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The in vitro generation of antigen-binding cells
CELLULAR
IMMUNOLOGY
52,
140- 153 (1980)
The in Vitro Generation of Antigen-Binding JEAN Department
Cells
E. MERRILL AND ROBERT F. ASHMAN’
of Microbiology and Immunology, and Rheumatology Division, Department UCLA School of Medicine, Los Angeles, California 90024
of Medicine,
Received March 29, 1979
To analyze early events following the contact of antigen with specific receptor-bearing cells, antigen-binding cells (ABC) specific for sheep erythrocytes (SRC) were generated in a primary Mishell-Dutton culture system. The maximal frequency of ABC (a five-fold increase over Day 0 involving both T and B cells) occurred on Day 4 in culture conditions which yielded optimal numbers of plaque-forming cells (PFC) specific for SRC 1day later. Assessment of the total &secreting cells as well as ABC and PFC for ox and burro erythrocytes showed that the ABC as well as the PFC response to SRC was antigen specific. By autoradiography of [3H]thymidine-pulsed cultures and by inhibiting cell division with hydroxyurea, the continuous contribution of cell division to the generation of ABC was demonstrated. As with PFC, the decline in ABC numbers began at the point where their generation by cell division slowed. After removal of hydroxyurea, ABC frequency and thymidine incorporation recovered substantially, but PFC did not. However, division of ABC was not the only process involved in ABC generation. Indeed, 20% of the Day 4 ABC arose from cells dividing in the first 24 hr of culture which were not detectable as ABC at that time, i.e., some ABC were derived from dividing non-ABC. Thus both cell division and maturation contributed to the increase in specific ABC seen after primary in vitro immunization. Both processes can be added to the list of early antigen-stimulated events in specific ABC which can be analyzed in the primary culture system in future studies of cell interactions.
INTRODUCTION Most of our current concepts of cell interactions in the immune response derive from experiments where lymphocyte populations defined and isolated by their surface markers are recombined so as to reveal their function. In most such experiments, the read-out of “function” is plaque-forming cells or antibody, the final step of antigen-driven B-cell maturation. Commonly, the models derived from such experiments place the B cell last, as if triggering of B cells cannot occur unless the antigen has hrst interacted with appropriate T cells and macrophages (1). But failure of B cells to complete terminal differentiation does not mean that normal triggering has not occurred. It is equally possible that B cells receive a normal activation signal directly from antigen-receptor contact, but that signals from other cells are required for progression beyond a certain later step. Testing this alternative model would require knowledge of the critical early events which follow 1 Reprint request should be sent to Dr. Ashman at Department of Microbiology and Immunology, 43319 Center for Health Sciences, UCLA School of Medicine, Los Angeles, Calif. 90024. 140 OOOS-8749/80/070140-14$02.00/O Copyright 0 1980by Academic F’ress, Inc. All rights of reproduction in any form reserved.
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antigen contact with that small fraction of lymphocytes which bear the correct specific receptors, the antigen-binding cells (ABC).’ In the mouse spleen the lymphocyte population which binds sheep erythrocytes (SRC) have been shown to contain both B and T cells (2), including the precursors for anti-SRC plaque-forming cells (3, 4), T helper cells (5,6), suppressor cells (7), and cytotoxic cells (8). We have defined several events which follow antigen contact with these cells: (i) capping, maximal by 30 min (9); (ii) accelerated phospholipid synthesis, evident within a few hours (IO); (iii) accelerated receptor turnover, evident within the first day (R. F. Ashman, manuscript in preparation)$ and (iv) decreased sensitivity of the antigen receptor to trypsin appearing around Day 3 (1 l), probably related to the decrease in surface IgD and gain of surface IgG (12, 13). Clearly the expansion of the ABC population is another significant early event that must be placed in this sequence. Although it is tempting to assume that ABC simply derive by division of previous ABC, proof of this assumption is fundamental to future progress in studying early events in ABC, particularly since traffic of ABC into or from the spleen and maturation of ABC without cell division remain important alternative explanations for the increase in ABC following in viva immunization. This paper establishes that the antigen-driven .increase in ABC can be analyzed without cell traffic in a primary culture system, providing the groundwork for our future investigations of early events. A substantial daily contribution of cell division to ABC generation from the third through the sixth day of culture was demonstrated, using both autoradiography and inhibition by hydroxyurea. But more surprisingly, a significant contribution to ABC expansion from early dividing cells which were not detectable as ABC after the first day was also shown, indicating that maturation of antigen-binding ability accompanied proliferation. MATERIALS AND METHODS Mice. Female C57B1/6 mice, 7 to 12 weeks old, were obtained from Jackson Laboratories, Bar Harbor, Maine. Antigen. Sheep erythrocytes (SRC) from a single sheep were obtained in Alsever’s solution from Gibco Diagnostics, Madison, Wisconsin, and stored less than 2 months before use. Cells from this sheep were selected from many lots of SRC for their ability to stimulate a response in a primary Mishell-Dutton culture system. Ox and burro erythrocytes were obtained from Colorado Serum Company Laboratories, Denver, Colorado. Spleen cell cultures. Spleens harvested from unimmunized mice were minced and passed through a wire screen. Primary in vitro immune responses to SRC were studied employing exactly the procedure and medium described by Mishell and Dutton (14) except that 50 mg/ml of gentamicin (Microbiological Associates, Bethesda, Md.) was used as the antibiotic. Cultures in Falcon tissue culture plates (No. 3001) contained 1.5 x 1O’spleen cells and 1O’SRC in 1 ml of medium. Two lots of fetal calf serum (FCS) giving high numbers of PFC in screening experiments were 2 Abbreviations used: ABC, antigen-binding cells; PFC, plaque-forming cells; SRC, sheep red blood cells (also sheep erythrocytes); BRC, burro red blood cells; ORC, ox red blood cells; HEG, Hepes-Eagle’s gelatin medium; FCS, fetal calf serum; MEM, Eagle’s minimum essential medium; HU, hydroxyurea; 13HlTdR, tritiated thymidine; TCA, trichloroacetic acid. 3 R. F. Ashman, J. Immunol. 124: 893, 1980.
142
MERRILL AND ASHMAN
used in all experiments. Cultures were rocked on a Bellco rocker at 37°C in Billups-Rothenberg chambers in a moist, nitrogen-rich atmosphere and fed daily with nutrient cocktail containing 30% FCS (14). Cells were harvested daily from cultures with and without antigen to assay for the generation of ABC and PFC. Viability was monitored by trypan blue exclusion and cell recovery by total cell counts in 1.0 M acetic acid. Assays for SRC-specijc ABC and PFC. ABC generated in vivo and in vitro were enumerated using the rosetting technique of Ashman (9). Lymphocytes binding more than four SRC were scored as rosettes (RFC) on a hemocytometer. By staining lipase-positive cells (15), it was determined that 60-90% of all macrophages and their precursors remained stuck to the plastic plate throughout the culture period and thus were absent from the suspensions analyzed for ABC. By morphological and cytochemical analysis, the macrophages remaining in suspension contributed less than 1% of the antigen-binding cells. IgM and IgG PFC specific for SRC were enumerated using the CunninghamSzenberg slide assay (16). The facilitating serum for detecting IgG PFC was the IgG fraction of a rabbit anti-mouse IgG (Cappel Laboratories Inc., Cochranville, Pa.) at a final concentration of 1500. Since this reagent did not inhibit direct IgM PFC, specific PFC (as in Figs. 1 and 2) equalled IgM plus IgG anti-SRC plaques. Specificity was determined using ox (ORC) and burro (BRC) erythrocytes as controls in the ABC and PFC assays. Assays for total non-SRC-specific PFC. To determine the total number of Ig-secreting cells in vitro over the 6-day course of culture, we employed a plaquing assay using protein A-coupled SRC or BRC as described by Gronowicz et al. (17). Protein A was prepared from Cowan I strain Staphylococcus aureus as described by Sjoquist et al. (18) and Kronvall(l9). Fresh guinea pig complement was used at a 1:60 final dilution. Facilitating sera were (i) rabbit anti-mouse IgM (a generous gift of Dr. Fritz Melchers) used at 1:800 final dilution, and (ii) rabbit anti-mouse IgG, generated against MOPC 21A (IgG,), used at tinal concentration of 1:135. In order to directly compare specific SRC-PFC to total Ig-secreting PFC using the same plaquing method, we modified the Jerne plaque assay to conform to the Gronowicz technique, omitting the protein A. BRC served as a control. Autoradiography of cultured cells. Cultures were incubated 17 hr just prior to harvest with 1 PCilculture of [3H]thymidine ([3H]TdR) (6.7 Ci/mmol, New England Nuclear, Boston, Mass.). Pulse-chased cultures were pulsed with 3 &i [3H]TdR (1.1 x lop4 mg), washed after 17 hr, and chased with 25 pugcold thymidine (Sigma Chemical Co., St. Louis, MO.) per 1ml culture in conditioned medium, and harvested daily until Day 6. This amount of cold TdR was 250-fold in excess of the [3H]TdR originally added, thus precluding [3H]TdR reutilization. This concentration of TdR was well below the 2 mM (484 pg/ml) necessary to inhibit DNA synthesis (20). Cells were harvested by washing twice in Eagle’s minimum essential medium (MEM) and resuspending in MEM plus 50% FCS. Lymphocytes were rosetted as above and smeared on subbed slides, prepared according to Rogers (21). These smears were methanol fixed, dipped in Ilford K2 nuclear emulsion (Ilford, Ltd., Essex, England), exposed for 4 days at 4°C developed in Kodak D19 developer for 5 min, and fixed for 8 min. The slides were stained with Cameo Quick Stain (Scientific Products, Detroit, Mich.) for 10 set, and at 1000x magnification any cell with more than’20 silver grains over its nucleus was scored as labeled. Background labeling
