Eur. J. Immunol. 1991.21: 2017-2023 VH and V, gene expression by CD5 B cells 2017

Luiz AndradeO, Franqois Huetz, Pascal Poncet, V6ronique Thomas-Vaslin, Michele Goodhardt. and Antonio Coutinho

Unit6 d’Immunobiologie and Unit6 de G6n6tique et de Biochimie du D6veloppemento, Institut Pasteur, Paris

Biased V H gene expression in murine CD5 B cells results from age-dependent cellular selection*

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and V, gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single- stranded DNA, trimethyl ammonium and bovine y-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased V ~ l l expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMFU3C reactivity, which is considerably higher in peritoneum than in spleen. No particular V, bias was observed in any of the populations studied with the possible exception of V,22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.

1 Introduction

In normal mice, CD5 B cells appear early in ontogeny but rapidly become outnumbered by “conventional7’, BM- derived B lymphocytes [l , 21. In adult life, CD5 B cells are undetectable in BM, LN and Peyer’s patches. They are found at very low frequencies in adult spleen, but are enriched among B cells located in the peritoneal [ l , 21 and pleuropericardial cavities [3]. In these cavities, CD5 B cells express a different Ab repertoire than conventional lym- phocytes [4-71, raising the possibility that they draw their specificities from a particular pool of VH and VL genes. These differences, however, could be due to cellular selection mechanisms, selectively operating either within the CD5 B cell population or in that particular location. One way of addressing this question directly is by compar- ing,Vgene family usage [8,9] and Ab repertoires of CD5+ and CD5- B cells in neonatal and adult animals [4, 51, i.e. before and after extensive selection has taken place.

In previous studies, we have demonstrated that the utiliza- tion of V ~ l l genes [lo], which correlate with anti-brome- lain-treated mouse RBC (BrMRBC) reactivity [ll], is increased in adult peritoneal CD5 B cells but not in newborn spleen cells [12], where CD5+ B cells are abun- dant [ l , 2].We have now determined the relative utilization

[I 87981

* This research was supported by grants from CEE, DRET,

0 Recipient of a doctoral fellowship from CNPq, Brazil.

Correspondence: Luiz Andrade, Unite d‘Immunobiologie, Institut Pasteur, 25-28, rue du Dr. Roux, F-75724 Paris Cedex 15, France

INSERM and ARC.

Abbreviations: Bd: C57BL/6 BCP: B cell precursors BGG: Bovine y-globulin BrMRBC: Bromelain-treated mouse RBC Pee: Peritoneal cells ssDNA: Single-stranded DNA

of ten and seven different VH and V, gene families, respectively, as well as the frequencies of B cell clonal precursors (BCP) against four different antigens. This analysis compared CD5+ and CD5- B cells from newborn and adult mice in two local environments (spleen and peritoneal cavity), with the aim of obtaining quantitative data on putative repertoire differences between these B cell subsets and, thus, indications on the question of cellular selection. The results demonstrate that both CD5+ and CD5- B cells start with equivalent VH and V, repertoires but the former are submitted to local, age-dependent cellular selection.

2 Materials and methods

2.1 Animals, cells and FCM analysis

Inbred C57BL/6 (B6), raised in our own colonies at the Pasteur Institute, were used as neonates (5 days old) or as adults (8-10 weeks old). Spleen cells were prepared by teasing the organ in cold BSS. Peritoneal cells (PerC) were collected by washing the peritoneal cavity with 5-10 ml chilled BSS supplemented with 3% FCS. To obtain high frequencies of adult splenic CD5 B cells we enriched them using a discontinuous gradient of Percoll with which most CD5 B cells are recovered in the low-density fraction (50%-60% interface). Cells were washed twice and sub- mitted to double staining as follows. Cell concentrations were adjusted to 20 x 106/ml and the suspensions incubated for 15 min on ice with FITC-labeled rat IgGza anti-mouse IgM (331.12) [13] and biotin-coupled rat IgGZ, anti-CDS (53-7.3) [14] at a final concentration of 20 pg/ml each. The cells were then washed three times in BSS supplemented with 3% FCS and incubated for 15 min with streptavidin coupled to PE (Becton Dickinson, Mountain View, CA). After washing, the cells were resuspended at 2 x 106/ml and sorted using a FACStar cell sorter (Becton Dickinson). All analysis were made on a Hewlett Packard (Palo Alto, CA) computer (HP900) using the Consert 30 program (Becton Dickinson) .

