an important measure as it reflects the purity of the cells used in the experiments, which differs somewhat from the purity suggested by rosette counts determined at the end of the purification. The original descriptions of haptenated gelatin panning on used levels of RFC as the assay for purity, and demonstrated around 40% purity (Haas and Layton, 1975). Later modifications were assayed for purification of PFC (Nossal and Pike, 1978) or cells proliferating in response to antigen (Pike and Nossal, 1984). Here the highest purity was achieved with cell yields of the order of 0.001%, containing PFC at up to 1 in 3.5 cells. Although our limiting dilution assays show approximately 20-fold lower PFC responses, our cell yields are up to 300-fold greater. The only other description of purification of long-term primed (> 8 weeks) memory ABC is that of
Yefenof et al. (1985) in which both the present technique and that of Snow et al. (1983) were used' and no differences were found in purity. In terms of technical difficulty, this technique requires no specialized equipment except a centrifuge that will accelerate and decelerate smoothly. The only exacting procedures are the pouring and harvesting of Percoll gradients and a single person can easily handle 4-8 gradients in a single preparation. Twenty-five mouse spleen equivalents can be processed in 1 day. As stated above, the yield is dependent on the level of haptenation used, but even at 10 mg TNBS per ml packed HRBC, this will routinely produce around 3 X 106 ABC/109 input cells, which is superior to all other techniques described when estimated using equivalent haptenation levels. We have been unable to improve on published techniques for the removal of bound antigen from the surface of ABC. Although trypsin-pronase treatment of ceils has previously been demonstrated to induce cell activation (Vischer, 1974) and affect other cell responses (Kern, 1985), Yefenof et al. (1985) demonstrated that both MABC and ABC recovered after overnight incubation are > 95% small, G O cells, suggesting that the enzyme treatment is not activating the cells. These data also support our findings that the use of aspirin during the rosetting stages of this technique is unnecessary (data not shown). We have shown a direct relationship between the hapten density on the HRBC, the total cell yield of the purification and the functional capacity of the recovered cells. This area has not been well studied by previous authors (Haas and Layton, 1975; Yefenof et al., 1985; Howard et al., 1986). Decreasing the haptenation rate allows for the purification of MABC essentially free from contaminating virgin ABC (Yefenof et al., 1985). Increased haptenation levels clearly result in higher total cell yield but not an equivalent increase in precursor frequencies. This is probably due to the purification of weakly cross-reactive cells capable of binding antigen but not responding to it to produce antibody measurable in the present limiting dilution assays. Thus, the final cell yield is not a good criterion for the assessment of a purification procedure; functional criteria must also be assessed.
57 I n s u m m a r y , we have described a system allow: ing for the efficient purification of relatively large n u m b e r s of antigen-specific m e m o r y a n d virgin B cells. This system facilitates the c o m p a r i s o n of B cell activation b y antigen, anti-Ig a n d mitogens such as lipopolysaccharide. M o d i f i c a t i o n of h a p t e n a t i o n ratios used also allow for the dissection of the responses of virgin a n d m e m o r y B cells a n d their respective activation requirements.
Acknowledgements W e t h a n k Ms. T. W i l s o n a n d Ms. M.M. Liu for technical assistance a n d Ms. G.A. Cheek for secretarial help.
References Aarden, L.A., R.B. Corley, A. Soderberg and I. Lefkovitz, 1980, Immunology 41, 399. Brody, T., 1970, J. Immunol. 105, 126. Brody, T. and B.W. Papermaster, 1970, J. Immunol. 105, 139. Calvert, J.E., N.R. Ling and R. Jefferis, 1981, Immunology 44, 89. Choi, T.K., D.R. Sleight and A. Nisonoff, 1974, J. Exp. Med. 139, 761. Davie, J.M. and W.E. Paul, 1971, J. Exp. Meal. 134, 495. Defranco, A.L., E.S. Raveehe, R. Asofsky, and W.E. Paul, 1982, J. Exp. Med. 155, 1523. Elliot, B.E., 1979, Immunological Methods, Vol. 1, eds., .I. Lefkovitz and B. Pemis (Academic Press, London) p. 241. Haas, W. and J.E. Layton, 1975, J. Exp. Med. 141, 1004. Howard, M., K. Nakanishi and W.E. Paul, 1984, Immunol. Rev. 78, 185. Julius, M.H. and L.A. Herzenberg, 1974, J. Exp. Med. 140, 904. Julius, M.H.,. T. Masuda and L.A. Herzenberg, 1972, Proc. Natl. Aead. Sci. U.S.A. 69, 1934.
Kenny, J.J., J.E. Merrill and R.F. Ashman, 1978, J. Immunol. 120, 1233. Kern, M,, 1985, J. Immunol. 134, 2260. Kettman, J. and R.W. Dutton, 1970, J. Immunol. 104, 1558. Layton, J.E., P.H. Krammer, T. Hamaoka, J.W. Uhr and E.S. Vitetta, 1985, J. Mol. Cell. Immunol. 2, 155. Lefkovitz, I. and H. Waldman, 1979, Limiting Dilution Analysis of Cells in the Immune System (Cambridge University Press, Cambridge) p. 60. Lefkovitz, I., L.A. Aarden and R.B. Corley, 1980, Immunology 41, 407. Nossal, G.J.V. and B.L. Pike, 1978, J. Immunol. 120, 145. Osaba, D., 1970, J. Exp. Meal. 132, 368. Pike, B.L. and G.J.V. Nossal, 1984, J. Immunol. 132, 1687. Pike, B.L., D.L. Vaux and G.J.V. Nossal, 1983, J. Immunol. 131,554. Ramanadham, N., S.V.S. Gollapudi and M. Kern, 1985, J. Immunol. 134, 4. Rutishauer, U., P. D'Eustaehio and G.M. Edelman, 1973, Proc. Natl. Acad. Sci. U.S.A. 70, 3849. Schlossman, S.F. and L. Hudson, 1973, J. Immunol. 110, 313. Scott, D.W., 1976, J. Exp. Med. 144, 69. Scott, M.J. and J.B. Fleischman, 1982, J. Immunol. 128, 2622. Snow, E.C., E.S. Vitetta and J.W. Uhr, 1983, J. Immunol. 130, 607. Stein, P., P. Dubois, D. Greenblatt and M. Howard, 1986, J. Immunol. 136, 2080. Sulitzeanu, D. and M. Axelrad, 1973, Immunology 24, 803. Tanenbaum, R. and D. Sulitzeanu, 1975, Immunology 29, 409. Trnffa-Bachi, P. and L. Wofsy, 1970, Proc. Natl. Acad. Sci. U.S.A. 66, 685. Vischer, T.L., 1974, J. Immunol. 113, 58. Viseher, T.L., 1984, Clin. Exp. Immunol. 55, 99. Vitetta, E.S., K. Brooks, Y.-W. Chen, P. Isakson, S. Jones, J. Layton, G.C. Mishra, E. Pure, E. Weiss, C. Word, D. Yuan, P. Tucker, J.W. Uhr and P.H. Krammer, 1984, Immunol. Rev. 78, 137. Wigzell, H. and B. Anderson, 1968, J. Exp. Med. 129, 23. Wigzell, H. and O. M~lkel~i,1970, J. Exp. Med. 132, 110. Wilson, J.D., 1973, Immunology 25, 185. Yefenof, E., V.M. Sanders, E.C. Snow, R.J. Noelle, K.G. Oliver, J.W. Uhr and E.S. Vitetta, 1985, J. Immunol. 135, 3777. Yefenof, E., V.M. Sanders, J.W. Uhr and E.S. Vitetta, 1986, J. Immunol. 137, 1.
