Eur. J. Immunol. 1989.19: 1117-1122 VH gene expression in Ly-1' B lymphocytes 1117

Immunoglobulin VH gene expression in Ly-1' and conventional B lymphocytes*

Luiz Andrade', Anthio A. Freitas, Fransois Huetz, Pascal Poncet and Ant6nio Coutinho

Unit6 d'hmunobiologie, Mpartement d'Immunologie, Institut Pasteur, Paris

1 Introduction

Lymphocyte populations in which Ly-1 B cells are differentially represented were studied for the expression of ten VH gene families, either by an RNA colony blot assay or by in situ hybridization of single cells, in BALB/c and C57BL/6 mice. The comparisons of cells from lymph nodes, Peyer's patches and adult spleen (poor in Ly-1 B cells) with cells from peritoneal cavity and neonatal spleen (rich in Ly-1 B cells) were confirmed by the analysis of adult peritoneal Ly-1- and Ly-1' B cells sorted on the fluorescence-activated cell sorter. The results indicate that the peritoneal Ly-1' B subset uses the whole spectrum of known VH gene families, and shows a preferential utilization of CP12 VH genes, most likely as a result of a selective process during life.

Recent studies have defined a subset of murine B cells that expresses the Ly-1 (CD5) surface glycoprotein and might be relevant in the establishment of auto-immune diseases [l-31. The Ly-1' B cells, in contrast with the majority of B cells in the animal, are present at high frequencies in neonates and adult peritoneal cavity, but are rare or undetectable in lymph nodes and Peyer's patches of adult mice [l , 4, 51. Further- more, Ly-1' B cells do not appear to be generated from stem cells present in the bone marrow of adult mice, but from their own self-renewal capacity [5, 61. On the basis of these and other studies, the Ly-1' B cell subset is believed to constitute a distinct developmental lineage, separable from the conven- tional, adult bone marrow-derived B lymphocytes [3]. Most importantly, in spite of their relatively small number, the Ly-1' B cells appear to be responsible for the production of a major fraction of the normal serum IgM [3, 7, 81, and may directly participate in the selection of B and T cell repertoires [9, 101.

In view of the indications for clonal repertoire differences between Ly-1' and conventional B cells [ll], and given the availability of methods that assess the totality of repertoires in terms of VH gene families [12] expressed, we have now com- pared Ly-1-enriched or -depleted B cell populations in this respect. Our results fail to reveal major VH repertoire differ- ences between conventional and Ly-1' B cells, with the excep- tion of the CP12 VH gene, which is greatly overrepresented in Ly-1' cells. This bias is most likely the result of repertoire selection during ontogeny, as splenocytes from neonatal mice which are enriched in cells of the same Ly-1' phenotype do not show this preference.

[ 74881

* This work was supported by a grant from DRET, ARC, AMF and EEC. Recipient of a doctoral fellowship from CNPq, Brazil.

Correspondence: Luiz Andrade, Unit6 d'Irnmunobiologie, Institut Pasteur, 25-27, rue du Docteur Roux, F-75724 Paris Cedex 15, France

Abbreviations: CFU-B: Colony-forming unit BrMRBC: Brom- eleinized mouse red blood cell LPS: Lipopolysaccharide PC: Peritoneal cavity

2 Materials and methods

2.1 Animals and cells

Inbred BALB/c and C57BL/6 (B6), raised in our own colonies at the Pasteur Institute, were used either at birth or between 8-10 weeks of age. Spleen, peripheral and mesenteric lymph nodes (LN), Peyer's patches (PP) and peritoneal cavity (PC) cell suspensions were prepared by either gentle teasing of the organs in cold balanced salt solution (BSS) or by injecting 10 ml of BSS supplemented with 3% fetal calf serum (FCS) into PC of mice killed by C02 asphyxiation, followed by mild massage of the abdomen and collection of the fluid.

2.2 Cell staining and multiparameter fluorescence-activated cell sorter (FACS) analysis

Cells were stained with fluorescein isothiocyanate-labeled rat IgG2, anti-mouse IgM 331.12 [ 131 and biotin-coupled rat IgGz, anti-Ly-1 53-7.3 [14, 151 followed by avidin-phycoerythrin. Cell concentration was adjusted to 20 x 106/ml and the suspen- sion incubated with the two antibodies (final concentration 20 pg/ml each) for 15 min on ice. The cells were then washed three times in BSS supplemented with 3% FCS and incubated for 15 min with streptavidin coupled with phycoerythrin (Bec- ton Dickinson, Mountain View, CA). After washing, the cells were resuspended at 2 x 106/ml and sorted using a FACStar cell sorter (Becton Dickinson. Mountain View. CA). All analyses were made on Hewlett Packard computer (HPYOO) using either Consort 30 or Lysis analysis programs (Becton Dickinson).