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 OO14-2980/91/O9O9-2017$3.50 + .25lO

2018 L. Andrade, F. Huetz, €! Poncet et al. Eur. J. Immunol. 1991. 21: 2017-2023

2.2 B cell colony assay

The different cell suspensions were cultured in a double- layer colony technique, as described by Wu and Paige [15]. Briefly, the bottom layer corresponded to 1 ml Iscove's modified Dulbecco's medium, supplemented with 5 m g / d delipidated BSA, 10% FCS, 0.3% melted Bacto Agar (Difco, Detroit, MI), 50 Fg LPS and 1% washed SRBC. Cells were resuspended in the same medium without LPS and SRBC, and plated as top layer. The cultures were incubated at 37 "C, in 5% COz atmosphere, for 5-6 days, and transferred by suction to Hybond N filters (Amersham Int., Les Ulis, France).

The RNA blots were prehybridized overnight at 42°C in 50% formamide, 5 x SSPE, 4 x Denhardt's, 0.2% SDS, and 100 pg/ml each, polyadenylic acid and denatured E. coli DNA. They were then hybridized with VH- and V,-specific probes for 48 h at 42°C. After hybridization, the filters were washed once with 2 x SSPE, 0.2% SDS for 30 min at 6O"C, and once with 1 x SSPE, 0.1% SDS for 30 min at 60°C, dried and autoradiographed for 3-14 days. The dots of CFU-B were enumerated, and the relative representation of each gene family plotted as the percen- tage of total VH or V, gene expression.

2.3 Probes

TheVH and V, gene family probes were gifts of Drs. F. Alt, S. Riley, J. Kearney, U. Krawinkel, L. Reininger, E. Marche and D. Hoostelaere. The ten VH probes used were v ~ 8 l X (7183), VH300-19 (Q52), VHs107 (S107), vHx24 (x24), v ~ P B 1 4 (J~O~),VHP~-~R~(~~-~O),VHPNP B4 (J558),v~ v ~ ~ ~ 3 . 8 ( V G A M ~ . ~ ) , V ~ 3 1 (V31), v ~ c P 1 2 ( V ~ l l ) , and have been described elsewhere [lo, 16-21]. The seven V, probes used were V,1 , V,4, V,8, V,9, V,21, V,22 and V,23 and have been described elsewhere [9,22-25].The C, probe is the genomic fragment described by Max et al. [23]. Probes were labeled by the random priming method [26], using [32P]dCTP (Amersham Int., Amersham, GB).

2.4 LD analysis

Limiting numbers of sorted CDS+IgM+ or CDS-IgM+ B cells were distributed as described [27] in microculture wells at various concentrations (48 replicates for each), containing 6 x lo5 irradiated (50 rad) rat thymus filler cells, in RPMI 1640 medium (Gibco, Paisley, Scotland) supple- mented with 5 x M 2-ME (Merck, Darmstadt, FRG), 10% FCS (Boehringer-Mannheim, Mannheim, FRG), 1 O m ~ Hepes buffer, pH7.3 (Sigma, St. Louis, MO), 25 pg/ml LPS from Salmonella abortus equii (Difco Labo- ratories, Detroit, MI), penicillin, streptomycin (Flow, Irvine, Scotland) and gentamycin (Gentalin, Unilabo, Levallois Perret, France). For each determination of LPS- reactive B cell frequency, four to six different cell concen- trations were used. Cultures were incubated at 37 "C, in a 5% C02 atmosphere for 8 days. At the end of the incubation period, culture SN were tested for the presence of total and antigen-specific IgM (see below).

