CELLULAR

IMMlJNOLOGY

143,449-466

(1992)

Monoclonal Antibodies Specific for Novel Murine Cell Surface Markers Define Subpopulations of Germinal Center Cells FRANCES

M.

PLATT,*.’ JOSEPH

JUDITH M.

A. CEBRA-THOMAS,~.~

DAVIE.*

AND

JOHN

CHARLES

M.

BALJM,~~’

P. MCKEARN*

A panel of mAbs has been generated which selectively, but not exclusively, recognizes populations of cells within germinal centers of immunized mice. All four mAbs stain B cell populations as defined by flow cytometry. The mAbs FH9.5 and C3.5 also stain T cell subsets (CD4+ and CD8’. respectively). Following density gradient centrifugation of spleen cells from immunized mice the majority of FH9.5’ and C3.5+ B cells are found in the low density. activated fractions. The cells bearing the epitope(s) recognized by the C6C3 and the A6A2 mAbs are less frequent, and from flow cytometric analysis the cells stained with these mAbs are B cells and myeloid cells. The surface markers defined by the four mAbs are not induced following mitogen stimulation of small resting B cells suggesting that these molecules are not general activation markers, Cell lines from a variety of hematopoietic lineages expressing the four markers have been identified. The cell surface molecule immunoprecipitated by the FH9.5 mAb is a polypeptide of 23-28 kDa. The C3.5 antigen is an 85- to 95-kDa protein. These mAbs will be useful in elucidating the complex events involved in B cell differentiation and maturation which occur within germinal centers. ca 1992 4cademic

Press. Inc.

1. INTRODUCTION

Germinal centers (GCs) are histologically conspicuous regions which develop within B cell follicles of peripheral lymphoid tissues in response to T helper cell-dependent antigens (1). They are composed of clustered B cell blasts undergoing antigen-driven activation and proliferation (1). GCs have been shown to develop in an oligoclonal manner (2) and to be major sites of isotype switching (3). In addition. they are also important sites for the generation of memory B cell (4) and plasma cell precursors ( 1). A complex series of cellular events takes place within the peripheral lymphoid tissues following antigen administration. In the absence of antigenic challenge the B cell follicles consist of small resting B cells that are slgM/slgD positive and express the MEL- 14 lymphocyte homing receptor (5). Following immunization it has been dem’ Present address: Department of Biochemistry, University of Oxford, South Parks Road. 3QU. UK. * Present address: Department of Biology, Princeton University, Princeton. NJ 08544-101. 3 Present address: Systemix, Palo Alto. CA 94303. 4 Abbreviations used: CC. germinal center(s): PNA, peanut agglutinin.

Oxford

OX I

449 000%8749/92

$5.00

Copyright 6 1992 by Academic Press, Inc All rights of reproductmn in any form reswed.

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onstrated (6) that a novel class of follicular dendritic cell traps antigen on its cell surface. The cell then disseminates antigen in an intact form on the surface of beaded processes which detach from the dendritic cell and disperse throughout the B cell follicle (6). These antigen-coated bodies are then available for uptake by either macrophages or antigen-specific B cells which are both capable of antigen presentation to T cells (7). Following activation of antigen-specific B cells, extensive proliferation is accompanied by maturation and a decrease in slgD expression and cell density. In addition, expression of the MEL- 14 homing receptor is lost (8) rendering the GC B cells nonmigratory. It has been speculated that during GC development somatic mutation of the Ig V genes of GC B cells takes place (9). The role that the specialized GC microenvironment plays in the regulation of these processes is poorly understood. An important area of study is the involvement of soluble cytokines and cell adhesion molecules in the control of B cell maturation within these sites. Recently it has been demonstrated that IL-5 is capable of driving both proliferation and maturation of small resting B cells to Ig-secreting cells in vitro (10). However, the precise sequence of signaling events required to drive B cell differentiation and maturation within the context of the GC microenvironment has yet to be determined and has not been mimicked in vitro. However, there has been a recent report of the analysis of single Peyer’s patch GC B cells in a T cell-dependent microculture system which supports the proliferation, isotype switching, and Ig secretion of a population of GC B cells (11). Quantitating the repertoire of cell surface molecules expressed by cells within GCs may aid in the elucidation of these processes. Subsequent analysis of the function of such membrane molecules may further define the elements required to drive B cell maturation. GC B cells are readily identifiable because they express high levels of the receptor for the plant lectin, peanut agglutinin (PNA (12)) and low levels of slgD. However, these criteria alone do not permit the subdivision of the GC B cell population into distinct groups that may represent functional subsets or define maturational state. A variety of monoclonal antibodies (mAbs) have been described which specifically distinguish between subsets of human GC cells ( 13- 16) but there is a lack of comparable reagents specific for murine GC cells. In this report we describe a panel of rat mAbs which bind to subsets of cells within murine GCs. The cell surface molecules recognized by these mAbs are apparently unique on the basis of cellular distribution and preliminary biochemical characterization. 2. MATERIALS

AND

METHODS

2.1. Animals Adult outbred rats were purchased from Sprague-Dawley, Inc. (Indianapolis, IN). Adult BALB/c mice were purchased from Charles River Laboratories Inc. (Wilmington, MA). CBA/N mice were obtained from The Jackson Laboratory (Bar Harbor, ME).

