Immunology Letters, 24 (1990) 261-264 Elsevier IMLET 01394

Evidence that FMC7 is a human B cell differentiation antigen Ger T. Rijkers, Ineke Dollekamp and Ben J. M. Zegers Department of Immunology, University Hospital for Children and Youth, "Het Wilhelmina Kinderziekenhuis", Utrecht, The Netherlands (Received 1 March 1990; accepted 20 March 1990)

1. Summary

FMC7 is a 105-kDa B cell restricted antigen which is expressed on about 50070 of adult human peripheral blood B cells. Seven to ten days following booster immunization with tetanus toxoid, peripheral blood contains a small population of B cell blasts with an increased density of FMC7. The majority of antitetanus toxoid antibody secreting cells (both IgM and IgG) are however found in FMC7- B cells. These data indicate that upon in vivo B cell activation FMC7 expression initially increases. B cells involved in antibody secretion have lost the FMC7 determinant. 2. Introduction

The process of B cell differentiation is accompanied by changes in the expression of Ig and non-Ig cell surface antigens [1]. One of these antigens is a 105-kDa glycoprotein, recognized by the FMC7 monoclonal antibody [2, 3]. We have previously described that the FMC7 determinant is expressed on 100°70 of neonatal B cells [4] and on about 50070 of adult peripheral B cells [5]. Based on reactivity with B cell malignancies, FMC7 is believed to define relative mature B cells [6]. In vitro studies have shown that antibody production to either T-cell dependent or type-2 T cell-independent antigens is localized in the B cell subpopulation carrying the FMC7 determinant [5]. In this paper we describe

Key words." FMC7; B cell differentiation antigen; Human Correspondence to." Ger T. Rijkers, Dept. of Immunology, Het Wilhelmina Kinderziekenhuis, P.O. Box 18009, 3501 CA Utrecht, The Netherlands. Tel. +31 30 320911; Fax +31 30 324825.

FMC7 expression during in vivo human B cell differentiation. 3. Materials and Methods

Adult donors were booster immunized with diphtheria-tetanus-poliomyelitis vaccine (National Institute for Public Health and Environmental Hygiene RIVM, Bilthoven, The Netherlands), containing 5 Lf tetanus toxoid. Blood was withdrawn by venepuncture before and on days 7, 14 and 28 following vaccination. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Ficoll Isopaque (1.077 g/cm3; Pharmacia, Uppsala, Sweden). For cell sorting, 1 to 2x 107 PBMC were incubated for 30 min at 4 °C with FMC7 (10 #g/ml; Seralab, Crawley Down, U.K.) in MEM/I% BSA. After washing, the cells were incubated with FITCconjugated goat anti-mouse IgM (Nordic, Tilburg, The Netherlands) for another 30min at 4°C, Stained cells were analyzed with a cytofluorograph 50 H (Ortho Diagnostic Systems, Beerse, Belgium). An argon laser (250 mW, 488 nm) was used for excitation of FITC. Fluorescence emission was collected using a 570 nm dichroic mirror in combination with a 525 nm band-pass filter. Axial light loss, right angle scatter, and green fluorescence were simultaneously recorded and stored in an Olivetti M24 PC. Viable cells were sorted under sterile conditions on basis of FMC7 expression (see Results). For two-color FACS analysis, 0.5 x 106 cells were incubated with FMC7 and FITC-conjugated goat anti-mouse IgM (as described above) followed by biotinylated B1 (CD20; Coulter Clone, Luton, U.K.) and streptavidin phycoerythrin (Becton Dickinson, Mountain View, CA). Stained cells were run on a

0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

261

10 3

10 2 .

0 101 ...: a tJ

100

=

100

= =l=lll

I

I

I =lllil

101 FMC7

I

102

J

=

=llil 10 3

(FITC)

Fig. 1. FMC7 expression on B cells following vaccination with tetanus toxoid. PBMC obtained from a healthy adult volunteer on day 7 following booster immunization with tetanus toxoid. PBMC were stained with FMC7 (FITC) and CD20 (B1; PE) and analyzed on a FACS analyzer. Data are presented as a contour plot with log green fluorescence on the x axis and log red fluorescence on the y axis.