Detection of total IgM and antigen-specific Ab was done by ELISA as previously described [28]. Plates were coated

overnight at 4°C with 5 pg/ml goat anti-mouse Ig Ab, 100 pg/ml single-stranded DNA (ssDNA), 10 pg/ml bovine y-globulin (BGG) or BGG coupled to trimethylammonium (TMA) by diazotation. Specific anti-BrMRBC IgM was detected in a complement-dependent hemolysis assay followed by photometric determination or hemoglobin concentrations, as reported [28]. Cultures were considered positive when the respective A was superior to the mean + 3 SD of the 48 cultures containing filler cells alone. In all cases, the results were conform to the first term of Poisson's distribution and were treated by the x2 minimiza- tion method.The results are expressed as absolute frequen- cies, i.e. total frequencies obtained in the antigen-specific assay divided by total LPS-reactive cell frequencies.

3 Results

3.1 FCM purification of CD5+ and CD5- B cells

As most CD5 B cells in adult spleen are large low-density cells [29], we have sorted for CD5 B cells from the low-density cell fraction previously isolated from total spleen cells on a Percoll gradient. This procedure gave a tenfold enrichment in adult splenic CD5 B cells (1y0 vs. 13%), and allowed for a final 65% of CD5 B cells in the sorted population. The degree of FCM purification of the isolated CD5+ peritoneal and newborn B cell populations represented >80% of total cells in the final sample (Fig. 1).

3.2 Both CD5+ and 0 5 - B cells show D-proximal VH preferences in newborn mice

The comparison of VH gene family usage in CD5+ and CD5- clonable B cells from newborn and adult C57BL/6 mice (Figs. 2 and 3), confirms the D-proximal VH predom- inance in neonatal animals [16, 30, 311, and reveals that such preference is observed in both B cell subsets. CD5+ B cells, both in newborn and adult show a somewhat higher expression of the 7183 and Q52 gene families. In newborn spleen the percentage of (7183 + Q52) VH was 35% among CD5- B cells and 41% among CD5+ B cells. These values decrease in adult spleen to respectively, 23% and 31%, and even more in adult peritoneal cavity (respectively, 14% and 22%). For total splenic cells the mean expression of (7183 + Q52) VH genes ranges from 41 k 6 in neonates to 21 k 3 in adults, and 1 5 f 3 in total PerC. Thus, all B lymphocytes from newborn mice show a biased nonran- dom, position-dependent preferential VH gene expression [12,16,30-321 ,which decreases with age. CD5 B cells seem to keep a slight overrepresentation of D-proximal VH genes in adults, as compared to conventional B cells.

3.3 VHll overrepresentation among CD5 B cells is restricted to adult mice and similar in spleen and peritoneum

As previously reported, peritoneal CD5 B cells from adult mice show a significant overexpression of the VHll gene family [12, 33, 341. This preferential utilization was also found in adult splenic CD5 B cells, although less markedly in absolute numbers (Fig. 3). The correlation of VHll

Eur. J. Immunol. 1991. 21: 2017-2023 VH and V, gene expression by CD5 B cells 2019

(C) SORTED LIJ- l o 8 CELLS

IBI SORTED LIJ- I - 8 CELLS

(Al TOTAL CELLS

Figure 1. 'If.pical two-color fluorescence and sorting of splenic cells from newborn and adult mice, and of adult PerC. (A) Staining

?! 0

5 0

z r pattern of total splenic or PerC before sorting; (B) sorted CD5- B cells; (C) sorted

9. CD5+ B cells. Gates used for sorting and i n g 1.' I d I" -m0 la' 10' 10' IP' -lmo 's' 10' so* I.* percentages oflymphocytesin these gates are

f indicated. lgn

t (9

U n

VH gem hmlly 2

Figure 2. VH gene usage in FCM-purified CD5+ and CD5- B cells from the spleen of newborn B6 mice. Bars correspond to the relative frequencies of eachVH family in the total pool of VH genes scored. A total of 383 colonies expressing detectable Vn gene sequences were counted. The cloning efficiencies were similar (1 : 10) or both CD5+ and (335- B cells plated.

expression with CD5 B cells is, however, equivalent in both locations. Thus, the ratio of VHll utilization in CD5+ vs. CD5- B cells was 7.3 for PerC and 8 for splenic cells (Table 1). In contrast, newborn splenic B cells showed very low V ~ l l utilization in both CD5+ and CD5- B cells, suggesting that overexpression of VHll genes in the CD5 B cell subset is dependent on cellular selection with age.