2.3 Probes

The C, and VH gene family probes were kind gifts of Drs. F. Alt, S. Riley, J. Kearney, U. Krawinkel and J. L. Reininger. The ten V, probes used were V, 81-X (7183), VH 300-19 (Q52), VH S107 (S107), VH X24 (X24), VH PBV14 (J606), VH P6-3RI (36-60), VH PNP B4 (J558), v~AM3.8 (VGAM3.8), VH V31 (V31/36.09), VH cP12 (VHll), and have been described elsewhere [16-221. The C, probe is the genomic fragment PGGN-1 described in Chih-Ping et al. [23]. Probes were labeled by the random priming method [24], using [35S] or [32P]dCTP (Amersham Int., Bucks., GB).

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 0014-2980/89/0606-1117$02.5010

1118 L. Andrade, A. A. Freitas, F. Huetz et al. Eur. J . Immunol. 1989.19: 1117-1122

2.4 In situ hybridization

In situ hybridization was performed using a modification of the method initially described by Haase et al. [25]. Briefly, cells were cytospun onto clean slides pre-treated with Denhardt's solution, air-dried, and fixed for 20 min in ice-cold PFG (0.5% paraformaldehyde, 0.5% glutaraldehyde, 1.6% glucose, 0.02% CaC12, 1% dimethylsulfoxide in 0.1 M phosphate buf- fer, [26]). After fixation, the cells were quenched by immer- sion for 20 rnin in cold 0.15 M ethanolamine (pH 7.5), and washed twice in cold phosphate-buffered saline (PBS). To allow longer periods of storage at room temperature, cells were dehydrated in ethanol, 70%, 95% and 100% (for 5 rnin at each step), treated with 100% xylene (5 min) and embed- ded in Paraplast.

Slides were prepared for hybridization by removal of paraffin with xylene (3 x 10 min), followed by rehydration of the cells in successive baths of ethanol at loo%, 95% and 70%, and pyrolized water (5 rnin each). After rehydration of the cells, slides were immersed in 0.2 M HCl for 20 min, incubated at 70°C in 2 X S s c (0.3 M NaCl, 0.03 M sodium citrate) for 30 min, and treated with proteinase K (1 pg/ml) for 15 min at 37°C. After treatment, cells were dehydrated and allowed to air-dry before hybridization. The hybridization mixture con- taining 50% deionized formamide, 600 mM NaCl, 10 mM Tris- HCl, pH 7.5, 1 mM EDTA, 1 mg/ml bovine serum albumin (BSA), 0.02% Ficoll, 0.02% polyvinylpyrolidone, 100 pg/ml sonicated, denatured salmon sperm DNA, 100 pg/ml poly(A), 2 mg/ml ribonucleic acid (Sigma Chemical Co., St. Louis, MO), and the 35S-labeled cDNA probe, was treated to 100°C for 1 min and cooled in ice for a few min. After addition of dithiothreitol to 1% of the final volume, 5 p1 of the hybridiza- tion mixture, containing approximately 60 000-70000 cpm of probe, was added to the cell pellet. Cells were then covered with a 12-mm diameter siliconized and baked cover slip and sealed with rubber cement. Slides were incubated for 72 h at room temperature in the dark. After hybridization, excess of nonspecific binding was removed by washing 2 x 5 min in HBW (50% formamide, 10 mM Tris-HC1, 1 mM EDTA, 600 mM NaCI), 5 min at room temperature in 2 x SSC, fol- lowed by successive washes in TNE (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCl) for 72 h with agitation. After a previous acetylation step, slides were then dipped in NTB2 Kodak emulsion (Rochester, NY) and exposed in the dark for 7 days at 4°C. After development, slides were stained in Giemsa and the cells analyzed by light microscopy.

2.5 B cell colony assay

Spleen and PC cell suspensions were cultured in double-layer colony technique, as described by Wu and Paige [27] Briefly, the bottom layer contained in 1 ml Iscove's modified Dulbec- CO'S medium, supplemented with 5 mg/ml delipidated BSA, 10%-15% FCS, 0.3% melted Bacto Agar (Difco, Detroit, MI), 50 pg lipopolysaccharide (LPS) and 1% washed sheep red blood cells (SRBC). Cells were resuspended in the same medium without LPS and SRBC, and plated as top layer. The cultures were incubated at 37"C, 5% C02 , for 5-6 days, and transferred by suction to Hybond N filters (Amersham).