2.2. Antibodies In addition to the mAbs generated for this study, several rat mAbs of defined specificity were used throughout these experiments: 187.1, anti-mouse kappa light chain (17); 14.8, anti-B cell lineage specific antigen “B220” (18); M5/114, anti-I-A, and IE (19); GK1.5, anti-CD4 (20); 53-6.7, anti-Ly-2 or CDS (21) (Becton-Dickinson,

mAbs

DEFINE

CC

CELL

SUBPOPULATIONS

Mountain View, CA); 30H 12, anti-Thy- 1.2 (2 1) (Becton-Dickinson): Mac- 1 (22); and J 1J, anti-Thy- 1.2 (23).

451 M l/70, anti-

2.3. Cell Lines The cell lines X16C-8.5, MOPC-3 15J, S49.1, P388D-1, WEHI-3B, 5774, PU-5-18, P8 15, BCL- I-3b3, and 70213 were obtained from the American Type Culture Collection (Rockville, MD). The pre-B cell lines 40E1, 38Bl 1, 22D6, 204-I-8, 220-8, 230-238, 204-3-1, 28C9, and 230-37 have been described previously (24). The B and T cell lines WEHI-279.75, WEHI1, 2PK3.5558 11, MPC- 11, MOPC21, S107, WR19-L, and BW5 147 (25) and the three IL-3-dependent lines, FDC-Pl, FL5.12, and MC/9 (26) were also used during these studies. The IIIC2.9 T cell line was generated in this laboratory. L 1OA and A20- 1 were provided by Dr. J. McCubrey (East Carolina University) and EL4/9 by Dr. M. Julius (McGill University). Cell lines were maintained in Iscove’s modified Dulbecco’s medium (IMDM. GIBCO-Life Technologies Inc., Grand Island, NY) supplemented with 5% fetal calf serum (Hazleton Research Products Inc., Denver, PA). The factor-dependent cell lines FDC-P 1, MC/ 9, and FL5.12 were supplemented with 5% WEHI-3B-conditioned medium as a source of IL-3. Cultures were maintained in a humidified incubator at 37°C with 5% COz. 7% 02, and 88% NZ. 2.4. Monoclonal

Antibody

Production

Sprague-Dawley outbred rats were immunized (iv) with either 1 X lo* 5606 plasmacytoma cells (passaged as ascites, Litton Bionetics Inc., Kensington, MD) or 1 X 10’ (BALB/c X CBA/N) F, spleen cells in PBS (0.1 M, pH 7.2). Four days later the spleens were fused with the nonsecreting mouse myeloma line Sp2/0-Ag14 (27) according to established techniques (28). Supematants from wells positive for hybridoma cell growth were screened histologically on 5-pm frozen spleen sections. The clones C6C3 and A6A2 resulted from the fusion of the 5606 immunized rat and the clones FH9.5 and C3.5 resulted from the fusion ofthe rat immunized with murine splenocytes. The FH9.5 mAb was of the IgG2a subclass while the other three mAbs were of the IgM isotype as determined by immunodiffusion using antisera specific for each rat Ig heavy chain isotype (Serotec Limited, Oxford, UK). 2.5. Antihod), Pur(jication

and Cmjugation

mAbs were purified from both serum-containing and serum-free culture medium using a protein G-Sepharose column for FH9.5 (IgG2a) (Zymed Laboratories Inc., South San Francisco, CA) or by ion exchange and gel filtration chromatography for the IgM mAbs. Purified mAbs were fluorescein-conjugated by dialyzing the protein against 0.1 M NaHC03/Na2C03 buffer at pH 9.0 and adjusting the concentration to 3-4 mg/ml. Twenty microliters of 5 mg/ml fluorescein isothiocyanate (FITC, Calbiothem, La Jolla, CA) in DMSO (Sigma) was added per milliliter of mAb solution and reacted in the dark for 1-2 hr at room temperature. Free FITC was removed from the mAb preparation by passage over a Sephadex G-25 column equilibrated with PBS. Biotin conjugation was performed using purified mAb at 1 mg/ml in 0.1 M sodium bicarbonate buffer, pH 8.4. Sixty microliters of a 2 mg/ml NHS-LC-biotin (Pierce,

452

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Rockford, IL) in DMSO (Sigma) was added per milliliter of mAb solution and stirred for 4 hr in the dark. Free biotin was removed by dialysis against PBS for 18 hr. 2.6. Immunizations

BALB/c mice were immunized (ip) with 100 ~1 of a 10% suspension of sheep red blood cells (SRBC, Colorado Serum Company, Denver, CO) in PBS. 2.7. Flow Cytometry