GENERATION
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143
was less than 5 grains per nucleus. There were no cells showing 5 to 20 grains per nucleus. Use ofhydroxyurea (HU) in vitro. Hydroxyurea (Sigma Chemical Company) was stored as crystals at 4°C in a darkened desiccator. It was dissolved in PBS and sterilized by filtration prior to use in culture. Direct effects of the drug in vitro were examined by adding it 24 hr prior to harvesting the cultures. To determine the reversibility of the drug, cells in cultures incubated for 24 hr with hydroxyurea (HU) were washed, resuspended in medium without drug, and harvested 24 hr later. No-drug control cultures were treated identically. Effect of HU on DNA and protein synthesis. To assess DNA synthesis, sample cultures with and without HU were incubated with 1 &i[3H]thymidine (6.7 Cilmmol, New England Nuclear) either during the 24 hr of exposure to drug or during the 24-hr post-wash recovery time. Determination of protein synthesis was accomplished by incubating cultures with 30 &i[3H]leucine (57 Ci/mmol, New England Nuclear) for 4 hr prior to harvest. Cells from these cultures were harvested and washed twice in PBS. DNA or protein was precipitated in 10% cold TCA for 1 hr. Precipitates were redissolved in 2 N NaOH and counted in a Beckman scintillation counter. RESULTS Primary Response Kinetics In viva, the primary IgM PFC response to SRC in C57B1/6mice peaked on Day 5, and IgG PFC peaked on Day 6 (data not shown). Antigen-binding cells (ABC) also peaked on Day 5, averaging 13-fold more than the Day 0 ABC and fivefold more than Day 5 IgM PFC. In primary Mishell-Dutton cultures, preliminary experiments showed optimal conditions for ABC and PFC generation to be the same: 1.5 x 10’lymphocytes and l-2 x 10’ SRC per culture. The frequency of specific PFC generated and the Day 5 peak were features similar in vivo and in vitro. But several aspects of the in vitro response differed from that seen in vivo: (i) Only IgM anti-SRC PFC were produced in vitro, (ii) ABC peaked on Day 4 in vitro and were declining at the time of
DAYS IN CULTURE
FIG. 1. Primary in vitro response to SRBC. ABC/lo3 lymphocytes in culture with antigen (W), without antigen (O), PFC per lo3 lymphocyte in cultures with antigen (O), without antigen (0), using Cunningham-Szenberg slide assay (28). Standard errors shown. In SRBC-stimulated cultures, ABC/lo3 specific for control BRC and ORC decreased from 0.4 to 0 by Day 2 and from 0.3 to 0 by Day 6, respectively; PFC/lOG specific for BRC decreased from 0.3 to 0 by Day 5.
MERRILL AND ASHMAN
‘“L-J 0
1
2 3 4 5 DAYS IN CULTURE
6
FIG. 2. Comparison of specific PFC to total Ig-secreting cells in primary in vitro response to SRC. SRC-specific PFC in cultures with antigen (0) without antigen (0), measured by the Gronowicz technique without protein A or facilitating antiserum (29); total Ig-secreting cells in culture with antigen (W), without antigen (U), measured by the Gronowicz technique. Standard errors shown.
maximum PFC (Day 5), (iii) the number of ABC increased only five-fold over Day 0, so there was little excess of ABC over PFC (Fig. 1). In control cultures with no SRC, the frequency of specific ABC declined from the Day 0 level after Day 4 (Fig. 1). However, since the absolute number of total cells per culture decreased from Day 0 (data not shown), the decline in ABC/culture was progressive from Day 0. SRC-stimulated cultures reached IO-fold higher SRC-PFC culture than antigen-free controls (Figs. 1 and 2). PFC as well as ABC for ORC and BRC declined progressively in all cultures (legend, Fig. l), but SRC-PFC even in antigen-free cultures rose somewhat (Figs. 1 and 2), an observation best explained by invoking the “J-substance” of FCS that cross reacts with a sheep erythrocyte membrane component (14, 22) rather than solely the mitogenic effect of FCS. Total Ig-Secreting
Cell Response
There was no detectable influence of SRC on the magnitude of the increase in nonspecific IgM-secreting cells or its kinetics (Fig. 2). This increase was therefore attributed solely to mitogenic activity in the medium. The percentage decrease of total PFC in the first 24 hr approximated the percentage decrease of specific PFC and total cell numbers. When protein A-BRC were used in place of protein A-SRC in this assay for nonspecific IgM-secreting cells, there was no significant difference in the number of PFC observed, ensuring that SRC-specific PFC were not interfering with the development of protein A-SRC PFC. Using a rabbit-facilitating antibody directed toward mouse IgG capable of detecting IgG PFC after in vivo immunization, we were unable to detect any IgG-secreting cells in nonimmune spleen, nor did they appear in the 6 days of culture. Autoradiographic
Demonstration
that ABC Arose from Dividing
Cells
Figure 3 shows data pooled from four autoradiography experiments in which non-ABC and ABC pulsed each day with [3H]TdR were scored. Labeled ABC in
GENERATION
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CELLS
145
60
9 50 % 'LJ 40 2 $ 30 s 20 10
01 0
1
2
3
4
5
6
DAYS IN CU4lURE
FIG. 3. Autoradiography of cells pulsed 17 hr with 1 &i titrated thymidine (sp act 6.7 Ci/mmol) immediately before harvest. P, Pulse, H harvest. Non-antigen-binding cells in cultures with antigen (Cl), in cultures without antigen (0); antigen-binding cells in cultures with antigen (W), in cultures without antigen (0). Percentage cells labeled is: number of labeled ABC/total number ABC; number labeled nonABC/total non-ABC. Standard errors shown.
FIG. 4. Antigen-binding cell in late telophase from a Day 4 culture. Cells in culture were incubated Days 3-4 with 1 &i 13H]TdR (sp act 6.7 Ci/mmol), harvested, rosetted, and smeared on subbed slides. After a 4-day exposure of Ilford K2 nuclear emulsion, the slides were developed in Kodak D19 developer. Photograph is 250~ magnification.
146
MERRILL AND ASHMAN
antigen-stimulated cultures increased from 0% of total ABC at Day 1 to 60% at Day 4 (when peak ABC numbers occurred, Fig. I), declining on Days 5 and 6 parallel to the decline in ABC absolute numbers. Due to the effects of mitogenic components of the medium, the percentage of ABC and non-ABC labeled in antigen-free cultures and non-ABC labeled in antigen-stimulated cultures increased together to a plateau of 20-30%. Thus antigen preferentially stimulated specific ABC (or their precursors) to division as early as Day 2. Figure 4 shows an ABC with grains over its telophase nucleus, illustrating the point that receptors can be maintained by ABC through mitosis. Figure 5 presents data pooled from two identical experiments where cells were labeled with [3H]TdR in the first 24 hr and then chased with thymidine showing absolute numbers of labeled ABC and non-ABC per culture. In the first 24 hr of culture, lo5 non-ABC (representing 1% of the total cells in culture) were labeled, but not one labeled ABC could be found even after scanning 6.6 x lo5 Day 1 cells and scoring lo4 non-ABC and 600 ABC for silver grains. The 3.2 x lo3 ABC which were labeled at Day 4 (representing 20% of the total Day 4 ABC) could only have been derived from non-ABC which were making DNA during the first 24 hr providing a direct demonstration that dividing non-ABC can mature to ABC. As in Fig. 3, the rapid early rise of labeled ABC from 0 per culture at Day 1 to 3 x lo3 per culture at Day 4 contrasted with the later more gradual increase in labeled non-ABC (Fig. 5); but in both cases the maximum number of labeled cells coincided with the Day 4 maximum ABC numbers (Fig. 1). Thereafter, labeled non-ABC plateaued, but labeled ABC fell abruptly, perhaps because they were maturing beyond the receptor-bearing stage. Dependence ofABC Generation on DNA Synthesis Demonstrated Inhibition
by Hydroxyurea
Since autoradiography had demonstrated that many ABC generated in vitro by antigen exposure arose from cells dividing in the previous day, the next step was to demonstrate that this cell division was obligatory, i.e., that interruption of DNA synthesis would interrupt the generation of new ABC.