3.4 Anti-BrMRBC specificity is overrepresented in adult, but not in newborn, CD5+ B cells

Previous studies have shown that the VHll gene family expression is intimately associated with anti-BrMRBC reactivity [lo, 11, 34, 351 and that adult CD5+ B cells produce anti-BrMRBC at high frequency [36] .We have now determined BCP frequencies against four different antigens (BrMRBCTMA-BGG, BGG and ssDNA) in sorted CD5+ and CD5- B cell populations from spleen and peritoneum of newborn and adult mice. The results confirm the correlation between the expression of VHll and the frequency of anti-BrMRBC clones (Table 2). In newborn spleen, neither in CD5+ nor in CD5- B cells could such BCP be found, while they are readily detectable in adult splenic or peritoneal B cells with, respectively, a 40- or 100-times higher frequency in CDS B cells than in conven- tional B cells (Table 1). BCP directed against TMA, which cross-react with BrMRBC [37] and phosphoryl choline (data not shown) are also more frequent in adult CD5+ than

2020 L. Andrade, F. Huetz, F! Poncet et al. Eur. J. Immunol. 1991.21: 2017-2023

0 y.

Figure3. VH gene usage in FCM- purified CD5+ and CD5- B cells from spleen and peritoneum of adult B6 mice. Bars correspond to the relative frequencies of each V, gene family in the total pool of VH genes scored. A total of 460 and 1121 colonies (in three independent experiments) expressing detectable VH gene sequences were counted, with cells from spleen and peritoneal cavity, respectively. The cloning efficiencies were similar for both CD5+ and 0 5 - Bcells and varied from 1 : 3 to 1 : 6.

n t. 0 m t 0 0 n m

c (I) m 3 n

? c 7 m X ?

n Y) 0 0

Table 1. Representation of CD5 B cells in different lymphoid compartments and their selection for VHll and anti-BrMRBC expression

'%, CD5 Ratios of CDS+/CDS -'I' B cells V l l l 1 Frequency of

expression anti-BrMRBC

scwl~orn Total splccn cclls (1 2 No precursors A1lul1

Pcritonc;il cells 45 7.3 IOO l.argc splccii cells 13 8 40

a Ratios were calculated by dividing either the percent values for V H l l expression or the frequency of anti-BrMRBC BCP determined for CD5+ cells, by the corresponding figures found for cD5- cells in each lymphoid population, as indicated.

c r 0 c

n > I -I >

in CD5- B cells. In contrast, the anti-TMA BCP frequency in newborns was quite high, but no differences were observed between CD5+ and CD5- B cells. Essentially no selection was observed in one or the other B cell subpopu- lation for anti-BGG and anti-ssDNA BCF?

3.5 No detectable bias in V, gene expression in CD5+ and CD5- B cells

L chain isotypes cannot be used as a selection parameter of B cell subsets, since about 90% of either CD5+ or CD5- B cells bear the 3t L chain (Andrade et al., submitted). To find other markers of selection in CD5+ B cells, we have now compared V, gene family utilization in sorted CD5- and CD5+ PerC and in total adult spleen cells. As shown in Table 3, the sevenV, probes used in this study account for >60% of all C,-expressing colonies screened; a value consistent with estimates of the number of V, gene homology groups [9,38]. In this study,V,l and V,22 were,

Table 2. Reciprocal absolute frequencies of anti-BrMRBC, anti-TMA-BGG, anti-ssDNA and anti-BGG in CD5+ and CD5- B cells from peritoneal cavity and spleen of newborn and adult B6 mice

B cell BrMRBC TMA-BGG BGG s\DNA population

Newborn Spleen CD5+ > I O O ~ H I O 10s 43 1 125

CD5 > IOOotM) 140 410 215, (89- 138)") (343-5X1) (9l-l'M)

(I 10- 196) (309-607) (164-333) Adult !jpIc.cn CD5. l(70

C'D5 ti O(M)

(135- 198)

(4880-7800) Pcritoncal cin it! Cl1.i - 9

(7- 13)

(483-2011) CIX 980

420 (326- 6 0 5 )

WK) (635- 1400)

4 (2- 9 )

31 ( 1 1 - 102)

ND 47

ND 172

77 5

107 8

(35- 73)

(134-241 )

(61-104) (4- 8)

(83-152) (7- 11) a) 95% confidence limit.