45
Elsevier JIM 04013
Isolation of antigen-binding virgin and memory B cells * C h r i s t o p h e r D. Myers, Virginia M. Sanders a n d Ellen S. Vitetta ** Department of Microbiology, Universityof Texas Health Science Center, Southwestern Medical School, Dallas, TX 75235, U.S.A,
(Received10 March 1986, accepted 31 March 1986)
One major obstacle in studying the activation of antigen-specific B cells is the small number of B cells reactive with a particular antigenic epitope. In this report, we describe a method by which large numbers of highly purified antigen-binding cells can be obtained. We have shown that by varying the haptenation level of the erythrocytes used for rosetting, we can purify antigen-binding B cells which have different affinities for the antigenic epitope. Thus, memory cells (which have receptors of higher affinity) can be prepared and these cells are essentially free of contaminating virgin cells. The effects of varying the haptenation levels on the red cells used for purifying the B cells can, in turn, be related to the precursor frequency of secreting cells following their activation with T cells and antigen. Key words: B cells, antigen-specific;Memorycell; Lymphocyte
Introduction It is estimated that 1 / 1 0 6 B cells express immunoglobulin (Ig) receptors specific for any given
* This work is supported by NIH Grants AI-11851 and AI21229. ** Address for correspondence: Dr. E.S. Vitetta, Department of Microbiology,Universityof Texas Health ScienceCenter at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235, U.S.A. Abbreviations: ABC, antigen-binding cells (virgin); ACT, hypotonic ammonium chloride; BSA, bovine serum albumin; BSS, balanced salt solution; BSS/5% FCS, balanced salt solution supplemented with 5% fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; DNP, dinitrophenyl; FCS, fetal calf serum; GAMIg, goat anti-mouse immunoglobulin; Gly-Gly, glycyl glycine buffer; HBSS, Hanks' balanced salt solution; HRBC, horse erythrocytes;MABC, memory antigen-binding cell; PBS, phosphate-bufferedsaline; PFC, plaque-forming cells; RFC, rosette-formingcells; IliA, radioimmunoassay; TD, thymus-dependent; Trt, helper T lymphocytes; TNBS, trinitrobenzene sulfonic acid; TNP, trinitrophenyl; TNP-HRBC,TNP-haptenatedhorse erythrocytes; TNPx-KLH,TNP-haptenated keyholelimpet hemocyaninwith X moleculesof TNP per 100000 Mr of KLH.
antigenic epitope. Following interaction of these antigen-specific B cells with antigen and carrierspecific T cells, the B cells expand and secrete antibody specific for this antigen. Proliferation and secretion are driven by a series of soluble cytokines (Howard et al., 1984; Vitetta et al., 1984). Because of their small number, antigenspecific B cells are difficult to isolate and study. As a result, the majority of B cell activation studies are performed with antigen-primed cells (only a small fraction of which are antigen-specific) or polyclonal activators such as anti-Ig or mitogens which activate a fraction of the B cells regardless of their antigen-binding specificity (Defranco et al., 1982). Nevertheless, a number of approaches have been developed for obtaining populations of B cells enriched for antigen-specific cells where activation by antigen, rather than mitogens or anti-Ig can be studied. Three major approaches have been utilized to enrich antigen-binding cells (ABC): (a) soluble fluoresceinated antigen can be bound to cells and the ABC can be separated from the non-ABC by
0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
46
flow cytometry (Julius and Herzenberg, 1974); (b) antigen can be immobilized on solid matrices, e.g., glass beads, plastic, gelatin, and ABC can be absorbed and eluted (Truffa-Bachi and Wofsy, 1970; Choi et al., 1974; Haas and Layton, 1975); and (c) for hapten-binding B cells, haptenated red cells can be bound to the B cells and the rosetted ABC can be purified by velocity and/or density sedimentation techniques (Elliot, 1979; Snow et al., 1983). Each of the above techniques has its advantages and drawbacks. We have utilized the third approach since relatively large numbers of resting ABC can be isolated within a 2 day period. In this report we describe a modification of the rosetting method, originally described by Snow et al. (1983) and Yefenof et al. (1985), which allows us to produce up to 2.5 x 107 B cells, 50-90% of which are antigen-specific. This technique can be applied to both memory and virgin B cells and is readily performed by a single person. Finally, by varying the haptenation density of the red cells used for rosetting, it is possible to isolate B cells bearing receptors of different affinities (Yefenof et al., 1985). This allows for the separation of memory cells (which bear receptors of high avidity) from virgin cells (Yefenof et al., 1985).
at 37 °C for 30 min with occasional gentle mixing. The reaction was stopped by diluting the cell suspension to 30 ml with cold BSS supplemented with 5% fetal calf serum (FCS) (BSS/5% FCS) followed by centrifugation at 4 ° C. Cells were then washed once in 30 ml glycyl glycine buffer (GlyGly) and three times in 30 ml BSS/5% FCS to obtain a cell pellet. Gly-Gly was prepared by dissolving 0.6% (w/v) glycyl glycine in a veronal buffer containing 1.82 mM sodium barbital, 3.11 mM barbital, 0.15 mM calcium chloride, 0.23 mM magnesium chloride and 145 mM sodium chloride.
Animals
BALB/c female mice, aged 8-16 weeks, were obtained from Cumberland Farms, and were maintained in our own animal facilities. For the preparation of memory antigen-binding cells, mice were primed with a single dose of 100 /tg of TNP-KLH in complete Freund's adjuvant i.p. 10-15 weeks prior to sacrifice. Age-matched untreated mice were used as controls in these experiments.