0123456 DAYS IN CULTURE
FIG. 5. Autoradiography of [3H]TdR pulse-chased cells. Cells pulsed with 3 @i VH]TdR (sp act 6.7 Ci/mmol), chased with 25 w/ml cold TdR. P, Pulse, H, harvest. Pulse, dark line; chase, light line. Labeled ABC/culture (m), labeled non-ABC/culture (0). Standard deviations shown.
GENERATION
OF ANTIGEN-BINDING
CELLS
147
Preliminary studies (not shown) using HU in the cultures for 24 hr at concentrations ranging from 0.10 to 10.0 mM showed that 0.35 mM HU substantially decreased DNA synthesis (measured as [3H]TdR incorporation), without affecting cell viability or total protein synthesis (measured as [3H]leucine incorporation into 10% TCA-precipitable material). Table 1 shows the effect of this dose on [3H]TdR incorporation, PFC, and ABC when cells were pulsed with HU 24 hr prior to harvest. From Day 2 on, 13H]TdR uptake was 90% inhibited. PFC were most sensitive to inhibition between Day 2 and Day 5, decreasing more than 70%. ABC were 60-70% inhibited between Days 2 and 4. Thus the greatest inhibition of ABC generation by HU coincided with the greatest proportion of autoradiographitally labeled ABC (Fig. 3). After the peak of ABC on Day 4 and the peak of PFC on Day 5, these cells rapidly lost their sensitivity to HU (Table 1). Yet 80-90% of [3H]TdR uptake was still inhibited on these days. The simplest explanation for this decrease of sensitivity to HU is that the peak ABC and PFC numbers occurred when the generation of new ABC or PFC by cell division began to decline. If the cells were pulsed for 24 hr washed, resuspended in HU-free medium, and allowed to recover for 24 hr [3H]TdR incorporation and ABC recovered substantially (especially Days 2-4), but PFC did not recover (Table l), even if 4 days were allowed for recovery (not shown). To determine whether the direct effect of HU on dividing ABC could fully account for the decrease in ABC, autoradiography was used to assess cultures treated with HU. The results are shown in Table 2. Hydroxyurea reduced the percentage of labeled ABC and labeled non-ABC to the same degree on all 3 days, demonstrating that antigen-stimulated ABC were not preferentially susceptible to HU (column 8). At Days 4 and 5, there was good correlation between two methods used to calculate the percentage reduction of total ABC (compare columns 3 and 9): (i) comparing ABC numbers in HU-treated and control cultures (column 3), and (ii) (as recorded in column 9) by multiplying precentage labeled ABC in HU-free cultures (column 6) by the present reduction of labeled cells after hydroxyurea (column 8). This correlation also held for the reduction of absolute numbers of ABC per culture on Days 3, 4, and 5 (compare columns 2 and 7). In other words, the decreasein labeled ABC accounted for the total decreasein ABC observed with HU. Thus there was no need to postulate either a dividing non-ABC required for the generation of ABC on Days 4 and 5, or an effect of HU on ABC generation other than its effect on DNA synthesis. DISCUSSION Since antigen receptors are displayed both on the resting B and T cells and (in lesser amounts) on some antibody-secreting cells (23, 24), one would predict that cells at intermediate stages in the response should also bind antigen. Since PFC generation is known to require cell division (25-27), and since PFC themselves have been shown to incorporate [3H]TdR (28-30), antigen-induced proliferation of ABC should be demonstrable. This paper (a) provides a direct demonstration that specific ABC can be generated by antigen exposure of spleen cells in vitro where cell traffic can be eliminated, (b) provides the first demonstration that cell division played a major (Fig. 2) and necessary (Table 1) role in this increase, (c) demonstrates by autoradiography that receptors could be retained by ABC in
1
2
3
4
5
6
0
1
2
3
4
5
5
83 ? 15
116 ” 34
72 2 16
55 t_ 10
43% 5
3,
cpm x 10-3/107 control cells’
1.03 2 0.06
0.57 t 0.03 0.45 r 0.02 -
0.13 2 0.03 0.15 2 0.07 0.10 k 0.01
0.29 iz 0.10
0.49 + 0.02
0.10 k 0.01 0.21 k 0.09
0.33 k 0.12
0.90 -c 0.07
0.53 k 0.01
0.69 k 0.05
0.89 -t 0.06
0.78 2 0.02
-
0.16 -c 0.10
0.18 -c 0.09
0.03 r+_0.02
0.52 2 0.13
0.56 t 0.03
ExperimentaUControl at end 24 hr of pulse post-W.O.
PFClculture
1.03 2 0.01
Experimental/Control at end 24 hr of pulse* post-W.0.d
[3H]TdR incorporation”
0.73 k 0.08 0.90 k 0.15 0.80 + 0.10
0.39 + 0.10 0.29 t 0.06 0.88 k 0.14
-
0.77 c 0.04
0.73 2 0.10
0.61 2 0.08
0.93 + 0.03
0.95 t 0.06
Experimental/Control at end 24 hr of pulse post-W.O.
ABC/culture
a 1 /.Ki tritiated thymidine (sp act 6.7 Ci/mmol) present 17 hr preceding harvest. b HU present in culture 24 hr immediately preceding harvest. c cpm x 10-3/107control cells, representing the average of six experiments. rl Drug present 24 hr in culture during period indicated, washed out, and cultures harvested after 24-hr recovery period (i.e., 24 hr after harvest indicated in b. t3H]TdR incubated in 24-hr recovery period immediately preceding harvest. Standard errors shown.
Culture harvested” (Day)
HU and VH]TdR added (Day)
Effect of 0.35 mM Hydroxyurea on Primary SRBC Response in Vitro
TABLE 1
8
% ;
F
E
E m .-.
0.4 x 104
0.6 x IO4 1
+
-
4
5
5
ABC
Non-ABC
ABC
-
x x x x
100 106 104 IO4
2
0.2 x 104
0.7 x 106
1.6 x lo4
1.6 x 10”
1.0x104
1.8 x 106
Per culture
3
40
21
69
32
82
31
%
x x x x
x x x x 106 lo6 lo4 lo4
10” 106 104 104
2.5 x 10” 2.3 x lo6 0.3 x 104 0.3 x 104 4
3.3 3.8 0.6 0.8
4.0 5.3 0.2 0.5
Unlabeled cells/culture x x x x
52 6
3.0 x 103 5
5 25 12.5 67
2 10.5 3 59
7.5 32 5
106 10” 104 104
lo” lo5 102 lo2
Percentage labeled’
0.2 x 106 1.1 x 106 0.2 x 103
0.2 x 1.3 x 0.1 x 1.5 x
0.8 6.2 0.7 72.0
Labeled cells/culture”
Autoradiography
7
2.8 x lo3
0.9 x 10”
1.4 x 104
1.1 x106
0.71 x 104
5.4 x lo5
Per culture
Reduction of labeled cells”
8
93
81
94
87
99
87
%
9
48
26
63
22
58
Percentage reduction of total cells’
Note. 0.35 mM hydroxyurea was present in culture 24 hr immediately preceding harvest on Days 3, 4, and 5 as shown. The effect of the drug on ABC and non-ABC was assayed by direct cell and rosette counts on a hemocytometer and by the reduction of [3H]TdR-labeled cells assessed by autoradiography. HU, Hydroxyurea. u Reduction of total cells: Column 2: cells per culture without HU minus those with HU (column 1). Column 3: percentage is column 2 as a percentage of cells without HU (column 1). * Labeled cells per culture: Column 5: assayed by scoring 12,000 cells with greater than 20 grains per nucleus. r Percentage labeled: Column 6: labeled ABC or non-ABC per culture (column 5) divided by total ABC or non-ABC per culture (column l), respectively. ’ Reduction of labeled cells: Column 7: labeled cells per culture without HU minus labeled cells with HU (column 5). Column 8: column 7 divided by labeled cells without HU (column 5). e Percentage of total cells: Column 9: percentage reduction of labeled cells (column 8) times percentage of cells labeled in cultures without drug (column 6). This represents an estimate of the percentage reduction of total ABC which derives from the loss of labeled ABC. This table represents the results from a single experiment.