Analysis

Preparation of cells for fluorescence analysis has been described previously (25). Briefly, 4 X lo5 mononuclear cells were incubated in individual wells of a 96-well Vbottomed plate (Dynatech Laboratories Inc., Chantilly, Virginia) with an optimal dilution of rat mAb for 30 min on ice. The cells were washed twice and incubated for 30 min with FITC-conjugated RG7/9 (mouse anti-rat K light chain) and RG7/11 (mouse anti-rat IgG2b antibody) (29). The cells were washed twice and resuspended in PBS containing 0.1% BSA (Sigma Chemical Co., St. Louis, MO), 0.02 A4 sodium azide (Sigma), and 2 yg/ml propidium iodide (Sigma). Samples were analyzed using a FACScan cytometer (Becton-Dickinson, Sunnyvale, CA). Dead cells were excluded from the analysis on the basis of differential uptake of propidium iodide. Data were collected on 1 X lo4 viable cells. The percentage of cells expressing a given surface marker was assessedby defining a region which included no overlap with cells incubated with second-step antibody alone or cells incubated with an isotype-matched rat mAb (with no binding specificity for murine cells) plus second-step anti-rat Ig. For two-color analysis, single-cell suspensions were stained with biotinylated rat mAb as described above or bioltinylated PNA (Sigma, 1:80 dilution) followed by phycoerythrin-conjugated avidin (Biomeda Corp., Foster City, CA) and FITC-conjugated rat anti-mouse Ig. The samples were then analyzed as described above. Contour plots from the analysis of 1 X lo4 viable cells are plotted on a 4-decade log,, scale of increasing green fluorescence on the x-axis and red fluorescence on the v-axis. The cursor positions are placed such that cells incubated with secondary antibody alone fall within the boundaries of the lower left-hand quadrant. Isotype-matched “negative” controls 49C2D6 (rat IgM anti-DNP) and YTH 76.9 (rat IgG2a anti-human HLA class 1 antigen, Serotec Ltd., Oxford, UK) were run in the assays and did not stain above the fluorescence intensity of the secondary antibody alone (data not shown). 2.8. Histology

Portions of spleens removed from animals for analysis by flow cytometry were immediately embedded in O.C.T. compound (Miles Scientific, Naperville, IL), frozen in liquid nitrogen, and were stored at -80°C. The 5-pm cryostat sections were cut and fixed for 2 min in absolute ethanol, air-dried, and stored at -80°C. Tissue sections were brought to room temperature and blocked with 15% horse serum (Hazleton) in PBS. The primary rat mAb was incubated at room temperature for 30 min, the slides were washed three times with PBS and were incubated with biotinylated goat anti-rat IgG (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) in PBS for 30 min at room temperature. The sections were washed and incubated with avidin-peroxidase (Vectastain ABC, Vector Laboratories Inc., Burlingame, CA) and washed three times

mAbs

DEFINE

GC

CELL

SUBPOPULATIONS

453

with PBS. The staining was visualized by the addition of 0.05% diaminobenzidine (Sigma) and 0.035% H202 in PBS for 5 min at room temperature. The slides were washed, dehydrated, and mounted. Isotype-matched negative control mAbs (see flow cytometry methods) were included as controls for nonspecific and Fc receptor binding.

2.9. Density Gradient Centrtjiigation Mononuclear cell preparation from BALB/c spleens were washed three times with PBS and incubated at 4 X lo7 cells/ml with the JlJ mAb (anti-Thy 1.2) for 30 min on ice. The cells were washed once with HBSS and resuspended at 4 X lo7 cells/ml in IMDM containing rabbit complement (Pel-Freez Biologicals, Rogers, AR) and incubated at 37°C for 1 hr. The cells were washed three times with HBSS (pH 7.2). Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradients were prepared by layering 2 ml of each of the densities of balanced Percoll (1.090, 1.085, 1.080. 1.075, and 1.065) and applying the cells in 1 ml of HBSS (0.5-1.0 X 10’ cells per gradient) to the top of the gradient. The gradients were spun at room temperature at 14OOg for 30 min. Dead cells which did not enter the 1.065 density layer were discarded. Cells at the density layer interfaces were analyzed by flow cytometry as described above.

2.10. LPS Blasts Splenic B cell populations were prepared by anti-Thy- 1 and complement treatment as described above. The small resting B cell population was purified by density gradient centrifugation harvesting only the cell population falling within the 1.090 density band and excluding those cells falling at the interface between the 1.090 and 1.085 density layers. The cells were washed three times with PBS and once with IMDM and were resuspended at 0.2 X lo6 cells/ml in the presence of 25 or 50 pg/ml of Escherichia co/i-derived lipopolysaccharide (LPS) (Difco Laboratories, Detroit, MI) and cultured at 37°C for 2 or 5 days. The cells were harvested, washed three times with PBS, stained, and analyzed by flow cytometry as described above.

2.11. Immunoprecipitation Two million cells in 0.5 ml of PBS were surface labeled with 1 mCi of 12? (Amersham, Arlington Heights, IL) in Iodogen (Pierce Chemical Company, Rockford, IL)-coated tubes according to the method of Markwell and Fox (30). The cells were washed three times with PBS/5 mM NaI and lysed in 1 ml of PBS/O.S% NP-40 (Sigma) containing 50 pg/ml TPCK (Sigma), 50 @g/ml TLCK (Sigma), and 100 pg/ml PMSF (Sigma) on ice for 30 min. Immunoprecipitations were performed with the mAb absorbed to rabbit anti-rat-coated Staphylococcus aweus Cowan (SAC) cells or directly coupled to cyanogen bromide-activated Sepharose beads (Sigma). The lysates were precleared with either rabbit anti-rat-SAC or normal rat serum (Sigma) coupled to Sepharose beads depending on the solid phase used for the immunoprecipitation. The precipitates were analyzed by SDS-PAGE and visualized by autoradiography. 3. RESULTS

3. I. Histological

Localization

OJ‘mAb Binding to GCs

From the mAbs generated for this study four (FH9.5, C3.5, C6C3, and A6A2) were selected for detailed characterization on the basis of their capacity to bind to subsets