Preparation of splenocytes Materials and methods
Haptenated horse erythrocyte (HRBC) preparation 5 ml HRBC in Alsever's solution (Colorado Serum Co., Denver, CO) were layered onto 5 ml of Ficoll-metrizoate, density 1.09 (10.08 g Ficoll 400 + 30 ml sodium metrizoate in 102 ml) and centrifuged at 2000 X g for 20 rain at 4°C. Cells at the interface were discarded and the cells in the pellet were washed twice at 4°C in 30 ml of balanced salt solution (BSS) by centrifugation for 5 rain at 1200 × g. This produced a pellet of 1.0 ml packed cells. An alternative to the Ficoll separation was to wash the cells three times in BSS discarding the leukocyte-rich buffy coat after each wash. Trinitrobenzene sulfonic acid (TNBS) (usually 4 or 20 mg for memory or virgin cells respectively) was dissolved in 10 ml cacodylate buffer, 0.28 M, pH 7.4, filter sterilized and added directly to the cell pellet. Cells were resuspended and incubated
All procedures were carried out at 4°C unless otherwise stated. Spleens from 2 to 20 mice were removed and a single cell suspension was prepared in BSS. The suspension was allowed to settle for 10 min on ice and the pellet was discarded. The remaining cells were centrifuged for 10 min at 300 X g and the cells in the pellet were resuspended in 0.5-3 ml (depending on number of spleens) hypotonic ammonium chloride (ACT) (0.8% ammonium chloride, 0.1% KH2PO4) and incubated at 37°C for 4 min to lyse red ceils. After incubation, the cells were immediately diluted with cold BSS and centrifuged. The cells in the pellet were then resuspended in 10 ml BSS/5% FCS per mouse spleen, passed through a 22 gauge needle to prepare a single cell suspension, and layered onto Ficoll-metrizoate (density 1.09 g/ml) using 20-25 ml of cells per 8 ml Ficoll in clear 50 ml tubes (Corning, cat. no. 25339). Tubes were centrifuged for 20 rain at 4°C at 2000 x g. Cells at the interface were ~ollected and washed (2-3 interfaces/50 ml wash) in 50 ml BSS/5% FCS. Cells
47 were pooled and washed at 4 ° C in 50 ml BSS/5% FCS. The first centrifugation after Ficoll was carried out at 660 x g and the second centrifugation was carried out at 300 x g. The A C T and Ficoll steps removed the majority of the mouse erythrocytes. The Ficoll step also removed non-viable cells and the majority of granulocytes.
Purification of ABC Splenocytes prepared as described above were resuspended in BSS/5% FCS at 2.25 X 107cells/ml and trinitrophenyl-haptenated horse erythrocytes ( T N P - H R B C ) were resuspended at a final concentration of 1.5% in BSS/5% FCS. All procedures were carried out at 4 ° C to prevent cell activation (Pike et al., 1983). Percoll gradients were prepared as described in Table I and contained 10 ml of 75% Percoll overlayered with 10 ml of 30% Percoll in alcohol-sterilized, 50 ml round bottomed tubes (Nalgene, cat. no. 3117-0500). 0.5 ml of the T N P - H R B C suspension was added to each ml of splenocytes and 10-20 ml of this mixture was immediately layered onto each gradient. The tubes were covered with foil and centrifuged at 4 ° C for 15 min at 300 x g, accelerating and decelerating gently. Pellets from these tubes were collected by careful aspiration of the supernatant and the cells in the pellet were resuspended immediately in 15 ml 1.09 Percoll per pellet and layered onto 15 ml, 75% Percoll in the same type of tube. These gradients were overlayed with 5 ml, 1.07 Percol~, the tubes were covered with foil and centrifuged at 120 x g for 35 min, again accelerating and decelerating gently. The
cell pellets were collected by aspiration, the cells were resuspended in 15 ml BSS/5% FCS and an aliquot was removed for rosette counting prior to centrifuging at 300 x g for 10 min at 4°C. Cells were then washed once in serum-free RPMI-1640 and were resuspended in 10 ml of serum-free RPMI-1640 containing 25 m M Hepes (Whittaker MA Bioproducts, cat. no. 12-115) with 1.5 m g / m l Trypsin (Sigma, cat. no. T-0134) and 1.5 m g / m l protease (Sigma, cat. no. P-5147). This mixture was incubated for 30 rain at 37°C with vigorous resuspension after 15 and 30 rain. The enzymatic reaction was 'stopped' by the addition of 2 ml of FCS and the cells were pelleted by centrifugation at 300 X g for 10 min at 4°C. Cells were then resuspended in 1.5 ml BSS/5% FCS per original Percoll tube and layered onto Ficoll, using 2 ml Ficoll and 3 ml cell suspension in sterile 12 x 75 polystyrene tubes. Gradients were centrifuged at 2000 x g for 20 min at room temperature and the cells at the interface were collected using a glass pasteur pipette which had been prewashed with FCS to reduce cell adherence. The cells were then washed twice in BSS/5% FCS by centrifugation at 4°C, first at 6 6 0 X g for 10 min and then at 300 × g for 10 min. All procedures between the admixtures of splenocytes with the T N P - H R B C and the enzyme treatment were carried out as rapidly as possible at 4 ° C to avoid cell activation. Finally, cells were resuspended in complete medium (RPMI-1640 plus 10% FCS, 2 m M glutamine, 5 x 1 0 -5 M 2-mercaptoethanol, 50 U / m l streptomycin and 50 /~g/ml penicillin) at 5 x 105/ml and cultured overnight in an atmo-
TABLE I PERCOLL PREPARATIONS a Percoll
75% 1.09 1.07 30%
Density
Volumes (ml)
Calculated (g/l)
Measured (g/l)
Percoll
H20
10 × HBSS
1.098 1.083 1.065 1.039
1.0975 1.0825 1.0650 1.0375
37.5 19.17 5.0 6.0
7.0 7.53 3.9 11.8
5.0 3.0 1.0 2.0
1.0 M Hepes pH 6.8 0.5 0.3 0.1 0.2
a Names and preparations of Percoll mixtures used in gradients. Names and percentages of Percoll are those used by Snow et al. (personal communication). Calculated densities are based on a 1.0 density for reagents other than Percoll and a Percoll density of 1.130. Densities were measured with a narrow range hydrometer and are accurate to +0.0025 g/l. The amounts described produce enough Percoll for two gradients.
48 sphere of 10% CO2, 7% O2 and 83% N 2 at 1 ml/well in 24-well cluster plates (Costar) to allow re-expression of surface Ig (Snow et al., 1983).