Column
2.7 x lo6 3.4 x 10”
+
4
Non-ABC
+ -
3.5 5.1 0.7 2.3
+
3
ABC
106 106 104 104
+ -
x x x x
4.1 5.9 0.2 1.2
+
3
Non-ABC
Cells/culture
0.35 mM HU
Harvest Day
Reduction of total cells”
Cell counts
Effect of Hydroxyurea on Antigen-Binding Cells
TABLE 2
52 cl PI
6 9 62
E
150
MERRILL
AND ASHMAN
the act of mitosis (Fig. 4), (d) shows by two independent techniques that the contribution of cell division to ABC generation was continuous throughout this rising phase of the response, peaking when the absolute numbers of ABC peaked, and falling abruptly when the numbers of ABC declined, overlapping broadly with terminal differentiation events in the same culture (Tables 1 and 2; Fig. 3), and (e) shows that the division of ABC was not the only source of new ABC. Since 20% of the Day 4 ABC derived from cells dividing in the first 24 hr which could not bind enough antigen to be recognized as ABC at that time, maturation of receptor displays also contributed to ABC generation (Fig. 5). The rise in ABC observed in vitro was indeed antigen-driven (i.e., did not occur in antigen-free medium, Fig. l), and antigen-specific (shown by lack of increase in ORC- and BRC-binding cells, Fig. 1 legend). In contrast, about 10% of the maximal increment in SRC-PFC was attributable to SRC- cross-reactive medium components (14, 22) (Fig. 2). There was no evidence from enumerating total Ig-secreting cells for mitogenic stimulation by SRC in our in vitro system (Fig. 2), even though in vivo SRC have been reported to stimulate non-SRC-specific Ig secretion (31). However, mitogenic effects of the medium were evident in the sevenfold increase in total Ig-secreting cells reaching a peak on Day 4, characteristically 1 day earlier than the anti-SRC response peak (32) (Fig. 2), and in the increase from 1 to 30% of [3H]TdR-labeled cells in nonantigen-stimulated cultures (Fig. 3). Though cytophilic IgM antibody generated in the culture could have contributed to the apparent increase in ABC, this contribution is probably small for several reasons: (i) ABC began to increase and peak 1 day earlier than PFC (Fig. l), (ii) Day 0 cells incubated in Day 5 supernates failed to show increased antigen binding, (iii) cells intentionally loaded with cytophilic anti-SRC antibody may bind SRC, but this binding is lost during the washing that precedes ABC counting (33). ABC at the peak of the C57BV6 response in vivo and in vitro were 40-50% T cells and 50-60% B cells (33) (data not shown), as were SRC-ABC in CBA mice (2,34), and ABC for DNP-KLH (35), or TIGAL (36). The proportion of T and B ABC observed in antigen-free Day-4 cultures and normal spleen cells was similar. Thus the increase in ABC (Fig. 1) included both T and B ABC, and further studies have examined the kinetics of this expansion in more detail (33). More than 3000 ABC per culture, up to 20% of the ABC at the peak of the response, arose from cells not detectable as ABC in the first 24 hr of culture (Fig. S), presumably because their receptor density or avidity was too low to retain detectable antigen. Nevertheless, their receptors must have been sufficient to enable antigen contact to induce division, leading to a later increase in their antigen binding ability into the detectable range by maturation. Variables which may have affected the detectability of ABC include changes in receptor density, turnover, or orientation in the membrane, and the deployment of new receptor-associated molecules such as Ia (37). Whereas autoradiography directly revealed the proportion of ABC which had divided in the previous 24-hr period (Fig. 3), HU inhibition demonstrated that this cell division was necessary for generation of ABC. Hydroxyurea prevents the reduction of ribonucleotides to deoxyribonucleotides by inhibiting the ribonucleotide diphosphate reductase (38). Its effect is immediate and partially reversible upon removal (39, 40). Since the cell cycle time for antibody-forming cells in an antigen-induced response has been reported to be 13 hr (41) and in a
GENERATION
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mitogen-induced system, 12.5 (42) or 18hr (43), the effect of HU on the dividing cell population was maximized by pulsing cultures for 24 hr immediately preceding harvest and assay. Lower doses of HU than 0.35 mM or shorter exposure time gave better reversibility, but showed less inhibition. Several observations support the conclusion that the effect of HU on ABC numbers is derived from inhibition of cell division in the ABC themselves: (i) If the decrease in ABC in HU-containing cultures had been due only to inhibition of DNA synthesis in a dividing non-ABC cooperating cell, rather than direct inhibition of division in the ABC themselves, then the percentage of reduction of autoradiographically labeled ABC (column 9, Table 2) should have been much less than the percentage reduction in total ABC (column 3, Table 2), but it was not. (ii) Nor was the decrease in labeled ABC per culture (column 7, Table 2) less than the decrease in total ABC per culture (column 2, Table 2). (iii) In the first 24 hr of antigen exposure, there was no significant [3H]TdR uptake (Table l), and few cells labeled by autoradiography (Figs. 3 and 5), consistent with previous reports of an 18- to 24-hr lag in DNA synthesis after antigen (44) and mitogen (45). During this lag period, HU had no effect on ABC numbers, showing that the decrease of ABC by HU was probably not the result of direct “receptor stripping.” (iv) The lower proportion of ABC after 0.35 mA4HU was not due to a gross inequality in cell cycle time between ABC and non-ABC, as the percentage reductions of dividing ABC and non-ABC were about equal (Table 2). Our data indicate that at Days 3-5,70-80% of PFC must arise by division (Table 1); some other reports have claimed all PFC do (29, 30, 46). The effect of HU on PFC was not reversible (Table l), a finding reported previously by Jaroslow and Ortiz-Ortiz (47) though not confirmed by Andersson and Melchers (48). Perhaps HU interfered with critical cellular cooperation steps required at specific times in the maturation of plaque-forming cells. If division did not occur at these times, precursors might be driven irreversibly into the memory pool instead of the antibody-secreting population. But we cannot rule out the alternative explanations that HU might have increased the population of suppressor cells, or might even have killed the antigen-induced PFC. After Day 4, the autoradiography data showed that more ABC were dividing (Fig. 3) than one would have predicted if ABC from Days 5 and 6 had come from the previous day’s ABC augmented by cell division. Furthermore, from the absolute values of ABC (not shown) in the experiments in Table 1, one can calculate that ABC were actually lost in the absence of cell division from Day 2 on, not just after Day 4 when cell division declined anyway. Taken together, these observations support the concept that progression of ABC to non-ABC was occurring, but do not permit a quantitative statement as to how much of this progression was due to conversion of ABC to nonantigen-binding PFC. Thus three processes govern the numbers of ABC present in culture: the generation of ABC by division until the fourth day of culture, the maturation of dividing non-ABC to ABC, and the transformation of ABC into non-ABC. This concept of the dynamic role of ABC in the in vitro primary response, derived from the current paper, forms the cornerstone of our ongoing efforts to define the antigen-induced early events in ABC, such as the kinetics of B and T ABC, changes in surface Ig isotype following the antigen contact (33), and the site of action of regulatory influences.
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ACKNOWLEDGMENTS The authors are grateful to Dr. Irving Weissman for a generous gift of anti-T serum, to Dr. Fritz Melchers for anti-p serum, to Dr. James Kenny for anti-y serum, and to Dr. W. R. Clark for reviewing the manuscript. Otto Martinez purified the protein A, Ada Sulitzeanu assisted with the immunofluorescence experiments, Zane Price took the photograph, and Judy Fung and Emma Hollins typed the manuscript. Supported by NIH Training Grant AL-0712601 to Jean Merrill, NIH Grant CA-12808 from the National Cancer Institute, and NIH Grant AI-14922 from the National Institute of Allergy and Infectious Disease.
REFERENCES 1. Feldmann, M., Beverley, B., Erb, P., Howie, S., Kontiainen, S., Maoz, A., Mathies, M., McKenzie, I., and Woody, J., Cold Spring Harbor Symp. Quant. Biol. 41, 112, 1976. 2. Ashman, R. F., and Raff, M. C., J. Exp. Med. 137,69, 1973. 3. Brody, T., J. Immunol. 105, 126, 1970. 4. Wilson, J. D., Immunology 21, 233, 1971. 5. Kontiainen, S., and Andersson, L. C., J. Exp. Med. 142, 1035, 1975. 6. Elliott, B. E., and Haskill, J. S., J. Exp. Med. 141, 600, 1975. 7. Maoz, A., Feldmann, M., and Kontiainen, S., Nature (London) 260, 324, 1976. 8. Elliott, B. E., Haskill, J. S., and Axelrad, M. A., J. Exp. Med. 414,584, 1975. 9. Ashman, R. F., J. Immunol. 111, 212, 1973. 10. Kenny, J. J., Martinez-Maza, O., Fehniger, T., and Ashman, R. F., J. Immunol. 122, 1278, 1979. 11. Ashman, R. F., Eur. J. Immunol. 6,421, 1975. 12. Kenny, J. H., Kessler, S. W., Ahmed, A., Ashman, R. F., and Scher, I., J. Immunol. 122,2037, 1979.