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mAbs

DEFINE

CC

CELL TABLE

Tissue Distribution

of CC Markers

within

I

the Lpmphoid

Tissues of Unimmunized Percent

Tissue

FH9.5

Bone marrow Spleen Lymph node Thymus Peyer’s patch

46 t 6.8 24t 12 29 f 5.0 3 2 0.6 Ilf I.1

455

SUBPOPULATIONS

c3.5 59 IS 19 2 I8

f k t f -t

BALB/c

positive C6C3

4.0 2.3 2.8 0.6 4.0

Mice

2 I 0 0 2

t i f k f

0.6 I.0 0.0 0.0 0.5

A6A2 2 2 0 0 I

f * t f +

0.0 1.0 0.0 0.0 0.4

h’ofe. The data are presented as the percentage of cells staining with the four mAbs in a variety of tissues. The percentages of positive values are expressed kstandard deviation for three mice. Data are derived from flow cytometric analysis on single-cell suspensions.

of cells within murine GCs. Figure 1 shows a series of cryosections (nonserial) from the spleens of either an unimmunized BALB/c mouse (panels a-f) or from an animal 9 days following immunization with SRBC (panels g-l). Staining with the anti-IgD mAb in the unimmunized mouse showed that the white pulp was dominated by primary follicles (a) which contained small resting B cells expressing high levels of slgD. There were no focal clusters of activated GC B cells present in the unimmunized spleen and PNA staining was therefore low (b). No significant levels of staining were seen within the B cell follicles when the four mAbs were used to stain the unimmunized spleen sections (c-f) and the isotype-matched controls were negative on both primary and secondary follicle sections (data not shown). In contrast, the immunized spleen showed extensive GC development within primary follicles. The IgD-positive cells were displaced into a peripheral zone (g) around the actively proliferating GC B cells. The GC B cells expressed high levels of PNA binding sites (h). Staining of immunized spleen with the four mAbs revealed their preferential binding to subpopulations of GC cells. The mAbs FH9.5 and C3.5 both stain cell populations within the interfollicular regions of nonimmune and immune spleen, although the pattern of staining is antibody specific.

3.2. In Vivo Tissue Distribution

cfthe GC A4arker.r

The distribution of the antigens recognized by the four mAbs within lymphoid tissues from unimmunized BALB/c mice was analyzed by flow cytometry (Table 1). The two markers defined by mAbs C6C3 and A6A2 were essentially undetectable within the lymphoid tissues from unimmunized mice. In contrast, FH9.5 and C3.5 stained approximately 50 and 60% respectively of bone marrow cells and were also

Flc. 1. Histological localization of mAb binding to GCs. The two panels represent tissue the spleen of an unimmunized BALB/c mouse (a-f) and from an immunized mouse 9 antigenic challenge (g-l). The B cell follicles are visualized with an anti-IgD mAb (a and g) of CC formation elucidated with PNA staining (b and h). The mAb staining patterns shown FH9.5 (c and i). C3.5 (d and j), C6C3 (e and k), and A6A2 (f and I).

sections from days following and the extent are with mAb

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expressed on a significant number of cells in spleen, lymph node, and Peyer’s patch. The FH9.5 surface marker was found on approximately 25% of cells in spleen and lymph node and approximately 10% of cells in the Peyer’s patch. The C3.5 mAb stained 15-20% of cells from spleen, lymph node, and Peyer’s patch. Thymus cells were found not to express detectable levels of C6C3 or A6A2 surface markers and only 2-3% of cells stained with FH9.5 and C3.5. This indicates that the molecules recognized by C6C3 and A6A2 are extremely restricted in their distribution, being predominantly associated with the surface of GC cells in immunized animals. In contrast, the surface determinants recognized by the FH9.5 and C3.5 mAbs are differentially expressed among cells within the lymphoid tissues.

3.3. Characterization

of Lymphoid

Cells Expressing the GC Markers

To establish which cell types normally express the GC markers, spleens from mice 9 days following immunization were analyzed by two-color flow cytometry. All four markers were expressed by B cell populations (Fig. 2). Both C6C3 and A6A2 mAbs stained B cell subsets and accounted for 8 and 5% of total splenic cells, respectively. FH9.5 and C3.5 both stained 12% of the total B cell population and were also found

FIG. 2. Two-color flow cytometry analysis of spleen cells from BALB/c mice 9 days following immunization. Contour levels were plotted with a threshold level of 5%. Anti-kappa staining (FITC conjugated) is on the ,x-axis and the four mAbs, FH9.5, C3.5, C6C3, and A6A2 (biotin conjugated and visualized with avidinphycoerythrin) on the Ja-axis. The percentage of cells falling within each quadrant is indicated.

mAbs

DEFINE

CC

CELL

457

SUBPOPULATIONS

on other cell types which were surface immunoglobulin negative. The distribution of the FH9.5 and C3.5 markers was studied on T cell populations using two-color flow cytometry (Fig. 3). FH9.5 staining was confined to the majority of CD4+ T cells but was absent from CD8 T cells. Conversely, C3.5 staining was absent from the CD4+ T cells but was present on the majority of CD8+ T cells. The expression of the two markers on T cell subsets was identical in both immune and nonimmune animals.