Percoll gradients The constituents of these gradients were based on those described by Snow et al. (1983). The major modifications were in the buffering system. Snow et al. (1983) used 10 x Dulbecco's modified Eagle's medium (DMEM) to prepare the isoosmotic Percoll with bicarbonate buffer. 10 x DMEM is no longer available commercially and the powder is technically difficult to dissolve at 10 x . Thus, we now use 10 x Hanks' balanced salt solution (HBSS). Bicarbonate buffering is intended for use in high CO 2 environments and, in Percoll gradients, often requires pH adjustment with HC1. To avoid problems with pH fluctuations, Hepes buffer, pH 6.8, is now used. Detailed procedures for the preparation of the gradients are described in Table I.
Rosetting techniques The enumeration of rosetted cells has been used as the major criterion for purity throughout this work since the number of rosettes has previously been shown to correlate with presence of ABC (Snow et al., 1983). 100 ffl samples for initial rosette counting were collected from the suspension layered onto the first Percoll gradients; these samples were centrifuged at 70 × g for 10 min at 4°C in 12 × 75 round bottomed tubes and the pellet was incubated at 4°C for 10 min to ensure rosette formation prior to resuspending and counting. Samples taken throughout the preparation were counted directly after collection without further washing. Rerosetting at later times was performed in a manner analogous to the initial rosetting procedure using at least 10 freshly prepared TNP-HRBC per leukocyte. All counting of rosettes was performed in the presence of crystal violet to aid in the enumeration of unrosetted cells. The results represent the mean of 4 counts of each sample.
Radioirnmunoassay (RIA) Direct measurement of the level of hapten expressed on the surface of TNP-HRBC was performed by RIA. Briefly, TNP-HRBC were sus-
pended at 2 x 109 cells per ml in 1% bovine serum albumin/phosphate-buffered saline (BSA)/ (PBS)-azide and 10/~1 of the cells were added per V-bottom ELISA microwell, precoated with 1% BSA/PBS azide. 10 #l/well monoclonal anti-dinitrophenyl (DNP) antibody (29.13 (Scott and Fleischman, 1982)) at 1.5 /~g/ml were added and cells were incubated for 1 h at room temperature before washing them three times in BSS containing 1% BSA. Plates were developed with 125I-goat anti-mouse immunoglobulin (GAMIg) (2× 105 cpm/well), incubated for 1 h at room temperature, washed four times and counted.
Indirect assay for haptenation The total amount of TNBS bound to the HRBC was assessed indirectly by measuring the amount of unreacted TNBS remaining after haptenation. Varying concentrations of TNBS were dissolved in cacodylate buffer, pH 7.4. 0.5 ml samples were removed and added to 2 ml Gly-Gly. After haptenation, cells were pelleted by centrifugation (without dilution) and 0.5 ml of the supernatant was removed and added to 2 ml Gly-Gly. Cells were then washed and utilized as normal. The absorbance at 340 nm (A340) of solutions in GlyGly was then measured and the amount of TNBS remaining was estimated from a standard curve.
Proliferation assays Proliferation assays were carried out as described by Snow et al. (1983). Freshly prepared TNP-ABC or splenic B cells were cultured for 16-20 h at 5 × 10 4 per microwell in 100 /~1 of complete medium. Various reagents, as listed in the Results section, were added to give a final volume of 200/~1. Cells were incubated for 3 days and then pulsed for 16 h with 1 /~Ci per well of [3H]thymidine (ICN Radiochemicals, Irvine, CA) and harvested on a MASH II automated cell harvester. The uptake of [3H]thymidine was assessed by scintillation counting.
Aspirin Acetyl salicylic acid (J.T. Baker, Philadelphia, PA) was dissolved in absolute alcohol at 167 mg/ml immediately prior to use. This stock solution was added, with shaking, to all reagents used between mixing the TNP-HRBC with the spleno-
49 cytes and the enzyme treatment, at a final concentration of 0.4 mg/ml (Snow et al., 1983). The Hepes buffering of Percoll gradients minimized the pH effects of aspirin and no further adjustments were made; however, BSS and BSS/5% FCS were neutralized after addition of aspirin using 1 M NaOH.
Limiting dilution assay Limiting dilution assays of TNP-ABC were performed as described by Yefenof et al. (1985). Briefly, after overnight incubation, ABC were harvested and their viability was assessed by trypan blue exclusion. Between 10 and 1000 viable cells were cultured with 5 x 104 irradiated, KLHprimed T cells and 0.4/tg/ml TNP33-KLH in 10 /zl per Terasaki well. After 5 days, cultured cells were washed and transferred to 96-well microtiter plates with TNP-sheep erythrocytes (SRBC) and guinea pig complement (absorbed with SRBC) in 75 /~1 BSS/5% FCS. Plates were centrifuged to form a cell lawn and incubated at 37°C for 1 h before scoring for the presence of PFC on a Zeiss inverted microscope at 40 x magnification. Sixty replicates were scored per condition. Precursor frequencies were assessed using the Poisson distribution as described by Lefkovitz and Waldmann (1979).
Results
Enrichment of TNP-ABC In evaluating this modified procedure for the purification of TNP-ABC, we have assessed total cell yield and the percentage of rosetting cells. Table II shows these values for several such cell preparations. These values are representative of our experience with a large number of similar purifications. In this procedure, 1 ml packed cell aliquots of HRBC were haptenated with either 10, 20, 40 or 120 mg of TNBS. BALB/c splenocytes from 12 spleens were collected and the erythrocytes were lysed by ACT treatment. Remaining red cells, dead cells and granulocytes were removed on a Ficoll gradient to give a total cell yield of 1.16 x 109cells which was subsequently divided into four
aliquots. The cell yield is subject to large variations. Spleens from 7-12-week-old BALB/c mice yield 9.2 x 107-t- 20% cells per spleen after lysis and Ficoll separation. Omission of the Ficoll step at this stage has no deleterious effect on the preparation but increases the total numbers of cells being processed. Another alternative is to utilize a Percoll gradient to purify high density resting cells (Snow et al., 1983) for which we now use a modification of the method of Layton et al. (1985). As shown in Table II, the yield of rosettes in the first centrifugation step is quantitative, i.e., the number of rosettes recovered in the pellet was close to the number originally layered onto the gradient. This is one major modification in the procedure described by Snow et al. (1983). This single step replaces the repeated rosetting and short centrifugation steps, but the cell yields and purity are similar. The second Percoll gradient is not significantly different from that used by Snow et al. (1983) and the resulting cell population is similar. There is a consistent small loss of rosettes in this step as can be seen in Table II; however, the purity of the rosetting cells recovered was t'outinely in the range of 50-90% Disruption of the rosettes and removal of contaminating TNP-HRBC remains a problem. We have been unable to improve upon the method of Snow et al. (1983) using proteolytic enzymes followed by Ficoll-metrizoate separation. This gives a significant cell loss (Table II) but a variety of other methods have shown no improvement (see below). Cells are routinely incubated overnight at 5 x 105/ml in complete medium, in Costar 24-well clusters at 1 ml/well or microwells at 100/d/well. After overnight culture, the majority of the cells plated are recovered and these cells are 80-95% viable.