FACS analyzer (Becton Dickinson); data were analyzed using Consort 30 software. Individual immunoglobulin or specific antibody secreting cells were enumerated with the use of the spot-ELISA [4]. Briefly, fiat-bottomed 96-well clusters were coated with affinity-purified goat antihuman IgM or IgG (10/zg/ml) or 0.4 Lf/ml tetanus toxoid. Sorted cells were added to the wells and incubated overnight at 37°C, 5% CO2. After washing, individual wells were incubated with either affinity-purified alkaline phosphatase conjugated goat anti-human IgM or IgG. After washing, 5bromo-4-chloro-3-indolylphosphate was added in 0.3°7o (v/v) gelling agarose. Spots were enumerated under an inverted microscope after overnight incubation. 4. Results and Discussion

Two-color immunofluorescence studies of PBMC of adult donors booster immunized with tetanus toxoid reveals a B cell population with an increased density of FMC7 (Fig. 1). The volume and light scatter characteristics of these FMC7 ÷÷ B cells are corn262

patible with this population being activated B cells (Fig. 2). FMC7 + + B cell blasts are not found before immunization and disappear from peripheral circulation 3 - 4 weeks following immunization (data not shown). Because peripheral blood of adult donors booster immunized with tetanus toxoid contains (on day 7 - 1 0 following immunization) activated B cells which secrete anti-tetanus toxoid antibodies, we have directly addressed the question whether the in vivo induced anti-tetanus toxoid antibody-secreting cells are contained within the high-density FMC7 population. To this end, PBMC at day 7 following tetanus toxoid immunization were stained with FMC7 and separated (by cell sorting) into FMC7-negative ( F M C 7 - ; mean fluorescence intensity (MFI) 4 - 5), FMC7-positive (FMC7 +; MFI 8 - 9 ) , and highdensity FMC7 (FMC7 + + ; MFI 2 1 - 23) cells. Separate fractions were assayed in a spot-forming cell assay for total immunoglobulin and for anti-tetanus antibody production. FMC7 + sorted cells contained the highest number of IgM-secreting cells. A direct comparison with FKC7- cells is however hampered by the fact that the number of B cells within each fraction differs. We could demonstrate that, although CD20 is lost from terminally differentiated plasma cells, the vast majority of IgM and IgG secreting cells, as detected in an SFC assay, are CD20 ÷ cells (Table 1). Data on antibody production by FMC7 sorted cells were therefore expressed on basis of CD20 expression (Fig. 3). It appears that virtually all IgM and IgG is produced by F M C 7 - CD20 + B cells. The same holds true for specific antibody production; both the IgM and the IgG-anti tetanus toxoid antibody-producing cells are localized within the F M C 7 - CD20 + B cell population (Fig. 3). It can therefore be concluded that FMC7 density increases during in vivo B cell differentiation. These cells, although having the physical characteristics of activated B cells (volume, light scatter), do not secrete antibodies. Virtually all antibody-secreting cells are characterized by the absence of (immunoreactive) FMC7 on the cell surface. These data point towards a role for FMC7 in human B cell differentiation. The nature of the determinant recognized by the FMC7 antibody has not yet been established. There are indications that the FMC7 antibody recognizes a conformational epi-

10 3

10 3

I

.•

i ..

:

102

102 .

~ ld ®

."

i

'

ld-

100

=

~ii:i:i:i

100 0

1

....... . .... •. •. i

i ,.. ......... ....., . .........

100 Volume

0

3

-

=

I'" " '

'

200

I

'

100 Volume

~

200

103.

J -N • •

" .::0::." "

,l~

i

i .

'.~

~::!~,~ • ....i!?itz'~

lo 2 .

.

.

.

.

.

i

,0 2-

1

(1.

"

0oJ 0 lo 1 (J

..,I

i ~:?~ , I l',,t

•

,.

" " '.

.

i

"

104

100.