3.4. Expression qfthe GC Murders on Activated and Resting Cell Populations jrom T Ceil-Depleted Spleen Cells

Derived

When T cell-depleted spleen cells from immunized animals were subfractionated on the basis of cell density (Fig. 4 and Table 2) there was a positive correlation between increased activation/maturation and FH9.5 or C3.5 marker expression. Figure 4 shows cytometry profiles from a representative experiment. The staining observed with secondary antibody alone indicated the extent to which autofluorescence increased as the cells became activated and progressively less dense. As an internal control for activation state of the cells in the various density fractions expression of Ia was also measured and correlated positively with increased activation. FH9.5 expression was low on small resting cells but increased as the cells activated and further differentiated (6% positive in the resting fraction compared to 40% positive in the low-density fractions). The pattern of C3.5 expression also correlated with the more activated B cell

FH9.5

L3T4 FIG. 3. Two-color contour plots of spleen cells from unimmunized BALB/c plotted with a threshold level of 5%. Cells were stained with either fluoresceinated Ly-2 (CDS) (.x--axis) and FH9.5 biotin or C3.5 biotin (),-axis).

mice. Contour levels were anti-L3T4 (CD4) or anti-

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CONTROL

FH9.5 FIG. 4. Single-color flow cytometric analysis of T cell-depleted spleen cells from immunized mice harvested from Percoll density gradients. Representative histograms are shown for cells stained with either secondary antibody alone, anti-Ia, FH9.5, or C3.5 and plotted against relative cell number on y-axis. The histograms derived from staining the various density fractions are overlayed and represent low density cells (1.0651.075, ---), Intermediate density cells (I .075- 1.080, . . . ) (1.080-I ,085, . . . ) and small dense cells (1.085-1.090. ---).

populations (4% of the resting cells expressed this marker while 25% of the low-density cells were positive). Table 2 summarizes the data from five experiments in which the level of contaminating T cells was approximately 2% in the high-density (small resting) fraction. The staining of cells by C6C3 and A6A2 fell within the variability of the assay and no significant differences were observed between these fractions.

3.5. Analysis of Expression of Surface Markers on PNA-Positive

Cells

To investigate whether or not the FH9.5+ and C3.5+ cells coexpressed binding sites for PNA, two-color flow cytometry was performed. Spleen cells from mice 9 days following primary immunization were double stained with the four mAbs FH9.5, C3.5, C6C3, A6A2 (green), and PNA (red). The data are summarized in Fig. 5. Following primary immunization a PNA high population was induced. These cells did not express detectable levels of the C3.5 marker. In contrast, a population of the PNA high cells was costained with FH9.5. This FH9.5+/PNA+ population characteristically was stained weakly with FH9.5. This is consistent with the histological data which

mAbs

DEFINE

CC

CELL TABLE

Distribution

2

of B Cells from Immunized Mice Expressing CC-Specific Surface Markers from Density Gradient Centrifugation and Analyzed by Flow Cytometry Percentage

Fraction Low density Intermediate Small.

459

SUBPOPULATIONS

B cells density

Density 1.065-1.075 I .075- 1.080 I .080- I.085 I .085-l ,090

B cells

dense B cells

FH9.5 43 42 I7 6

+ 8.9 f 6.9 * 12.0 f 3.6

of recovered

cells expressing

c3.5 24 + I6 f 9 f 4&

C6C3 5.1 7.X 3.4 I.1

Recovered

6 7 3 I

* k f *

4.6 6.6 2.7 1.75

A6A2 2 -+ 3i 1t 2 +

1.8 2.3 0.5 1.75

h’orc. Analysis of fractions derived from density gradient centrifugation. The results are the means from five experiments with BALB/c mice 7 days following primary immunization with SRBC. The percentage of cells stained with the four mAbs was determined by flow cytometric analysis and values are expressed *standard deviation.

demonstrated that the FH9.5 staining associated with germinal center cells is weaker than that associated with the other cell types expressing the marker in the interfolicular regions. Approximately 50% of the cells stained by the C6C3 and A6A2 mAbs expressed binding sites for PNA.

FL1

~Fluoresccnce

1 -

FH9.5

A6A2

C6C3

FIG. 5. Two-color flow cytometric analysis of splenic cells from mice 9 days postprimary immunization. Contour levels are plotted at the 5% level. PNA staining is on the JJ-axis (PNA-biotin visualized with avidinphycoerythrin) and C3.5. FH9.5. C6C3. and A6A2 (FITC conjugated) staining is on the ,y-axis.

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3.6. Failure of LPS to Induce Expression of the Markers on Small Resting B Cells In order to evaluate whether expression of the four cell surface markers could be induced in response to mitogenic stimulation, small resting B cells were purified and cultured in the presence of LPS. In a series of experiments using two concentrations of LPS and analysis of blasts at 2 and 5 days following stimulation, no significant induction of expression of the GC markers was observed (Fig. 6) although there was induction of Ia (data not shown). There was a subtle shift in fluorescence intensity with FH9.5 but the levels of expression were low relative to those seen in vivo. This suggests that the markers are not simply activation markers which can be induced in response to mitogenic stimulation.

3.7. Distribution

Qfthe GC Markers on in Vitro Cell Lines

A panel of in vitro cell lines representing multiple bone marrow-derived lineages was stained with the four mAbs and analyzed by flow cytometry (Table 3). The markers were not expressed on the pre-B cell panel with the exception of weak staining of 28C9 by the C6C3 and A6A2 mAbs. Several mature B cell lines and plasmacytoma/myeloma lines expressed the marker recognized by the FH9.5 mAb. The C3.5 expression was more restricted and did not appear to be coexpressed on the cell lines which expressed

i 3 i

FH9.5

c3.5

C6C3

A6A2

“u

FIG. 6. Single-color flow cytometry analysis of LPS blasts. Representative histograms from are shown with the secondary only control ( p) overlayed with the four mAbs (. . * ).