Enrichment of TNP-(memory)ABC (MABC) Few modifications of the above methodology are required for purification of memory cells. Mice are primed 10-15 weeks prior to use with 100/~1 TNP-KLH at 1 mg/ml in complete Freund's adjuvant and the TNP-HRBC are haptenated at the
50 TABLE II PURIFICATION OF TNP-ABC a Haptenation (mg/ml packed cells)
Virgin cells 10
Memory cells 20
40
120
4
Number of cells (after Ficoll) ( x 10- 8)
2.9
2.9
2.9
2.9
11.0
Initial rosettes (%)
1.25
2.28
5.64
6.45
1.1
Rosettes after 1st Percoll step Yield (rosettes) ( x 10 -6) Purity (%)
3.7 45.6
6.1 40.7
13.0 51.2
14.8 51.6
8.1 12.1
Rosettes after 2nd Percoll step Yield (rosettes) ( × 10-6) Purity (%)
2.9 67.0
4.1 73.0
10.1 73.2
13.4 74.2
7.0 54.0
1.3
2.8
3.7
7.5
7.1
0.8 84
1.6 80
3.1 85
7.5 83
5.3 84
56
52
51
49
30
YieM After Ficoll step ( X 10 -6) Yield after overnight incubation Viable cells recovered ( x 10 -6) Viability (%) Rerosetting percentage
a Four virgin cell purifications were performed in parallel using the same initial splenocyte preparation. The purification of MABC was carried out with splenocytes from immunized mice at a separate time. Aliquots of cells were removed at all stages of the purification and their content assessed. The above data are from representative experiments. In a series of 10 consecutive purifications of virgin ABC, we recovered 5.0 × 105 (+_ 2.9 × 105) cells after Ficoll for each 108 cells initially rosetted with a mean purity of 70.8% (+8.3%). After overnight incubation, 3.4 × 105 (+1.9 x l0 s) cells at 54.4% (+9.6%) purity was recovered. In a similar series of 10 purifications of memory ABC, the cell yield was 5.1 x 103 (+ 3.2 x 105) per 108 cells rosetted with a mean purity of 59.6% (+ 6.7%). Overnight viability counts and rerosetting percentages were not routinely measured for MABC.
lower rate of 4 m g / m l p a c k e d cell v o l u m e (Yefenof et al., 1985). A l l steps used in the p u r i f i c a t i o n are i d e n t i c a l a l t h o u g h these rosettes are t r e a t e d even m o r e gently w h e n r e s u s p e n d e d b e t w e e n gradients. A s can b e seen in T a b l e II, the level of purific a t i o n achieved with the first Percoll step is lower with m e m o r y R F C t h a n virgin R F C and, a l t h o u g h the second Percoll g r a d i e n t i m p r o v e s this yield, the final p u r i t y of M A B C is i n v a r i a b l y lower t h a n t h a t of virgin A B C . F o l l o w i n g overnight culture, viability of M A B C is good, b u t the recovery of rosettes tends to b e lower t h a n t h a t o b t a i n e d with virgin ABC. Finally, in M A B C purifications, the n u m b e r of T N P - H R B C in the rosette-rich fraction is always higher t h a n in c o r r e s p o n d i n g fractions f r o m virgin A B C , b u t this is rectified b y the F i c o l l
step. The m e m o r y cells purified in this w a y have b e e n extensively c h a r a c t e r i z e d b y Y e f e n o f et al. (1985, 1986). Analysis o f the parameters of the different procedures used T N P - H R B C to splenocyte ratio Snow et al. (1983) d e s c r i b e d rosette f o r m a t i o n using T N P - H R B C at 0.5% p a c k e d r e d cells a n d leukocytes at 15 x 1 0 6 / m l . This yields a T N P H R B C : s p l e n o c y t e ratio of b e t w e e n 6 a n d 7 to 1. Since c o n t a m i n a t i o n with T N P - H R B C at the end of the p r o c e d u r e c a n b e a p r o b l e m , we e v a l u a t e d p r o c e d u r e s using fewer r e d cells in the p r e p a r a t i o n of the T N P - A B C . A s c a n b e seen in T a b l e III,
51 T A B L E III R E D CELL R E Q U I R E M E N T S a
%
TNP-HRBC per white cell
Final cell yield ( X 106 )
Rosettes
% of rerosettes (after cell culture)
6.7 5 4 3 2 1
2.03 2.11 1.84 1.03 0.50 0.30
70 63 66 53 70 50
70 74 63 68 50 58
a Cell preparations were performed and contained identical numbers of splenocytes with varying numbers of T N P - H R B C at 10 m g T N B S / m l of cells. The yield of rosette-forming cells, purity and rerosetting efficiency were measured. Similar results were obtained in a second experiment.
below a ratio of 5 : 1 for T N P - H R B C : splenocytes, the absolute cell yield began to fall although purity was not affected down to a 2 : 1 ratio. From these data we have chosen to continue using a 0.5% packed cell volume of TNP-HRBC.
Percoll gradients (a) Medium and buffering system.
As stated above, 10 x D M E M is no longer available and cannot be prepared reliably from powder. Therefore, we have examined other combinations based on HBSS, RPMI-1640 and 10 × PBS buffered either with bicarbonate or Hepes. Hepes buffer consistently gave better buffering than bicarbonate and, when prepared at p H 6.8, overcame the basic nature of the Percoll. HBSS was chosen since it consistently gave results equivalent to the b i c a r b o n a t e / D M E M mixture. RPMI containing high concentrations of glucose was not used because of cell clumping with Hepes buffer and Percoll (data not shown). (b) Inclusion of FCS in Percoll. It was thought that the long period during which the cells were maintained in serum-free conditions would be deleterious both to cell viability and purity of the preparation due to non-specific aggregation of cells. Consequently, a series of purifications was carried out using 5% FCS in all Percoll layers. In six experiments, the final cell yield was increased by 17.5% + 11.3% without any significant effect on either final purity or the % of rerosetting cells after overnight incubation. However, there was a significant increase in red cell
contamination after the Ficoll step. Therefore, the use of FCS was not adopted. (c) Percoll densities. The bottom layer of Percoil provides the major purification step. Therefore, we tested the effects of changing the density of this layer. Increasing the density of the bottom layer to 78% Percoll caused a decrease in cell yield, whereas 81% Percoll completely prevented rosettes from reaching the pellet, even when additional centrifugations were ~erformed (data not shown). Using the procedures described in Table I, no significant variations in yield were obtained using 4 different batches of commercially available Percoll (data not shown). (d) Centrifugation time. In calibration experiments, almost all rosettes reach the cell pellet within 15 min on the first Percoll gradient and 25 min on the second gradient. However, on a routine basis, we have found it necessary to centrifuge the second gradient for 30-35 min to ensure consistently good cell recoveries. Since both gradients are separating cells based on both bouyant density and sedimentation velocity, it is important not to centrifuge the cells too long since the number of unrosetted leukocytes and erythrocytes in the pellet increases with time. (e) Cell loading. The majority of data presented are based on purifications in which splenocytes were resuspended at 1.5 × 107cells/ml and 10-12 ml were loaded on each gradient. However, recent data show that up to 25 ml of cells at the same concentration can be loaded onto each gradient without significant effects on the yield or
52
purity of the cells recovered. Thus, a purification based on 12-15 spleens can be performed using only four gradients in each stage.