Eur. J. Immunol. 1991. 21: 2017-2023 VH and V, gene expression by CD5 B cells 2021

results can only be explained by a marked age-dependent cellular selection.

Table 3. V,, gene family usage by splenic B lymphocytes, CD5+ and CD5- peritoneal B cells

V, gene family Spleen PerC PerC W 5 + ) (CD5-)

V,l/C, 1641820b) (20% )

v,wc, 95/82Ob) (12%)

V,8/C, 13U820b) (16%)

V,9IC, 901520 (17%)

v,2 1/c, 131300 (4%)

v,22/c, 01520

v,23/c, 331520 (6%)

w300 (32%) 151300 (5%)

36l300 (12%) 201300 (7%) ND

6l300 (2%) 8/3w (3%)

1031300

33/30

501300

231300 (8%) ND

z300 (0.6%) 10/300 (3%)

(34%)

( 1 l Y O )

(17%)

v, 0.75 0.61 0.73 S Y

The degree of FCM purification of the peritoneal C D 5 - and CD5+ B cell populations studied was more than 80%. The cloning efficiencies were similar (1 : 6) for both peritoneal CD5- and CD5+ cells as well as for total splenic B cells. The results shown represent the ratio of the number of colonies expressing each V, family and the number of G+ colonies in triplicate blots. For VJ, V,4 and V,8 gene expression, the data of two experiments were pooled.

respectively, the most and the least frequent V, gene families transcribed in all cell populations analyzed (20% -34% vs. 0% -2%). Other V, families were expressed at intermediate frequencies. Furthermore, no significant differences were observed in V,, expression between CD5+ B and CD5- B cells other than a doubtful increased utilization of V,22 among CD5+ B cells (2% vs. 0.6%). This might be related to the exclusive expression by peritoneal CD5 B cells of the VX22-bearing T15 idiotype [39]. However, the low frequency of positive cells does not allow any firm conclusions.

4 Discussion

Knowing that the repertoire of adult peritoneal CD5+ B cells is different from that of splenic B cells [l-3,6,12], we aimed at establishing whether these differences result from genetic restrictions in germ-line repertoire expression or from cellular selection.

We have compared Ab repertoire and VH and V, gene utilization in CD5+ vs. CD5- FCM-sorted B cells from newborn and adult animals; i.e. before and after extensive selection might take place. We have also compared such populations isolated from the spleen and peritoneal cavity of adult animals, to assess putative local factors operating in selection. Our results show that CD5+ and CD5- B cells express indistinguishable repertoires at birth. If, as current- ly accepted, production of CD5+ B cells from uncomitted precursors is limited to the neonatal period [l, 21, these

Just like “conventional” B cells, newborn CD5 B cells show an accentuated utilization of D-proximal VH gene families, and are submitted to a parallel shift towards utilization of other families in adults. This so-called process of “nonnal- ization”,whichin conventional splenic B cells results in aVH gene family distribution proportional to the estimated number of genes of each family [40,41], is altered in CD5+ B cells by the overexpression of vH11, and perhaps a slight preference for D-proximal VH families, as compared to CD5- B cells. This maintenance of some D-proximal gene preference in CD5 B cells during the life of the mouse may be related to the persistence of such clones selected early in ontogeny. Recent studies have established a marked pre- ference for the expression of 7183 VH genes in pre-B cells from adult BM [32,42]. Since adult BM is devoid of CD5 B cells [l, 21, these observations argue against the notion that overexpression of D-proximal families results from a genetic restriction in the “CD5 differentiation pathway”. Instead, these preferences characterize VH repertoires of newly emerging cells. Furthermore, analysis of VH gene family usage in mice homozygous for the recessive mutation motheaten, which have an extreme overrepresentation of CD5 B cells, show a marked underrepresentation of VH 7183 in the actual repertoire as compared to normal mice [431-