5-day

cultures

mAbs

DEFINE

CC

CELL TABLE

Distribution

of CC Markers

WEHI-279.75 BCc,-3B3 WEHI1 A20- I 2PK3 LIOA Xl6C-8.5 MOPC-3 15J 5558-l 1 MPC-1 I MOPC-2 1 s107

on in I’ituo Cell Panel c3.5

D-Jn-Cu

-

-

Vu-D-J&u

-



B cell

.-

-.

+ +

* -

++

Plasma/myeloma

+ + T cells

MC/9

Mast cell

FL5.12

Lymphoid

-

*

-

f

ChC3

A6A2

-

Vu-D-Jn-Cu

WR19-I. IIIC2.9 BW5 I47 S49.1 EL4/9 (BII)

P388d-1 WEHI-3B 5774 FDC-P I PUS l-8 P815

3

FH9.5

Cell lines 40El 38BI 1 22D6 204- l-8 220-X 230.238 204.3- 1 28C9 230-37 70213

461

SUBPOPULATIONS

+ -

+

+

+

+

t -f+ +

+ + t +

+

+ ++i -

progenitor

Macrophage/monocyte

++ ++t

+ t

.V’olc The fluorescence intensity was characterized by flow cytometry and the data were expressed as the shift in mean channel fluorescence relative to cells stained with an isotype-matched control mAb. The data are summarized as follows: Channel shift (logarithmic scale): O-9 (-). IO-19 (&). 20-39 (+). 40-60 (++). >60 (+ ++). The table summarizes the results of at least three independent experiments evaluating levels of expression of CC markers by single-color flow cytometric analysis.

FH9.5. In addition, C3.5 was more confined to the plasmacytoma/myeloma cell lines. Cell lines which expressed the C6C3 and A6A2 markers included some members of the mature B cell panel. It is of interest that the 5606 cell line used as the immunogen in the fusion which produced A6A2 and C6C3 does not stain with these two mAbs

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(data not shown). It is likely that the mAbs were raised against cell surface antigens on cells derived from the mouse used for the passage of the J606 cells. All four markers were found on different T cell lines and were highly restricted within this compartment of the cell panel. There was extensive expression of the markers among cell types with a myeloid phenotype. It is of interest that both in terms of staining pattern and intensity C6C3 and A6A2 were virtually indistinguishable and suggests that they may recognize a common cell surface epitope. The cell line panel served to identify cell lines expressing high levels of the markers which could be used to investigate their biochemical nature.

3.8. Immunoprecipitation

of the FH9.5 and C3.5 Surface Markers

The FH9.5 cell surface antigen was immunoprecipitated from WEHIand WR19L cells (Fig. 7). In both cases the mAb precipitated a single protein species with an apparent molecular weight of 23-28 kDa. The protein migrated identically under reducing and nonreducing conditions (data not shown) indicating that the molecule is a single chain polypeptide. The FH9.5 antibody failed to precipitate protein(s) from

KD 98 69 46 30 *

1812-

6autoradiograph from surface-iodinated WR19L and WEHIcell lines FIG. 7. A 12.5% SDS-PAGE immunoprecipitated with either FH9.5 or an isotype-matched control mAb 5OCICl linked to SAC. A specific band is precipitated from both cell lines and runs with an apparent molecular weight of 23-28 kDa.

mAbs

DEFINE

CC

CELL

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the EL4/9 T cell line which was determined to be “negative” by FACS analysis (data not shown). When a cell suspension from a whole spleen was surface iodinated and immunoprecipitated with the FH9.5 mAb, an identical single band was identified (data not shown). This demonstrates that among the various cell types in spleen which express this surface marker, the protein is identical at the level of resolution by SDS-PAGE. Immunoprecipitation with the C3.5 mAb was performed using surface-labeled BW5 147 cells (Fig. 8). When compared to an isotype-matched negative control it was apparent that the C3.5 surface marker was an 85- to 95-kDa protein. This molecule migrated identically under reducing and nonreducing conditions (data not shown). 4. DISCUSSION Several key events in the differentiation and maturation of B cells take place within the GCs of peripheral lymphoid tissues (l-7). It is apparent that the follicular environment is complex, providing not only specialized dendritic cells to trap and disseminate antigen, but also the necessary signals required to orchestrate the processes associated with B cell activation and maturation. GCs also serve as the environment in

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FIG. 8. A 15% SDS-PAGE autoradiograph from with Sepharose beads coupled to either C3.5 or an is precipitated by C3.5 and runs with an apparent been overexposed to confirm the absence of a band

surface-iodinated BWS 147 cell line immunoprecipitated isotype-matched control mAb 49C2D6. A specific band molecular weight of 85-95 kDa. (The 49C2d6 lane has corresponding to that in the C3.5 lane).