Effects of aspirin Aspirin (Acetylsalicylic acid) was originally included in all purification steps where TNP-HRBC and splenocytes were together to avoid activation of the B cells (Snow et al., 1983). Since that time we have made further changes in the purification procedure and the effects of aspirin have been re-examined. In our current experiences aspirin has no effect on purification (Table IV), proliferation of TNP-ABC or anti-TNP PFC responses (data not shown). In addition, the presence or absence of aspirin has no effect on phospholipid metabolism induced by antigen in these cells (Myers et al., manuscript in preparation). Since Snow et al. (1983) found that the addition of aspirin was essential in avoiding cell activation during the isolation procedures, we can only conclude that the lower hapten density and shorter period of B cell contact with hapten avoids the problem of activation seen previously. Thus, all preparations are currently performed in the absence of aspirin.
Disruption of rosettes Snow et al. (1983) describe disruption of rosettes by treating the rosettes with trypsin and protease. We have titrated the enzyme concentration and confirmed that 1.5 m g / m l of each enzyme is opti-
T A B L E IV E F F E C T OF ASPIRIN ON T H E P U R I F I C A T I O N OF TNPABC a Parameter
With aspirin
Without aspirin
T N P - A B C Yield (n = 5) % Purity (n = 5) % Rerosetting (n = 2)
1.05 × 107 65.8 55.0
0.97 × 107 65.2 59.7
a Results of cell preparations carried out in parallel with and without aspirin. Data represent mean values for the number of experiments listed. Standard deviations for the % purity were less than 6% and a close correlation was seen in cell yields of all experiments.
TABLE V TITRATION OF PROTEOLYTIC ENZYMES a Enzyme concentration (mg/ml)
Rosettes remaining (%)
Recovery of cells after culture ( × 10- s )
Rerosetting cells (%)
3.0 1.5 0.75 0.38 0.19
0.39 0.60 2.50 2.62 2.32
8.85 8.50 9.02 8.20 9.10
59.5 64.5 56.0 56.8 53.6
a T N P - A B C were taken immediately after the final Percoll step and incubated for 30 min at 3 7 ° C with various concentrations of trypsin and protease. The rosettes remaining immediately after enzyme treatment were counted and the cells were Ficolled and cultured. Viable cell yields and rerosettes were measured 18 h later. Similar results were obtained in a second experiment. Significant cytotoxicity was seen at 4.5 m g / m l of trypsin and protease.
mal for disruption of rosettes (Table V) with 750 ~tg/ml yielding a percentage of unbroken rosettes and 4.5 m g / m l causing significant cytotoxicity to the lymphocytes. There are clearly a number of different effects occurring to induce this disruption since the enzymes alone would not be expected to remove all the surface Ig (Calvert et al., 1981). To further examine the effects of enzyme treatment on the cells, TNP-ABC were prepared and incubated overnight as usual. 74% of these cells rerosetted. Aliquots of these rosettes incubated at 37°C for 30 min in BSS contained 50% rosettes after treatment, but when incubated at 37°C for 30 min in RPMI (which is isotonic for human cells and hypotonic for murine cells), the level of rosettes dropped to 24%. Similarly, an aliquot of TNP-ABC was incubated in enzymes in the usual manner and then rosetted with fresh TNP-HRBC producing 11% rosettes. 15% rosettes were present when fresh TNP-ABC were rosetted with enzyme-treated TNP-HRBC. This suggests that the enzyme treatment removes some surface Ig from the TNP-ABC and some TNP from the TNP-HRBC, but at least part of the effect is due to low tonicity of the incubation medium. Enzyme treatment has been shown to activate some cells (Vischer, 1974, 1984) and to change the activation responses of B cells (Kern, 1985; Ramanadham et al., 1985). Therefore, other proce-
53 dures for disrupting rosettes were examined. Osmotic shock with A C T was found unsuitable in two experiments due to lowered viability after overnight incubation. Monomeric antigen in the form of TNP-lysine was shown to be effective at disrupting rosettes prepared with H R B C haptenated with 12 mg TNBS per ml packed cell volume (Fig. 1). Disruption was time and dose-related with an optimal concentration of 1 m g / m l in complete medium (Fig. 1). When utilized in BSS, this concentration caused cytolysis, probably due to lack of adequate buffering (data not shown). However, in agreement with previous data (Wigzell and Maleka, 1970), this method failed to disrupt rosettes prepared with red cells haptenated with higher concentrations of TNBS (20 m g / m l , Fig. 1). The monomeric antigen probably remained on the cell surface since the percentage of rerosettes formed by cells disrupted with TNP-lysine followed by overnight incubation was only 10% compared with 65% for the rosettes disrupted with proteolytic enzymes.