An interesting finding here is that the high values of VHll expression in adult peritoneal CD5 B cells (22%) are not found in CD5 B newborn spleen cells (2%) demonstrating postnatal cellular selection. In the adult spleen compart- ment, the utilization of V ~ l l by CD5 B cells, although low (4%) is still eight times higher than that found in CD5- B cells (Table 1). This observation could be due to migra- tion of C D 5 B cells from the peritoneum to the spleen in adult mice. We are aware that cross-contaminations in the cell populations analyzed could somewhat alter these results. From the purity of each preparation, however (see Fig. l) , it can be seen that if this were the case the differences in n b l e 1 would be even greater and our conclusions would be reinforced.

These differences in repertoire of the three CD5 B cell populations studied here were confirmed by analyzing antibody reactivities, particularly anti-BrMRBC BCP fre- quencies. Compared to the case with CD5- B cells, there is a very high selection of such specificity in peritoneal CD5+ B cells, which is lower in adult spleen and absent in newborn spleen.This is in agreement with results of Hardy et al. [34], who showed that anti-Brh4RBC Ab accounted for 30% of peritoneal CD5 B hybridomas and only 3% of hybridomas orginated from splenic CD5 B cells. In contrast toVH11 expression, therefore, local factors in the coelomic cavities seem to contribute to the selection of anti- Brh4RBC clones, which use VH genes other than vH11. There are likely to be the recently described vH12 genes which show preferential expression in PerC [44].

The absence of such reactivities in newborns might be due to lack of either the selective ligand or the specific BCPThe former appears unlikely since selective expansion of donor VH1l-expressing B cells could be obtained in neonates upon transfer of adult splenic B cells (Thomas-Vaslin et al.,

2022

submitted). The absence of anti-BrMRBC BCP in 5- day-old newborns [36,45,46] suggests that this whole set of results can be explained by the positive selection and expansion in the peritoneum of rather infrequently pro- duced VHll gene-expressing cells with anti-BrMRBC reactivity [4, 6, 11, 341.

L. Andrade, F. Huetz, I! Poncet et al. Eur. J. Immunol. 1991. 21: 2017-2023

Since no anti-BrMRBC BCP could be detected in newborn spleen cells, we determined the anti-TMA BCP frequency, which is also preferentially represented in CD5 B cells and cross-react with BrMRBC [37,47-491. Anti-TMA BCP are indeed more frequent in CD5+ than in CD5- B cells of adult mice, but their frequency is comparable in both newborn subpopulations. This finding reinforces the con- clusion of age-dependent selection.

In contrast to V, genes, no biased distribution of V, gene families in peritoneal CD5 B cells was observed. Even if some interfady cross-hybridization with certain probes cannot be excluded [38], we are confident that no marked restriction in V, gene expression applies to CD5 B cell populations. These results are in agreement with a recent study on V, gene family expression performed by in situ hybridization [50]. Interestingly, V,9 and V,4 genes that have previously been reported to associate preferentially withVHll andVH12, respectively, in anti-BrMRBCAb [lo, 33,34,51] are as frequently expressed by CD5+ and CD5- B cells in spleen or peritoneal cavity. It follows that, in contrast to VHll that seems to be exclusively used for anti-BrMBBC in peritoneal cavity [ll], the corresponding V, genes are more broadly used [52].

We conclude from these results that the differences in Ab repertoires expressed by CD5 B cells in adult or very old mice [53] do not result from a genetically programmed restriction, but rather from cellular selection mechanisms operating within a B cell subset with slow turnover rates.

We would like to thank Dr. A. Freitas for discussions and suggestions, Ms. A. Demond for preparing the manuscript and Ms. M . I? Lernbezat and I? Cavelier for technical assistance in the preparation of probes. We are grateful to Drs. U. Krawinkel, J. Kearney, E Alt, S. Riley, L. Reininger, E. Marche and L. A. D’Hoostelaere for VH and V, probes.

Received August 4, 1990, in revised form May 2, 1991.

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