13. Kanowith-Klein, S., Vitetta, E. S., Kom, E., and Ashman, R. F., J. Immunol. 122: 2349, 1979. 14. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. 15. Tucker, S. B., Pierce, R. V., and Jordan, R. E., J. Immunol. Methods 14,267, 1977. 16. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. 17. Gronowicz, E., Coutinho, A., and Melchers, R., Eur. J. Immunol. 6,588, 1976. 18. Sjoquist, J., Hjelm, H., and Meloun, B., Eur. J. Biochem. 29, 572, 1972. 19. Kronvall, G., Stand. J. Immunol. 2,31, 1973. 20. Xeros, N., Nature (London) 194, 682, 1962. 21. Rogers, A. W., (Ed.),ln “Techniques of Autoradiography,” p. 269. Elsevier, Amsterdam/London/ New York, 1967. 22. Dutton, R. W., and Mishell, R. I., In “Society of General Physiologists” (L. Goldstein, Ed.), pp. 19-46. Prentice-Hall, Englewood Cliffs, N.J. 1%7. 23. McConnell, I., Nature New Biol. 233, 177, 1971. 24. Bankert, R. B., Wolf, B., and Pressman, D., Cell. Immunol. 27, 111, 1976. 25. Nossal, G. J. V., and Pike, B. L., Immunology 30, 189, 1976. 26. Mishell, R. I., and Dutton, R. W., Science 153, 1004, 1966. 27. Biozzi, G., Stiffel, C., and Mouton, D., In “Immunity, Cancer and Chemotherapy” (E. Mihich, Ed.), p. 103. Academic Press, London/New York, 1%7. 28. Perkins, E. H., Sado, T., and Makinodan, T., J. Zmmunol. 103, 688, 1%9. 29. Koros, A. M., Mazur, J. M., and Mowery, M. J., J. Exp. Med. 128,235, 1968. 30. Hiinig, T., Schimpl, A., and Wecker, E., J. Exp. Med. 139, 754, 1974. 31. Moticka, E. J., Zmmunology 27, 401, 1974. 32. Melchers, F., and Andersson, J., Eur. J. Immunol. 4, 687, 1974. 33. Merrill, J. E., and Ashman, R. F., J. Immunol. 123: 434, 1979. 34. Wilson, J. D., Immunology 25, 185, 1973. 35. Kiefer, H., Eur. J. Immunol. 5, 624, 1975. 36. Roelants, G. E., and Ryden, A., Nature (London) 247, 104, 1974. 37. Lonai, P., and Steinman, L., Proc. Nat. Acad. Sci. USA 74, 5662, 1977. 38. Krakoff, I. H., Brown, N. C., and Reichard, P., Cancer Res. 28, 1559, 1%9. 39. Gale, G. R., Biochem. Pharmacol. 17,235, 1968. 40. Nedrud, J., Touton, M., and Clark, W. R., J. Exp. Med. 142, 960, 1975.
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Tannenberg, W., Malaviya, J. K., and Malaviya, A. W., J. Exp. Med. 128, 895, 1968. Gerhart, S., Mills, G., Monticone, V., and Paetkau, V., J. Zmmunol. 117, 1314, 1976. Andersson, J., Coutinho, A., Lemhardt, W., and Melchers, R., Cell. 10: 27, 1977. Mills, J.,Zn “Fifth Leukocyte Culture Conference” (J. L. Harris, Ed.), p. 51. Academic Press, New York, 1970. Melchers, P., and Andersson, J., Transplant. Rev. 14, 101, 1973. Dutton, R. W., and Mishell, R. I., .Z. Exp. Med. 126, 443, 1967. Jaroslow, B. N., and Ortiz-Ortiz, L., Cell. Zmmunol. 2, 164, 1971. Andersson, J., and Melchers, F., Eur. .I. Zmmunol. 4, 535, 1974.
IMMUNOLOGY
52,
140- 153 (1980)
The in Vitro Generation of Antigen-Binding JEAN Department
Cells
E. MERRILL AND ROBERT F. ASHMAN’
of Microbiology and Immunology, and Rheumatology Division, Department UCLA School of Medicine, Los Angeles, California 90024
of Medicine,
Received March 29, 1979
To analyze early events following the contact of antigen with specific receptor-bearing cells, antigen-binding cells (ABC) specific for sheep erythrocytes (SRC) were generated in a primary Mishell-Dutton culture system. The maximal frequency of ABC (a five-fold increase over Day 0 involving both T and B cells) occurred on Day 4 in culture conditions which yielded optimal numbers of plaque-forming cells (PFC) specific for SRC 1day later. Assessment of the total &secreting cells as well as ABC and PFC for ox and burro erythrocytes showed that the ABC as well as the PFC response to SRC was antigen specific. By autoradiography of [3H]thymidine-pulsed cultures and by inhibiting cell division with hydroxyurea, the continuous contribution of cell division to the generation of ABC was demonstrated. As with PFC, the decline in ABC numbers began at the point where their generation by cell division slowed. After removal of hydroxyurea, ABC frequency and thymidine incorporation recovered substantially, but PFC did not. However, division of ABC was not the only process involved in ABC generation. Indeed, 20% of the Day 4 ABC arose from cells dividing in the first 24 hr of culture which were not detectable as ABC at that time, i.e., some ABC were derived from dividing non-ABC. Thus both cell division and maturation contributed to the increase in specific ABC seen after primary in vitro immunization. Both processes can be added to the list of early antigen-stimulated events in specific ABC which can be analyzed in the primary culture system in future studies of cell interactions.
INTRODUCTION Most of our current concepts of cell interactions in the immune response derive from experiments where lymphocyte populations defined and isolated by their surface markers are recombined so as to reveal their function. In most such experiments, the read-out of “function” is plaque-forming cells or antibody, the final step of antigen-driven B-cell maturation. Commonly, the models derived from such experiments place the B cell last, as if triggering of B cells cannot occur unless the antigen has hrst interacted with appropriate T cells and macrophages (1). But failure of B cells to complete terminal differentiation does not mean that normal triggering has not occurred. It is equally possible that B cells receive a normal activation signal directly from antigen-receptor contact, but that signals from other cells are required for progression beyond a certain later step. Testing this alternative model would require knowledge of the critical early events which follow 1 Reprint request should be sent to Dr. Ashman at Department of Microbiology and Immunology, 43319 Center for Health Sciences, UCLA School of Medicine, Los Angeles, Calif. 90024. 140 OOOS-8749/80/070140-14$02.00/O Copyright 0 1980by Academic F’ress, Inc. All rights of reproduction in any form reserved.
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antigen contact with that small fraction of lymphocytes which bear the correct specific receptors, the antigen-binding cells (ABC).’ In the mouse spleen the lymphocyte population which binds sheep erythrocytes (SRC) have been shown to contain both B and T cells (2), including the precursors for anti-SRC plaque-forming cells (3, 4), T helper cells (5,6), suppressor cells (7), and cytotoxic cells (8). We have defined several events which follow antigen contact with these cells: (i) capping, maximal by 30 min (9); (ii) accelerated phospholipid synthesis, evident within a few hours (IO); (iii) accelerated receptor turnover, evident within the first day (R. F. Ashman, manuscript in preparation)$ and (iv) decreased sensitivity of the antigen receptor to trypsin appearing around Day 3 (1 l), probably related to the decrease in surface IgD and gain of surface IgG (12, 13). Clearly the expansion of the ABC population is another significant early event that must be placed in this sequence. Although it is tempting to assume that ABC simply derive by division of previous ABC, proof of this assumption is fundamental to future progress in studying early events in ABC, particularly since traffic of ABC into or from the spleen and maturation of ABC without cell division remain important alternative explanations for the increase in ABC following in viva immunization. This paper establishes that the antigen-driven .increase in ABC can be analyzed without cell traffic in a primary culture system, providing the groundwork for our future investigations of early events. A substantial daily contribution of cell division to ABC generation from the third through the sixth day of culture was demonstrated, using both autoradiography and inhibition by hydroxyurea. But more surprisingly, a significant contribution to ABC expansion from early dividing cells which were not detectable as ABC after the first day was also shown, indicating that maturation of antigen-binding ability accompanied proliferation. MATERIALS AND METHODS Mice. Female C57B1/6 mice, 7 to 12 weeks old, were obtained from Jackson Laboratories, Bar Harbor, Maine. Antigen. Sheep erythrocytes (SRC) from a single sheep were obtained in Alsever’s solution from Gibco Diagnostics, Madison, Wisconsin, and stored less than 2 months before use. Cells from this sheep were selected from many lots of SRC for their ability to stimulate a response in a primary Mishell-Dutton culture system. Ox and burro erythrocytes were obtained from Colorado Serum Company Laboratories, Denver, Colorado. Spleen cell cultures. Spleens harvested from unimmunized mice were minced and passed through a wire screen. Primary in vitro immune responses to SRC were studied employing exactly the procedure and medium described by Mishell and Dutton (14) except that 50 mg/ml of gentamicin (Microbiological Associates, Bethesda, Md.) was used as the antibiotic. Cultures in Falcon tissue culture plates (No. 3001) contained 1.5 x 1O’spleen cells and 1O’SRC in 1 ml of medium. Two lots of fetal calf serum (FCS) giving high numbers of PFC in screening experiments were 2 Abbreviations used: ABC, antigen-binding cells; PFC, plaque-forming cells; SRC, sheep red blood cells (also sheep erythrocytes); BRC, burro red blood cells; ORC, ox red blood cells; HEG, Hepes-Eagle’s gelatin medium; FCS, fetal calf serum; MEM, Eagle’s minimum essential medium; HU, hydroxyurea; 13HlTdR, tritiated thymidine; TCA, trichloroacetic acid. 3 R. F. Ashman, J. Immunol. 124: 893, 1980.