Haptenation ratio of TNP-HRBC Snow et al. (1983) reported haptenation of horse erythrocytes (HRBC) by the method of Kettman and Dutton (1970) using 120 mg TNBS per packed ml HRBC. As can be seen in Table II, the cell yield is highly dependent on haptenation ratio. We have re-examined this phenomenon by a number of parameters. Table II shows that the rate of initial rosette formation increases rapidly with increasing hap-
tenation levels. We have no explanation for the large differences in levels of initial rosettes described now versus previously. We have found that inclusion of aspirin at the time of rosetting causes a slight decrease in the % of initial rosettes, but this does not explain the observed difference. Examination of the rerosetting potential of the cell preparations described in Table II is presented in Table VI. As can be seen, rerosetting with cells haptenated at the same level as used in the purification were consistently below the levels of purity seen at the time of preparation. Higher levels of haptenation revealed a modest increase in the level of rerosetted cells, while decreased haptenation resulted in a marked decline in the percentage of rosettes. Similar findings have been demonstrated with T N P - M A B C (Yefenof et al., 1985). This finding suggests that TNP-ABC represent a population of cells bearing surface Ig with widely varied affinities for TNP, and that those cells prepared with the lower haptenation ratio represent the higher affinity population. To further address this point, the data shown in Fig. 2 were collected. Here the response of hapten bound to the T N P - H R B C , the hapten available on the surface of the T N P - H R B C and number of rosettes formed with splenocytes from unimmunized mice (initial rosetting level) were titrated versus increasing amounts of TNBS added to the HRBC. It should be noted that even at the lowest haptenation levels, this reaction does not go to completion, so one must take great care with the time and temperature of haptenation, as well as TNBS concentration, in order to obtain consistent
TABLE VI REROSETTING OF TNP-ABC a Haptenation ratio of TNP-HRBC used in preparation (mg TNBS/ml HRBC)
Haptenation ratio of TNP-HRBC used for rerosetting (%) 10
20
40
120
10 20 40 120
55.9 + 5.7 28.9 + 4.2 19.1 ± 5.1 6.9 ± 2.1
70.7 ± 2.1 51.6 ± 4.5 22.8 ± 7.5 13.2 ± 2.8
75.7 + 1.8 62.4 ± 2.2 51.2 ± 5.2 35.0 ± 9.0
76.5 + 5.0 65.0 ± 7.0 57.2 + 3.7 49.2 ± 6.8
a Cells from the preparation described in Table II were incubated overnight and then rerosetted with freshly prepared TNP-HRBC at the four haptenation ratios used in the preparation. Results represent mean + standard deviation from four separate counts on each sample. These results are representative of many similar experiments.
54
results. Secondly, as suggested by the previous data, the percentage of cells which initially form rosettes is related to the level of haptenation and thus markedly effects of final yield. The final test of the purification procedure is to determine the level of precursors responding to antigen and T cells. We examined this response using a limiting dilution assay for plaque-forming cells (PFC) in response to a thymus-dependent antigen, TNP30-KLH and the KLH-primed, irradiated helper cells (TH). As shown in Fig. 3, the precursor frequency in the TNP-ABC population is inversely proportional to the level of haptenation of the TNP-HRBC, suggesting that the cell
70 60 50
yield achieved with the more highly haptenated TNP-HRBC represents cells of lower affinity which do not respond to antigen in a PFC assay. Calculations based on the three experiments used for Fig. 3 show that the total yield of precursors (i.e., overnight viable cell count × precursor frequency) was constant at all haptenation levels at 4.2 × 10 4 (___0.5 × 10 4) with no obvious trends. The largest variation between the lowest and highest yield within a single experiment was twofold. With cells prepared usiJ~g; TNP-HRBC haptenated using 10 mg per ml packed cell volume, all points in the limiting dilution assay fell on a straight line. However, cells prepared with more heavily haptenated TNP-HRBC had a tendency to fall to the right of the line when higher input cell numbers were used. This phenomenon is similar to that described by Aarden et al. (1980) and Lefkovits et al. (1980) suggesting that a significant number of T suppressor cells are present in the populations purified with the more highly haptenated
40 U) LU
F-
50 (I)
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-
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1.5
60
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-I 6
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20 ~o--o o
1.0 I
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10 20 50 40 50 PERIOD OF INCUBATION (rain)
60
Fig. 1. Disruption of rosettes by TNP-lysine. TNP-ABC were prepared using HRBC haptenated with either 20 or 12 mg of TNBS per ml packed cells. After overnight incubation, cells were rerosetted with similarly prepared TNP-HRBC. At time 0, various concentrations of TNP-lysine were added and the number of rosettes remaining during incubation at 4°C was assessed. Cells were resuspended by brief, genre vortexing prior to removing for sampling. This treatment had little effect on cells lacking TNP-lysine. Experiments were performed in complete medium. Similar results were obtained in two other experiments, zx represents TNP-ABC prepared and rerosetted with TNP-HRBC haptenated with 20 nag TNBS/ml packed cell volume incubated in TNP-lysine at 1.5 mg/ral. The remaining curves were derived from data using TNP-ABC prepared and rerosetted with TNP-HRBC haptenated with 12 mg TNBS/ml packed cell volume and incubated in TNP-lysine at 0.5 mg/ml ( 0 ) , 0.68 mg/ml (O), 0.80 mg/ml ([]), and 1.0 mg/ml (11).
F-
20
t0
5
z5
t2
06o30t5'c
0
[TNBS](mg/ml)
Fig. 2. TNP-HRBC were prepared using various levels of TNBS to prepare the HRBC. The dilutions of TNBS used were also used to set up a standard curve after reaction with Gly-Gly. After haptenation, the supernatants were sampled and the absorbance at 340 nm (A340) of this Gly-Gly reaction (A) was compared with the standard curve (,x) (insert) to ascertain the amount of TNBS bound (n). The level of TNP expressed on the cell surface after haptenation was measured by RIA using monoelonal anti-DNP antibody (29.13) and an 125I-anti-mouse Ig (11). TNP-HRBC produced were then used in rosetting experiments both with freshly prepared splenocytes (initial rosetting) (e) and TNP-ABC prepared the previous day using 20 mg/ml TNBS on the TNP-HRBC (©). Similar results were obtained in two other experiments.
55 100 _J ..J
~ w
w Z
o_
5o
I
1-
lo6
100 200
400 600 INPUT CELLS PER WELL
800
1000
Fig. 3. Cells were purified using TNP-HRBC haptenated with 10 (C)), 20 (@), 40 (rq), or 120 (11) mg TNBS/ml packed cell volume (c.f. Table II). After overnight incubation, cells were harvested and plated at between 10 and 1000 viable cells/well in limiting dilution assay (see materials and methods section). Presence of PFC was scored on day 6 and plotted as the log of the proportion of negative wells vs. cells per well. Results represent mean±standard deviation from three separate experiments with 60 wells/point each. Points falling to the right of the least squares line probably represent suppressor effects at high cell concentration (see results section) and least squares lines were calculated using 4 (O), 3 (@,D) or 2 (11) points and the origin to give precursor frequencies of 1/72 with 10 mg TNBS, 1/167 with 20 mg TNBS, 1/418 with 40 mg TNBS and 1/649 with 120 mg TNBS. Correlation coefficients were all better than 0.98.
TNP-HRBC. However, further studies will be required to substantiate this hypothesis.