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which memory B cells develop, thus maintaining the capacity to generate secondary responses following reexposure to Ag (3 1, 32). Memory B cells require periodic reexposure to antigen in order to persist long term (33). Follicular dendritic cells within GCs (6) may play a role in the long-term maintenance of the memory cell population by periodically exposing intact antigen trapped on their cell surface. It is therefore clear that several B cell populations exist within the developing GC with the capability of following at least two independent routes of subsequent differentiation. At present the only available reagent that will selectively enrich for GC B cells is PNA, but this does not discriminate between B cell subpopulations that are functionally or maturationally distinct. We have therefore generated and characterized four mAbs which all share one feature, namely the capacity to selectively stain subpopulations of cells within GCs. These four mAbs stained GC in tissue sections from antigen-primed mice GCs but not the primary follicles in a naive animal. When the staining patterns of the mAbs were compared to the pattern of PNA staining, it was apparent that only a fraction of a given GC was stained. This provides evidence that there are within the GCs subpopulations of cells which can be distinguished on the basis of differential surface marker expression. The mAbs also provide a means of selectively enriching for those populations to determine whether they define distinct functional subsets or maturational states. From an examination of the interfollicular areas on the tissue sections, it is evident that FH9.5 also stains platelets and megakaryocytes (F. M. Platt, J. M. Davie, and J. P. McKearn, unpublished data). The C3.5 mAb stains scattered cells within the interfollicular regions of the splenic white pulp and these cells express the highest levels of the surface antigen that has been observed. When two-color flow cytometry was performed on spleen cells, the FH9.5 staining segregated with a population of CD4+ cells while C3.5 stained the majority of CD8+ cells. The brightly staining CDE+ cells were observed to be intensely stained on the tissue sections. The two markers recognized by the C6C3 and A6A2 mAbs are much rarer and from two-color flow cytometry are only expressed on the surface of a subset of B cells. In addition all four mAbs stain a population of Mac- I+ cells in spleen (data not shown). When a series of lymphoid tissues were examined for expression of these markers the levels of C6C3 and A6A2 were low in all tissues tested from unimmunized mice. Taken together with the fact that the maximum frequency of cells expressing these markers following immunization is 5-8%, this indicates that these cell surface markers are rare and highly restricted to a subpopulation of GC B cells. In contrast, the FH9.5 and C3.5 markers are expressed by a high frequency of cells in the bone marrow and to a lesser extent in the peripheral lymphoid tissues. However, very few thymus cells (O-3%) stained with either mAb. Both FH9.5 and C3.5 markers are present on mutually exclusive peripheral T cell populations suggesting that expression of these markers is regulated during T cell development. The signals required to induce expression of these markers will be of interest. The two mAbs, FH9.5 and C3.5, may also provide useful reagents for the study of peripheral T cell populations in vivo and in vitro. An in vitro cell panel was screened using cell types representing multiple hematopoietic lineages. The staining observed with the four mAbs was predominantly absent on the pre-B cells and was most abundant on mature B cells and plasmacytoma/ myeloma cells. In addition many members of the myeloid panel stained with the

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mAbs and thus supports the in vivo data in which the low frequency of Mac-l staining cells in the spleen costained with the four mAbs (data not shown). Selected cell lines were surface iodinated and the FH9.5 and C3.5 surface molecules were immunoprecipitated. The FH9.5 antigen is a single chain polypeptide with an apparent molecular weight of 23-28 kDa. The C3.5 antigen is a single chain polypeptide of 85-95 kDa. When the immunoprecipitation data are viewed in conjunction with the in vivo tissue distribution data, neither marker appears to have been previously described. To date, attempts to immunoprecipitate C6C3 and A6A2 have not been successful. This may simply reflect the relative affinity of the two antibodies or they may be analogous to the GC-specific glycolipids described in the human system ( 16) and therefore not detected under conditions described here. We have studied the B cell compartment in more detail defining the types of B cells expressing the four markers in immunized mice. When B cells are fractionated on the basis of cell density there is a dramatic enrichment for FH9.5 and C3.5 B cells in the population with lowest cell density which expresses the highest levels of la. In contrast, the small resting B cell population had a very low percentage of cells expressing either marker. Due to the rarity of cells expressing either of the C6C3 and A6A2 markers we could not demonstrate that the distribution of cells stained with these mAbs was significantly different between the various density fractions. In addition, the PNAh’ population of B cells in immunized mice was examined to determine whether or not the surface markers defined by the four mAbs are coexpressed on this population. The PNAhi population is C3.5 negative but a subpopulation of the PNAh’ cells costain with FH9.5. C6C3. and A6A2. The fact that C3.5’ cells do not express the PNA binding site raises the possibility that not all GC B cells are PNA+ or that there is a temporal acquisition and loss of surface markers during the GC reaction. Additional kinetic studies will be required to address this issue. The C3.5+ B cells are present in nonimmune animals although the frequency of B cells bearing this marker increases by approximately 4% following immunization and there is a parallel increase in the frequency of Ia+ cells (data not shown) indicating that C3.5+ B cells are induced following antigen administration. It is known that memory B cell development cannot be induced in an antigenically naive animal in response to T helper cell-independent antigens (34). It is therefore of interest that mitogen treatment of small resting B cells in vitro is insufficient to induce expression of any of the four markers. This clearly distinguishes these surface molecules from the B cell activation markers described in the human (14, 35, 36). It also suggests that signals derived from the microenvironment are required for the induction of expression of the surface molecules defined by these mAbs. The signaling requirements necessary for the induction of expression of these markers may prove to be highly complex and it can be speculated that it may involve a combination of direct cell signaling in addition to the interaction of soluble factors with their receptors. It is interesting to speculate on the putative functions of molecules selectively expressed on the surface of cells within GCs. Expression of receptors for soluble mediators such as cytokines and interleukins may play a key role in cellular maturation and differentiation within these sites. In addition, the fact that these cells are nonmigratory also implicates cell adhesion molecules functioning within the GC microenvironment. Not only would adhesion molecules ensure a relatively sessile, nonmigratory cell population but would also promote intimate cell contact which may be a prerequisite for cellular interactions mediated by direct cell-to-cell contact. The system described in