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used in all experiments. Cultures were rocked on a Bellco rocker at 37°C in Billups-Rothenberg chambers in a moist, nitrogen-rich atmosphere and fed daily with nutrient cocktail containing 30% FCS (14). Cells were harvested daily from cultures with and without antigen to assay for the generation of ABC and PFC. Viability was monitored by trypan blue exclusion and cell recovery by total cell counts in 1.0 M acetic acid. Assays for SRC-specijc ABC and PFC. ABC generated in vivo and in vitro were enumerated using the rosetting technique of Ashman (9). Lymphocytes binding more than four SRC were scored as rosettes (RFC) on a hemocytometer. By staining lipase-positive cells (15), it was determined that 60-90% of all macrophages and their precursors remained stuck to the plastic plate throughout the culture period and thus were absent from the suspensions analyzed for ABC. By morphological and cytochemical analysis, the macrophages remaining in suspension contributed less than 1% of the antigen-binding cells. IgM and IgG PFC specific for SRC were enumerated using the CunninghamSzenberg slide assay (16). The facilitating serum for detecting IgG PFC was the IgG fraction of a rabbit anti-mouse IgG (Cappel Laboratories Inc., Cochranville, Pa.) at a final concentration of 1500. Since this reagent did not inhibit direct IgM PFC, specific PFC (as in Figs. 1 and 2) equalled IgM plus IgG anti-SRC plaques. Specificity was determined using ox (ORC) and burro (BRC) erythrocytes as controls in the ABC and PFC assays. Assays for total non-SRC-specific PFC. To determine the total number of Ig-secreting cells in vitro over the 6-day course of culture, we employed a plaquing assay using protein A-coupled SRC or BRC as described by Gronowicz et al. (17). Protein A was prepared from Cowan I strain Staphylococcus aureus as described by Sjoquist et al. (18) and Kronvall(l9). Fresh guinea pig complement was used at a 1:60 final dilution. Facilitating sera were (i) rabbit anti-mouse IgM (a generous gift of Dr. Fritz Melchers) used at 1:800 final dilution, and (ii) rabbit anti-mouse IgG, generated against MOPC 21A (IgG,), used at tinal concentration of 1:135. In order to directly compare specific SRC-PFC to total Ig-secreting PFC using the same plaquing method, we modified the Jerne plaque assay to conform to the Gronowicz technique, omitting the protein A. BRC served as a control. Autoradiography of cultured cells. Cultures were incubated 17 hr just prior to harvest with 1 PCilculture of [3H]thymidine ([3H]TdR) (6.7 Ci/mmol, New England Nuclear, Boston, Mass.). Pulse-chased cultures were pulsed with 3 &i [3H]TdR (1.1 x lop4 mg), washed after 17 hr, and chased with 25 pugcold thymidine (Sigma Chemical Co., St. Louis, MO.) per 1ml culture in conditioned medium, and harvested daily until Day 6. This amount of cold TdR was 250-fold in excess of the [3H]TdR originally added, thus precluding [3H]TdR reutilization. This concentration of TdR was well below the 2 mM (484 pg/ml) necessary to inhibit DNA synthesis (20). Cells were harvested by washing twice in Eagle’s minimum essential medium (MEM) and resuspending in MEM plus 50% FCS. Lymphocytes were rosetted as above and smeared on subbed slides, prepared according to Rogers (21). These smears were methanol fixed, dipped in Ilford K2 nuclear emulsion (Ilford, Ltd., Essex, England), exposed for 4 days at 4°C developed in Kodak D19 developer for 5 min, and fixed for 8 min. The slides were stained with Cameo Quick Stain (Scientific Products, Detroit, Mich.) for 10 set, and at 1000x magnification any cell with more than’20 silver grains over its nucleus was scored as labeled. Background labeling
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was less than 5 grains per nucleus. There were no cells showing 5 to 20 grains per nucleus. Use ofhydroxyurea (HU) in vitro. Hydroxyurea (Sigma Chemical Company) was stored as crystals at 4°C in a darkened desiccator. It was dissolved in PBS and sterilized by filtration prior to use in culture. Direct effects of the drug in vitro were examined by adding it 24 hr prior to harvesting the cultures. To determine the reversibility of the drug, cells in cultures incubated for 24 hr with hydroxyurea (HU) were washed, resuspended in medium without drug, and harvested 24 hr later. No-drug control cultures were treated identically. Effect of HU on DNA and protein synthesis. To assess DNA synthesis, sample cultures with and without HU were incubated with 1 &i[3H]thymidine (6.7 Cilmmol, New England Nuclear) either during the 24 hr of exposure to drug or during the 24-hr post-wash recovery time. Determination of protein synthesis was accomplished by incubating cultures with 30 &i[3H]leucine (57 Ci/mmol, New England Nuclear) for 4 hr prior to harvest. Cells from these cultures were harvested and washed twice in PBS. DNA or protein was precipitated in 10% cold TCA for 1 hr. Precipitates were redissolved in 2 N NaOH and counted in a Beckman scintillation counter. RESULTS Primary Response Kinetics In viva, the primary IgM PFC response to SRC in C57B1/6mice peaked on Day 5, and IgG PFC peaked on Day 6 (data not shown). Antigen-binding cells (ABC) also peaked on Day 5, averaging 13-fold more than the Day 0 ABC and fivefold more than Day 5 IgM PFC. In primary Mishell-Dutton cultures, preliminary experiments showed optimal conditions for ABC and PFC generation to be the same: 1.5 x 10’lymphocytes and l-2 x 10’ SRC per culture. The frequency of specific PFC generated and the Day 5 peak were features similar in vivo and in vitro. But several aspects of the in vitro response differed from that seen in vivo: (i) Only IgM anti-SRC PFC were produced in vitro, (ii) ABC peaked on Day 4 in vitro and were declining at the time of
DAYS IN CULTURE
FIG. 1. Primary in vitro response to SRBC. ABC/lo3 lymphocytes in culture with antigen (W), without antigen (O), PFC per lo3 lymphocyte in cultures with antigen (O), without antigen (0), using Cunningham-Szenberg slide assay (28). Standard errors shown. In SRBC-stimulated cultures, ABC/lo3 specific for control BRC and ORC decreased from 0.4 to 0 by Day 2 and from 0.3 to 0 by Day 6, respectively; PFC/lOG specific for BRC decreased from 0.3 to 0 by Day 5.
MERRILL AND ASHMAN
‘“L-J 0
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2 3 4 5 DAYS IN CULTURE
6
FIG. 2. Comparison of specific PFC to total Ig-secreting cells in primary in vitro response to SRC. SRC-specific PFC in cultures with antigen (0) without antigen (0), measured by the Gronowicz technique without protein A or facilitating antiserum (29); total Ig-secreting cells in culture with antigen (W), without antigen (U), measured by the Gronowicz technique. Standard errors shown.
maximum PFC (Day 5), (iii) the number of ABC increased only five-fold over Day 0, so there was little excess of ABC over PFC (Fig. 1). In control cultures with no SRC, the frequency of specific ABC declined from the Day 0 level after Day 4 (Fig. 1). However, since the absolute number of total cells per culture decreased from Day 0 (data not shown), the decline in ABC/culture was progressive from Day 0. SRC-stimulated cultures reached IO-fold higher SRC-PFC culture than antigen-free controls (Figs. 1 and 2). PFC as well as ABC for ORC and BRC declined progressively in all cultures (legend, Fig. l), but SRC-PFC even in antigen-free cultures rose somewhat (Figs. 1 and 2), an observation best explained by invoking the “J-substance” of FCS that cross reacts with a sheep erythrocyte membrane component (14, 22) rather than solely the mitogenic effect of FCS. Total Ig-Secreting
Cell Response
There was no detectable influence of SRC on the magnitude of the increase in nonspecific IgM-secreting cells or its kinetics (Fig. 2). This increase was therefore attributed solely to mitogenic activity in the medium. The percentage decrease of total PFC in the first 24 hr approximated the percentage decrease of specific PFC and total cell numbers. When protein A-BRC were used in place of protein A-SRC in this assay for nonspecific IgM-secreting cells, there was no significant difference in the number of PFC observed, ensuring that SRC-specific PFC were not interfering with the development of protein A-SRC PFC. Using a rabbit-facilitating antibody directed toward mouse IgG capable of detecting IgG PFC after in vivo immunization, we were unable to detect any IgG-secreting cells in nonimmune spleen, nor did they appear in the 6 days of culture. Autoradiographic
Demonstration
that ABC Arose from Dividing
Cells
Figure 3 shows data pooled from four autoradiography experiments in which non-ABC and ABC pulsed each day with [3H]TdR were scored. Labeled ABC in
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FIG. 3. Autoradiography of cells pulsed 17 hr with 1 &i titrated thymidine (sp act 6.7 Ci/mmol) immediately before harvest. P, Pulse, H harvest. Non-antigen-binding cells in cultures with antigen (Cl), in cultures without antigen (0); antigen-binding cells in cultures with antigen (W), in cultures without antigen (0). Percentage cells labeled is: number of labeled ABC/total number ABC; number labeled nonABC/total non-ABC. Standard errors shown.