Discussion
Over the past 20 years, steady progress has been made in the purification of antigen binding B cells. Various approaches have been attempted, each with its own advantages and disadvantages and each producing populations with varied properties. The earliest attempt to purify antigen-binding cells was reported by Wigzel and Andersson (1968). Primed lymph node cells were passed over antigen-coated beads and the adsorbed cells were eluted by mechanical agitation. Significant retention of antigen-specific PFC was achieved, but the eluted fraction showed only 2.5-fold enrichment in PFC and considerably decreased viability. There was also a large amount of non-specific adsorp-
tion of cells to the beads resulting in significant cell loss. Immunoadsorbents based on polyacrylamide (Truffa-Bachi and Wofsy, 1970), agarose (Davie and Paul, 1971) and Sephadex (Schlossman and Hudson, 1973) reduced nonspecific binding and permitted recovery of cells by digestion of the matrix. Other solid-phase systems that were investigated include binding to plastic tubes (Choi et al., 1974) and nylon fibers (Rutishauer et al., 1973). By 1976, a system utilizing anti-fluorescein conjugated to Sephadex G-200 could bind a fluoresceinated protein antigen and act as an immunoadsorbent for antigen-binding B cells. Scott (1976) achieved a 1% yield of input cells containing 30% ABC with this technique. Haas and Layton (1975) described a solid-phase immunoadsorbent system in which ABC were adsorbed to hapten-derivatized gelatin-coated petri dishes. This system had the advantages of low non-specific adherence, simple recovery of bound cells by melting the gel at 37°C and easy regulation of the hapten density. Dependent on the haptenation level, between 0.25% and 2% of input cells were recovered (c.f. Table II, Fig. 2). However, cells released by melting the gelatin apparently retained bound antigen causing specific antigen non-responsiveness. Collagenase treatment reversed this effect and allowed detection of up to 42% RFC in the purified population. Various modifications of this approach have produced increased purities of the recovered populations (Nossal and Pike, 1978; Pike and Nossal, 1984). The method holding the greatest promise for achieving 100% pure populations of ABC is cell sorting. However, starting with less than 1% positive cells, extremely long sorting periods are required. Thus, this method is applicable to some in vivo biological assays (Julius et al., 1972; Julius and Herzenberg, 1974) but is impractical for most in vitro cell culture and biochemical assays. The other major approach to ABC purification, rosetting, was described by Brody (1970) and Brody and Papermaster (1970). Using rate zonal sedimentation in BSA gradients, yields were as low as 0.1-0.01% of input cells with purity usually below 10%. Early experiments with velocity sedimentation produced less than 5% RFC (Osaba, 1970; Wilson, 1973), but by 1979, Elliot had developed a two-step velocity sedimentation tech-
56 nique selecting for large rosettes giving rise to 75-90% pure RFC. However, this technique required specialized equipment, was technically exacting and limited in cell handling capacity. Density gradient centrifugation of rosettes on Ficoll (Sulitzeanu and Axelrad, 1973) or BSA (Tanenbaum and Sulitzeanu, 1975) produced puffties up to 20% and modification of this technique by Kenny et al. (1978) to include one buoyant density and one sedimentation velocity centrifugation on Ficoll produced 50-100% RFC from primed spleens and 20-40% RFC from virgin splenocytes. This approach was further improved by Snow et al. (1983) using Percoll gradients to achieve 60-70% RFC from unprimed spleens. The above methods all suffer to a greater or lesser extent from the same problems, namely non-specific cell contamination, poor overall yields, and difficulties in removing bound antigen. These problems have been identified and discussed by the authors of the papers dealing with each method. The current technique must be compared with other available methods. With regard to purity, Kenny et al. (1978) and Snow et al. (1983) demonstrated significant enrichment of ABC from unimmunized splenocytes by RFC measurement and our results of - 70% RFC equal or surpass their preparations. The present study, along with those of Snow (1983) and Yefenof (1985, 1986) are the only ones in which rerosetting has been reported. We feel that this is an important measure as it reflects the purity of the cells used in the experiments, which differs somewhat from the purity suggested by rosette counts determined at the end of the purification. The original descriptions of haptenated gelatin panning on used levels of RFC as the assay for purity, and demonstrated around 40% purity (Haas and Layton, 1975). Later modifications were assayed for purification of PFC (Nossal and Pike, 1978) or cells proliferating in response to antigen (Pike and Nossal, 1984). Here the highest purity was achieved with cell yields of the order of 0.001%, containing PFC at up to 1 in 3.5 cells. Although our limiting dilution assays show approximately 20-fold lower PFC responses, our cell yields are up to 300-fold greater. The only other description of purification of long-term primed (> 8 weeks) memory ABC is that of
Yefenof et al. (1985) in which both the present technique and that of Snow et al. (1983) were used' and no differences were found in purity. In terms of technical difficulty, this technique requires no specialized equipment except a centrifuge that will accelerate and decelerate smoothly. The only exacting procedures are the pouring and harvesting of Percoll gradients and a single person can easily handle 4-8 gradients in a single preparation. Twenty-five mouse spleen equivalents can be processed in 1 day. As stated above, the yield is dependent on the level of haptenation used, but even at 10 mg TNBS per ml packed HRBC, this will routinely produce around 3 X 106 ABC/109 input cells, which is superior to all other techniques described when estimated using equivalent haptenation levels. We have been unable to improve on published techniques for the removal of bound antigen from the surface of ABC. Although trypsin-pronase treatment of ceils has previously been demonstrated to induce cell activation (Vischer, 1974) and affect other cell responses (Kern, 1985), Yefenof et al. (1985) demonstrated that both MABC and ABC recovered after overnight incubation are > 95% small, G O cells, suggesting that the enzyme treatment is not activating the cells. These data also support our findings that the use of aspirin during the rosetting stages of this technique is unnecessary (data not shown). We have shown a direct relationship between the hapten density on the HRBC, the total cell yield of the purification and the functional capacity of the recovered cells. This area has not been well studied by previous authors (Haas and Layton, 1975; Yefenof et al., 1985; Howard et al., 1986). Decreasing the haptenation rate allows for the purification of MABC essentially free from contaminating virgin ABC (Yefenof et al., 1985). Increased haptenation levels clearly result in higher total cell yield but not an equivalent increase in precursor frequencies. This is probably due to the purification of weakly cross-reactive cells capable of binding antigen but not responding to it to produce antibody measurable in the present limiting dilution assays. Thus, the final cell yield is not a good criterion for the assessment of a purification procedure; functional criteria must also be assessed.
57 I n s u m m a r y , we have described a system allow: ing for the efficient purification of relatively large n u m b e r s of antigen-specific m e m o r y a n d virgin B cells. This system facilitates the c o m p a r i s o n of B cell activation b y antigen, anti-Ig a n d mitogens such as lipopolysaccharide. M o d i f i c a t i o n of h a p t e n a t i o n ratios used also allow for the dissection of the responses of virgin a n d m e m o r y B cells a n d their respective activation requirements.
Acknowledgements W e t h a n k Ms. T. W i l s o n a n d Ms. M.M. Liu for technical assistance a n d Ms. G.A. Cheek for secretarial help.
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