The RNA blots were prehybridized overnight at 42 "C in 50% formamide, 5 x SSPE, 4 x Denhardt's, 0.2% sodium dodecyl

sulfate (SDS), and 100 pglml each, polyadenylic acid and denatured E. coli DNA, and hybridized with C, and the ten VH family-specific cDNA fragments for 48 h at 42°C. After hybridization, the filters were washed once with 2 x SSPE, 0.2% SDS for 30 rnin each at 42"C, then twice with 2 x SSPE, 0.2% SDS for 30 rnin at 60"C, and once with 1 x SSPE, 0.1% SDS for 30 rnin at 60°C, dried and autoradiographed for 3-14 days. The dots of colony-forming unit (CFU)-B were enumerated, and the relative representation of each gene fam- ily plotted as the percentge of total VH gene expression.

3 Results

3.1 Experimental strategy

The present investigation follows VH gene family usage in different populations, either poor in or enriched for Ly-l+ B cells. Adult spleen either from normal or irradiated donors, LN and PP (poor in Ly-l+ B cells) were compared to adult PC and newborn SP (containing high frequencies of B cells ex- pressing the Ly-1 marker), as well as Ly-1-depleted to Ly-1+- sorted B cell populations from adult PC.

VH gene expression was studied in CFU-B using a RNA col- ony blot assay or in single cells by in situ hybridization [25, 271. With the colony blot assay, we screened over 5000 CFU-B colonies derived from spleen and PC, while the in situ hybridi- zation method allowed the study of 2000 single cells from the various organs in the adult BALB/c. Since both our techniques require relatively high levels of specific message for the unam- biguous detection of positive cells, the various lymphoid popu- lations were activated with LPS in culture.

The specificity of ten different VH probes was determined by testing their hybridization in both assays to cell lines express- ing known VH genes of the various families. In all cases, hybridization of each VH probe occurred only with the specific cell lines. In each experiment, the total number of cells or colonies hybridizing with the different VH gene probes, was between 70%-95% of the total number of cells or colonies hy- bridizing with a C, probe. For this reason, VH gene family usage in the different cell populations studied, was expressed as the percentage of the relative distribution of each VH gene family in the total number of positive cells or colonies detected with all VH gene probes.

3.2 VH gene family usage in various lymphoid organs from adult BALB/c mice

In a first set of experiments, we have analyzed the usage of ten different VH gene families by in situ hybridization in LPS- activated cells from several lymphoid- tissues: spleen, peripheral and mesenteric LN, PC and PP of adult BALB/c mice. Results in Table 1 show that the utilization of S107 and 52 VH genes was slightly greater in PC as compared to the other lymphoid tissues. In the same experiment X24 genes are also more expressed in PC than in spleen and LN, but they show an even higher representation in PP. The major differ- ence found in this set of experiments, however, was in the expression of VH gene CP12, which was 3-27 times more utilized in peritoneal B cells, when compared to B cells obtained from the other lymphoid tissue analyzed.

Eur. J. Immunol. 1989.19: 1117-1122 1119

Table 1. VH gene family usage in LPS-activated cells from different organs of adult BALB/c mice studied at a single-cell level by in siru hybridization@

VH gene expression in Ly-1' B lymphocytes

VH gene families Organ V31 5558 5606 VGAM S107 36-60 X24 Q52 7183 CP12 a) VH family usage in LPS-stimulated PC, spleen

(SPL), LN, mesenteric LN (MLN), and PP cells PC O.lb' 20.0 1.6 7.2 14.2 2.9 10.1 22.9 SPL 0.2 24.7 10.6 22.7 6.8 6.6 2.9 14.4 LN 0.3 47.8 3.5 14.2 2.8 4.2 3.5 13.8 MLN 0.1 37.3 9.2 20.6 10.3 6.4 2.8 12.1 PP 0.1 22.7 16.8 20.7 9.8 1.8 18.4 5.3

3.3 Comparison of VH gene utilization in splenic and peritoneal clonable B cells from adult BALB/c and C57BL/6 mice

In a second series of experiments, we studied the patterns of VH gene family usage in clonable B cells from PC and spleen. These results show that the expression of X24 and CP12 VH families is higher in PC from BALB/c (7%-18% and 3%, respectively; Fig. l), and from C57BL/6 (6%-11% and 4%, respectively; Fig. 2), as compared to spleen (2%-6% and 0.5%, respectively) from both strains (Figs. 1 and 2).

Irradiated (750 rad) and bone marrow-reconstituted BALB/c and C57BL/6 mice, believed to contain reduced levels of Ly-1' B cells, showed similar splenic VH patterns of expression as normal donors, except for the X24 VH family which was not found in irradiated BALB/c mice (Figs. 1 and 2).