FIG. 4. Antigen-binding cell in late telophase from a Day 4 culture. Cells in culture were incubated Days 3-4 with 1 &i 13H]TdR (sp act 6.7 Ci/mmol), harvested, rosetted, and smeared on subbed slides. After a 4-day exposure of Ilford K2 nuclear emulsion, the slides were developed in Kodak D19 developer. Photograph is 250~ magnification.
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antigen-stimulated cultures increased from 0% of total ABC at Day 1 to 60% at Day 4 (when peak ABC numbers occurred, Fig. I), declining on Days 5 and 6 parallel to the decline in ABC absolute numbers. Due to the effects of mitogenic components of the medium, the percentage of ABC and non-ABC labeled in antigen-free cultures and non-ABC labeled in antigen-stimulated cultures increased together to a plateau of 20-30%. Thus antigen preferentially stimulated specific ABC (or their precursors) to division as early as Day 2. Figure 4 shows an ABC with grains over its telophase nucleus, illustrating the point that receptors can be maintained by ABC through mitosis. Figure 5 presents data pooled from two identical experiments where cells were labeled with [3H]TdR in the first 24 hr and then chased with thymidine showing absolute numbers of labeled ABC and non-ABC per culture. In the first 24 hr of culture, lo5 non-ABC (representing 1% of the total cells in culture) were labeled, but not one labeled ABC could be found even after scanning 6.6 x lo5 Day 1 cells and scoring lo4 non-ABC and 600 ABC for silver grains. The 3.2 x lo3 ABC which were labeled at Day 4 (representing 20% of the total Day 4 ABC) could only have been derived from non-ABC which were making DNA during the first 24 hr providing a direct demonstration that dividing non-ABC can mature to ABC. As in Fig. 3, the rapid early rise of labeled ABC from 0 per culture at Day 1 to 3 x lo3 per culture at Day 4 contrasted with the later more gradual increase in labeled non-ABC (Fig. 5); but in both cases the maximum number of labeled cells coincided with the Day 4 maximum ABC numbers (Fig. 1). Thereafter, labeled non-ABC plateaued, but labeled ABC fell abruptly, perhaps because they were maturing beyond the receptor-bearing stage. Dependence ofABC Generation on DNA Synthesis Demonstrated Inhibition
by Hydroxyurea
Since autoradiography had demonstrated that many ABC generated in vitro by antigen exposure arose from cells dividing in the previous day, the next step was to demonstrate that this cell division was obligatory, i.e., that interruption of DNA synthesis would interrupt the generation of new ABC.
0123456 DAYS IN CULTURE
FIG. 5. Autoradiography of [3H]TdR pulse-chased cells. Cells pulsed with 3 @i VH]TdR (sp act 6.7 Ci/mmol), chased with 25 w/ml cold TdR. P, Pulse, H, harvest. Pulse, dark line; chase, light line. Labeled ABC/culture (m), labeled non-ABC/culture (0). Standard deviations shown.
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Preliminary studies (not shown) using HU in the cultures for 24 hr at concentrations ranging from 0.10 to 10.0 mM showed that 0.35 mM HU substantially decreased DNA synthesis (measured as [3H]TdR incorporation), without affecting cell viability or total protein synthesis (measured as [3H]leucine incorporation into 10% TCA-precipitable material). Table 1 shows the effect of this dose on [3H]TdR incorporation, PFC, and ABC when cells were pulsed with HU 24 hr prior to harvest. From Day 2 on, 13H]TdR uptake was 90% inhibited. PFC were most sensitive to inhibition between Day 2 and Day 5, decreasing more than 70%. ABC were 60-70% inhibited between Days 2 and 4. Thus the greatest inhibition of ABC generation by HU coincided with the greatest proportion of autoradiographitally labeled ABC (Fig. 3). After the peak of ABC on Day 4 and the peak of PFC on Day 5, these cells rapidly lost their sensitivity to HU (Table 1). Yet 80-90% of [3H]TdR uptake was still inhibited on these days. The simplest explanation for this decrease of sensitivity to HU is that the peak ABC and PFC numbers occurred when the generation of new ABC or PFC by cell division began to decline. If the cells were pulsed for 24 hr washed, resuspended in HU-free medium, and allowed to recover for 24 hr [3H]TdR incorporation and ABC recovered substantially (especially Days 2-4), but PFC did not recover (Table l), even if 4 days were allowed for recovery (not shown). To determine whether the direct effect of HU on dividing ABC could fully account for the decrease in ABC, autoradiography was used to assess cultures treated with HU. The results are shown in Table 2. Hydroxyurea reduced the percentage of labeled ABC and labeled non-ABC to the same degree on all 3 days, demonstrating that antigen-stimulated ABC were not preferentially susceptible to HU (column 8). At Days 4 and 5, there was good correlation between two methods used to calculate the percentage reduction of total ABC (compare columns 3 and 9): (i) comparing ABC numbers in HU-treated and control cultures (column 3), and (ii) (as recorded in column 9) by multiplying precentage labeled ABC in HU-free cultures (column 6) by the present reduction of labeled cells after hydroxyurea (column 8). This correlation also held for the reduction of absolute numbers of ABC per culture on Days 3, 4, and 5 (compare columns 2 and 7). In other words, the decreasein labeled ABC accounted for the total decreasein ABC observed with HU. Thus there was no need to postulate either a dividing non-ABC required for the generation of ABC on Days 4 and 5, or an effect of HU on ABC generation other than its effect on DNA synthesis. DISCUSSION Since antigen receptors are displayed both on the resting B and T cells and (in lesser amounts) on some antibody-secreting cells (23, 24), one would predict that cells at intermediate stages in the response should also bind antigen. Since PFC generation is known to require cell division (25-27), and since PFC themselves have been shown to incorporate [3H]TdR (28-30), antigen-induced proliferation of ABC should be demonstrable. This paper (a) provides a direct demonstration that specific ABC can be generated by antigen exposure of spleen cells in vitro where cell traffic can be eliminated, (b) provides the first demonstration that cell division played a major (Fig. 2) and necessary (Table 1) role in this increase, (c) demonstrates by autoradiography that receptors could be retained by ABC in
1
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[3H]TdR incorporation”
0.73 k 0.08 0.90 k 0.15 0.80 + 0.10
0.39 + 0.10 0.29 t 0.06 0.88 k 0.14
-
0.77 c 0.04
0.73 2 0.10
0.61 2 0.08
0.93 + 0.03
0.95 t 0.06
Experimental/Control at end 24 hr of pulse post-W.O.
ABC/culture
a 1 /.Ki tritiated thymidine (sp act 6.7 Ci/mmol) present 17 hr preceding harvest. b HU present in culture 24 hr immediately preceding harvest. c cpm x 10-3/107control cells, representing the average of six experiments. rl Drug present 24 hr in culture during period indicated, washed out, and cultures harvested after 24-hr recovery period (i.e., 24 hr after harvest indicated in b. t3H]TdR incubated in 24-hr recovery period immediately preceding harvest. Standard errors shown.
Culture harvested” (Day)
HU and VH]TdR added (Day)
Effect of 0.35 mM Hydroxyurea on Primary SRBC Response in Vitro
TABLE 1
8
% ;
F
E
E m .-.
0.4 x 104
0.6 x IO4 1
+
-
4
5
5
ABC
Non-ABC
ABC
-
x x x x
100 106 104 IO4
2
0.2 x 104
0.7 x 106
1.6 x lo4
1.6 x 10”
1.0x104
1.8 x 106
Per culture
3
40
21
69
32
82
31
%
x x x x
x x x x 106 lo6 lo4 lo4
10” 106 104 104
2.5 x 10” 2.3 x lo6 0.3 x 104 0.3 x 104 4
3.3 3.8 0.6 0.8
4.0 5.3 0.2 0.5
Unlabeled cells/culture x x x x
52 6
3.0 x 103 5
5 25 12.5 67
2 10.5 3 59
7.5 32 5
106 10” 104 104
lo” lo5 102 lo2
Percentage labeled’
0.2 x 106 1.1 x 106 0.2 x 103
0.2 x 1.3 x 0.1 x 1.5 x
0.8 6.2 0.7 72.0
Labeled cells/culture”
Autoradiography
7
2.8 x lo3
0.9 x 10”
1.4 x 104
1.1 x106
0.71 x 104
5.4 x lo5
Per culture
Reduction of labeled cells”
8
93
81
94
87
99
87
%
9
48
26
63
22
58
Percentage reduction of total cells’
Note. 0.35 mM hydroxyurea was present in culture 24 hr immediately preceding harvest on Days 3, 4, and 5 as shown. The effect of the drug on ABC and non-ABC was assayed by direct cell and rosette counts on a hemocytometer and by the reduction of [3H]TdR-labeled cells assessed by autoradiography. HU, Hydroxyurea. u Reduction of total cells: Column 2: cells per culture without HU minus those with HU (column 1). Column 3: percentage is column 2 as a percentage of cells without HU (column 1). * Labeled cells per culture: Column 5: assayed by scoring 12,000 cells with greater than 20 grains per nucleus. r Percentage labeled: Column 6: labeled ABC or non-ABC per culture (column 5) divided by total ABC or non-ABC per culture (column l), respectively. ’ Reduction of labeled cells: Column 7: labeled cells per culture without HU minus labeled cells with HU (column 5). Column 8: column 7 divided by labeled cells without HU (column 5). e Percentage of total cells: Column 9: percentage reduction of labeled cells (column 8) times percentage of cells labeled in cultures without drug (column 6). This represents an estimate of the percentage reduction of total ABC which derives from the loss of labeled ABC. This table represents the results from a single experiment.