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this study may prove to be useful in elucidating the signaling pathways required to induce expression of these markers and also shed light on the functional role(s) that these surface markers play in the process of B cell differentiation and maturation. REFERENCES I. Nieuwenhuis, P., and Opstellen, D., Am. J. Anat. 170, 421, 1984. 2. Kroese, F. G. M., Wubbena, A. S., Seijen, H. G., and Nieuwenhuis, P., Eur. J. Immunol. 17, 1069, 1987. 3. Kraal, G., Weissman, I. L., and Butcher, E. C., Nature 298, 377, 1982. 4. Coico, R. F., Bhogal, B. S., and Thorbecke, G. J., J. Immunol. 131, 2254, 1983. 5. Gallatin, W. M., Weissman, I. L., and Butcher, E. C., Nature 304, 30, 1983. 6. Szakal, A. K., Kosco, M. H., and Tew, J. G., J. Immunol. 140, 341, 1988. 7. Kosco, M. H., Szakal, A. K., and Tew, J. G., J. Immunol. 140, 354, 1988. 8. Kraal, G., Hardy, R. R., Gallatin, W. M., Weissman, 1. L., and Butcher, E. C., Eur. J. Immunol. 16, 829, 1986. 9. MacLennan, 1. C. M., and Gray, D., Immunol. Rev. 91, 61, 1986. 10. Karasuyama, H., Rolink, A., and Melchers, F. J., J. Exp. Med. 167, 1377, 1988. 1 I. George, A., and Cebra, J. J., Proc. Natl. Acad. Sri. USA 88, I 1, I99 I. 12. Rose, M. L., Birbeck, M. S. C., Wallis, V. J., Forrester, J. A., and Davies, A. J. S., Nature 284, 364, 1980. 13. Kokai, Y., Ishii, Y., and Kikuchi, K., C/in. Exp. Immunol. 64, 382, 1986. 14. Thorley-Lawson, D. A., Schooley, R. T., Bhan, A. K., and Nadler, L. M., Cell 30, 415, 1982. 15. Frisman, D., Slovin, S., Royston, I., and Baird, S., Blood 62, 1224, 1983. 16. Fyfe, G., Cebra-Thomas, J. A., Mustain, E., Davie, J. M., Alley, C. D., and Nahm, M. H., J. Immunoi. 139,2187, 1987. 17. Yelton, D. E., Desaymard, C., and Scharff, M. D., Hybridoma 1, 5, 198 1. 18. Kincade, P. W., Lee, G., Watanabe, T., Sun, L., and Scheid, M. P., J. Immunol. 127, 2262, 198 1. 19. Bhattacharya, A., Dorf, M. E., and Springer, T. A., J. Immunol. 127, 2488, 198 1. 20. Dialynas, D. P., Quan, 2. S., Wall, K. A., Pierres, A., Quintans, J., Loken, N. R., Pierres, M., and Fitch, F. W.. J. Immunol. 131, 2445, 1983. 21. Ledbetter, J. A., and Herzenberg, L. A., Immunol. Rev. 47, 63, 1979. 22. Springer, T., Galfre, G., Secher, D. S., and Milstein, C., Eur. J. Immunol. 8, 539, 1978. 23. Bruce, J., Symington, F. W., McKearn, T. J., and Sprent, J. J., Immunol. 127, 2496, 1981. 24. McKearn, J. P., and Rosenberg, N., Eur. J. Immunol. 15, 295, 1985. 25. McKearn, J. P., Baum, C., and Davie, J. M., J. Immunol. 132, 332, 1984. 26. McKearn, J. P., McCubrey, J. A., and Fagg, B., Proc. Natl. Acad. Sci. USA 82, 7414, 1985. 27. Shulman, M., Wilde, C. D., and Kohler, G., Nature 276, 269, 1978. 28. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W., and Howard, J. C., Nature 266, 550, 1977. 29. Springer, T. A., Bhattacharya, A., Cardoza, J. T., and Sanchez-Madrid, F., Hybridoma 1, 257, 1982. 30. Markwell, M. A. K., and Fox, C. F., Biochemistry 17, 4807, 1978. 31. Zan-Bar, I., Strober, S., and Vitetta, E. S., J. Immunol. 123, 925, 1979. 32. Yefenof, E., Sanders, V. M., Snow, E. C., Noelle, R. J., Oliver, K. G., Uhr, J. W., and Vitetta, E. S., J. Immunol. 135, 3777, 1985. 33. Gray, D., and Skarvall, H., Nature336, 70, 1988. 34. Liu, V. J., Oldfield, S., and MacLennan, I. C. M., Eur. J. Immunol. 18, 355, 1988. 35. Thorley-Lawson, D. A., Nadler, L. M., Bhan, A. K., and Schooley, R. T., J. Immunol. 134, 3007, 1985. 36. Lazarovits, A. I., Moscicki, R. A., Kurnick, J. T., Camerini, D., Bhan, A. K., Baird, L. G., Erikson, M., and Colvin, R. B., J. Immunol. 133, 1857, 1984.

Monoclonal antibodies specific for novel murine cell surface markers define subpopulations of germinal center cells.

A panel of mAbs has been generated which selectively, but not exclusively, recognizes populations of cells within germinal centers of immunized mice. ...
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