100" 10°

101 FMC7 (FITC)

102

103

n

10 °

u liuuln

i

i

u

u iJlul

101 FMC7 (FITC)

I

102

i

u UlUlUU

103

Fig. 2. B cells with increased FMC7 expression and contained within the blast cell population. PBMC were stained as described in legend to Fig. 1 and analyzed. Small (left-hand panels) and large cells (right-hand panels) were gated on the basis of volume and side scatter characteristics (upper panels). The lower panels show corresponding two-color fluorescence contour plots of size-gated cells. Contour plots are divided into quadrants such that > 9 9 % o f non-stained cells are located in the lower left quadrant. 263

10 6

10 6 e,3 m i

_

~.)

(3 rn + 10 5 0 ¢%1 c~ 0 ~0104

....

o 10'

!

(..) 1.1_ (/)

103

/I PBMC

10 3 L FMC7-

FMC7 ÷

F M C 7 ÷+

~ PBMC

FMC7-

FMC7 +

j-~ F M C 7 +÷

Fig. 3. Immunoglobulin and anti-tetanus toxoid secreting ceils in B cells sorted on basis of FMC7 expression. Peripheral blood mononuclear cells (PBMC) were separated by cell sorting into FMC7 - , FMC7 +, and FMC7 ÷ + cells. The number of IgM-secreting(open bars) and IgG-secretingcells (SFC; hatched bars) (left-hand panel), and the number of IgM anti-tetanus toxoid (open bars) and IgG antitetanus toxoid (hatched bars) SFC (right-hand panel) is determined and expressed per 106 CD20 ÷ B cells in the respective fractions.

Acknowledgements

TABLE 1 Anti-tetanus toxoid spot-forming cells are CD20+ . Cells

% CD20 ÷

Anti-tetanus toxoid

SFC/10 6 lymphocytes IgM (%) PBMC a

9.0

CD20-depleted PBMC

1.7

12 (100)

0

(0)

IgG (%) 2,432 (100)

512

References

(21)

a Peripheral blood mononuclear cells (PBMC) were obtained from adult donors 7 days following booster immunization with tetanus toxoid. Anti-tetanus toxoid spot forming cells(SFC) were determined in PBMC and in PBMC depleted for CD20+ cells. Shown are data from one representative experiment out of 3 independent experiments performed.

tope f o r m e d by m u l t i p l e C D 2 0 molecules (E Staal et al., s u b m i t t e d for p u b l i c a t i o n ) . T h e f i n d i n g t h a t C D 2 0 expression also is increased o n F M C 7 ÷÷ blasts (Fig. 1) w o u l d be c o m p a t i b l e with this view. Since we find a large v a r i a t i o n in F M C 7 expression in cells o f u n i f o r m CD20 density, it c o u l d be speculated that the spatial d i s t r i b u t i o n o f CD20 molecules o n the surface o f a B cell determines reactivity with the F M C 7 antibody. W h e t h e r or n o t this t u r n s out to be true, the expression o f the d e t e r m i n a n t recognized by the F M C 7 a n t i b o d y d y n a m i c a l l y changes d u r i n g the process o f B cell activation a n d as such this d e t e r m i n a n t c a n be considered a B cell different i a t i o n antigen. 264

We t h a n k Erik Rozemuller (Dept. o f Clinical Imm u n o l o g y , State University Hospital, Utrecht) for p e r f o r m i n g cell sorting a n d L i n d a G r a m b e r g for expert secretarial assistance.

[1] Freedman, A. S. and Nadler, L. M. (1987)Sem. Hematol. 24, 230. [2] Brooks, D. A., Beckman, I. G. R., Bradley, J., McNamara, P. J., Thomas, M. E. and Zola, H. (1981) J. Immunol. 126, 1373. [3] Zola, H. (1987) Immunol. Today 8, 308. [4] Rijkers, G. T., Dollekamp, E. G. and Zegers, B. J. M. (1987) Scand. J. lmmunol. 25, 447. [5] Bloem, A. C., Chand, M. A., Dollekamp, E. G. and Rijkers, G. T. (1988) J. Immunol. 140, 768. [6] Catovsky, D., Cherchi, M., Brooks, D., Bradley, J. and Zola, H. (1981) Blood 58, 406.