Column
2.7 x lo6 3.4 x 10”
+
4
Non-ABC
+ -
3.5 5.1 0.7 2.3
+
3
ABC
106 106 104 104
+ -
x x x x
4.1 5.9 0.2 1.2
+
3
Non-ABC
Cells/culture
0.35 mM HU
Harvest Day
Reduction of total cells”
Cell counts
Effect of Hydroxyurea on Antigen-Binding Cells
TABLE 2
52 cl PI
6 9 62
E
150
MERRILL
AND ASHMAN
the act of mitosis (Fig. 4), (d) shows by two independent techniques that the contribution of cell division to ABC generation was continuous throughout this rising phase of the response, peaking when the absolute numbers of ABC peaked, and falling abruptly when the numbers of ABC declined, overlapping broadly with terminal differentiation events in the same culture (Tables 1 and 2; Fig. 3), and (e) shows that the division of ABC was not the only source of new ABC. Since 20% of the Day 4 ABC derived from cells dividing in the first 24 hr which could not bind enough antigen to be recognized as ABC at that time, maturation of receptor displays also contributed to ABC generation (Fig. 5). The rise in ABC observed in vitro was indeed antigen-driven (i.e., did not occur in antigen-free medium, Fig. l), and antigen-specific (shown by lack of increase in ORC- and BRC-binding cells, Fig. 1 legend). In contrast, about 10% of the maximal increment in SRC-PFC was attributable to SRC- cross-reactive medium components (14, 22) (Fig. 2). There was no evidence from enumerating total Ig-secreting cells for mitogenic stimulation by SRC in our in vitro system (Fig. 2), even though in vivo SRC have been reported to stimulate non-SRC-specific Ig secretion (31). However, mitogenic effects of the medium were evident in the sevenfold increase in total Ig-secreting cells reaching a peak on Day 4, characteristically 1 day earlier than the anti-SRC response peak (32) (Fig. 2), and in the increase from 1 to 30% of [3H]TdR-labeled cells in nonantigen-stimulated cultures (Fig. 3). Though cytophilic IgM antibody generated in the culture could have contributed to the apparent increase in ABC, this contribution is probably small for several reasons: (i) ABC began to increase and peak 1 day earlier than PFC (Fig. l), (ii) Day 0 cells incubated in Day 5 supernates failed to show increased antigen binding, (iii) cells intentionally loaded with cytophilic anti-SRC antibody may bind SRC, but this binding is lost during the washing that precedes ABC counting (33). ABC at the peak of the C57BV6 response in vivo and in vitro were 40-50% T cells and 50-60% B cells (33) (data not shown), as were SRC-ABC in CBA mice (2,34), and ABC for DNP-KLH (35), or TIGAL (36). The proportion of T and B ABC observed in antigen-free Day-4 cultures and normal spleen cells was similar. Thus the increase in ABC (Fig. 1) included both T and B ABC, and further studies have examined the kinetics of this expansion in more detail (33). More than 3000 ABC per culture, up to 20% of the ABC at the peak of the response, arose from cells not detectable as ABC in the first 24 hr of culture (Fig. S), presumably because their receptor density or avidity was too low to retain detectable antigen. Nevertheless, their receptors must have been sufficient to enable antigen contact to induce division, leading to a later increase in their antigen binding ability into the detectable range by maturation. Variables which may have affected the detectability of ABC include changes in receptor density, turnover, or orientation in the membrane, and the deployment of new receptor-associated molecules such as Ia (37). Whereas autoradiography directly revealed the proportion of ABC which had divided in the previous 24-hr period (Fig. 3), HU inhibition demonstrated that this cell division was necessary for generation of ABC. Hydroxyurea prevents the reduction of ribonucleotides to deoxyribonucleotides by inhibiting the ribonucleotide diphosphate reductase (38). Its effect is immediate and partially reversible upon removal (39, 40). Since the cell cycle time for antibody-forming cells in an antigen-induced response has been reported to be 13 hr (41) and in a
GENERATION
OF ANTIGEN-BINDING
CELLS
151
mitogen-induced system, 12.5 (42) or 18hr (43), the effect of HU on the dividing cell population was maximized by pulsing cultures for 24 hr immediately preceding harvest and assay. Lower doses of HU than 0.35 mM or shorter exposure time gave better reversibility, but showed less inhibition. Several observations support the conclusion that the effect of HU on ABC numbers is derived from inhibition of cell division in the ABC themselves: (i) If the decrease in ABC in HU-containing cultures had been due only to inhibition of DNA synthesis in a dividing non-ABC cooperating cell, rather than direct inhibition of division in the ABC themselves, then the percentage of reduction of autoradiographically labeled ABC (column 9, Table 2) should have been much less than the percentage reduction in total ABC (column 3, Table 2), but it was not. (ii) Nor was the decrease in labeled ABC per culture (column 7, Table 2) less than the decrease in total ABC per culture (column 2, Table 2). (iii) In the first 24 hr of antigen exposure, there was no significant [3H]TdR uptake (Table l), and few cells labeled by autoradiography (Figs. 3 and 5), consistent with previous reports of an 18- to 24-hr lag in DNA synthesis after antigen (44) and mitogen (45). During this lag period, HU had no effect on ABC numbers, showing that the decrease of ABC by HU was probably not the result of direct “receptor stripping.” (iv) The lower proportion of ABC after 0.35 mA4HU was not due to a gross inequality in cell cycle time between ABC and non-ABC, as the percentage reductions of dividing ABC and non-ABC were about equal (Table 2). Our data indicate that at Days 3-5,70-80% of PFC must arise by division (Table 1); some other reports have claimed all PFC do (29, 30, 46). The effect of HU on PFC was not reversible (Table l), a finding reported previously by Jaroslow and Ortiz-Ortiz (47) though not confirmed by Andersson and Melchers (48). Perhaps HU interfered with critical cellular cooperation steps required at specific times in the maturation of plaque-forming cells. If division did not occur at these times, precursors might be driven irreversibly into the memory pool instead of the antibody-secreting population. But we cannot rule out the alternative explanations that HU might have increased the population of suppressor cells, or might even have killed the antigen-induced PFC. After Day 4, the autoradiography data showed that more ABC were dividing (Fig. 3) than one would have predicted if ABC from Days 5 and 6 had come from the previous day’s ABC augmented by cell division. Furthermore, from the absolute values of ABC (not shown) in the experiments in Table 1, one can calculate that ABC were actually lost in the absence of cell division from Day 2 on, not just after Day 4 when cell division declined anyway. Taken together, these observations support the concept that progression of ABC to non-ABC was occurring, but do not permit a quantitative statement as to how much of this progression was due to conversion of ABC to nonantigen-binding PFC. Thus three processes govern the numbers of ABC present in culture: the generation of ABC by division until the fourth day of culture, the maturation of dividing non-ABC to ABC, and the transformation of ABC into non-ABC. This concept of the dynamic role of ABC in the in vitro primary response, derived from the current paper, forms the cornerstone of our ongoing efforts to define the antigen-induced early events in ABC, such as the kinetics of B and T ABC, changes in surface Ig isotype following the antigen contact (33), and the site of action of regulatory influences.
152
MERRILL AND ASHMAN
ACKNOWLEDGMENTS The authors are grateful to Dr. Irving Weissman for a generous gift of anti-T serum, to Dr. Fritz Melchers for anti-p serum, to Dr. James Kenny for anti-y serum, and to Dr. W. R. Clark for reviewing the manuscript. Otto Martinez purified the protein A, Ada Sulitzeanu assisted with the immunofluorescence experiments, Zane Price took the photograph, and Judy Fung and Emma Hollins typed the manuscript. Supported by NIH Training Grant AL-0712601 to Jean Merrill, NIH Grant CA-12808 from the National Cancer Institute, and NIH Grant AI-14922 from the National Institute of Allergy and Infectious Disease.
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