The most significant difference in V H gene family distribution was observed upon comparison of B cell colonies derived from FACS-purified Ly-1' and Ly-1- B cells from adult PC of

C P l ?

V31

J558

JCCG

- VGAM38

I

k S107

>= 36 60

W BALWCSPL 0 lrr BALWC Sl'l 0 BALB/CPEC

x2.1

0 5 2

7183

0 1 0 2 0 3 0 4 0 5 0 % o f total V, expression

Figure 1. VH gene family distribution in splenic and peritoneal clon- able B cells from normal and irradiated bone marrow-reconstituted 'BALB/c mice. Each bar corresponds to the relative frequency of each YH family in the total pool of VH genes expressed. A total of 1206 splenic (six individual experiments) and 807 peritoneal (three indi- vidual experiments) colonies expressing detectable VH gene sequences were scored.The VH J558NH 7183 ratios, defined in [27] as a parame- ter to identify strain-specific differences in VH gene family usage, were similar in both peritoneal and spleen populations and ranged from p = 0.52 t o p = 0.83. The cloning efficiencies (1110) were also similar in both spleen and PC populations plated.

2.3 18.7 from BALB/c mice. The numbers correspond to 5.3 5.8 the relative frequency of each VH family in total 9.3 0.2 pool of VH genes expressed. A total of at least 0.2 1.0 500 positive cells from a pool of 2-3 mice were 2.7 1.8 scored in each experiment.

b) Percent values.

BALB/c and C57BL/6 mice. The degree of FACS purification of the isolated Ly-1' and Ly-1- peritoneal B cells studied is shown in Fig. 3. In both cases, the cell population studied represented 80% or more of the final sorted sample of PC cells. The expression of CP12 VH gene was markedly increased in Ly-1' B cells (20%-25% vs. 3%-7% in Ly-l+ or Ly-1- cells, respectively) in both strains of mice (Figs. 4 and 5).

3.4 Comparison of VH gene usage of splenic clonable B cells from adult and newborn C57BL/6 mice

The developmental appearance of Ly-1' B cells during the early stages of life, when conventional B cells are still rare, is one of the characteristics of the Ly-1' B cell subset [ 5 ] . In order to determine whether the pattern of VH gene family usage changes during the development of the mouse, and whether neonatal Ly-l+ B cells show the same preferential usage of the CP12 V H genes observed in the adult PC, we next investigated VH family usage in splenic clonable B cells from 2-day-old neonates and adult C57BL/6 mice (Fig. 6).

CP12 P V3 1

J558

JGOG

& 5107

>* UGSPL

0 BG PLC 36 60 0 Irr BGSPI

UGSPL 0 Irr BGSPI 0 BG PEC

X24

0 5 2

7183

0 1 0 2 0 3 0 4 0 5 0 % o f t o t a l V, expression

Figure 2. VH gene family distribution in splenic and peritoneal clon- able B cells from normal and irradiated bone marrow-reconstituted C57BL/6 mice. A total of 1253 splenic- (four individual experiments, J558/7183 p = 4.55 k 2.3) and 722 peritoneal- (three individual experi- ments, J558/7183p = 7.3 k 2.5) derived colonies expressing detectable VH gene sequences were scored. The cloning efficiencies were similar (1/6) in both spleen and PC cells plated.

1120 L. Andrade, A. A. Freitas, F. Huetz et al. Eur. J. Immunol. 1989.19: 1117-1122

(C)

7

FLI - - - ? i L I ---> F L I - - ->

Figure 3. Typical two-color fluorescence analysis and sorting of peritoneal cells. (A) Staining pattern of adult peritoneal cells before sorting; (B) sorted Ly-1' B cells; (C) sorted Ly-1- B cells. Gates used for sorting and percentages of lymphocytes in these gates are indicated.

cp12 V31 ? J558

g J606

5 VGAM3.8 - - .- -

WCPECCONV BIC PEC LY1

S107 al m ,x 36-60

X24

0 5 2

7183 I . I . I . I . I - J

0 1 0 2 0 3 0 4 0 5 0 % of t o t a l V, expression

Figure 4. VH gene usage in FACS-purified Ly-1' and Ly-1- B cells from adult PC of BALBlc mice. The lymphocyte populations studied are indicated in Fig. 3. A total of 300 colonies expressing detectable V, gene sequences were scored. The cloning efficiencies were similar (1/10) for both Ly-1' and Ly-1- B cells.

CP12

V3 1

5558

g J606

5 VGAM3.8 - - .- L

7183 *----

Our observations, using the CFU-B RNA colony blot assay, confirm previous studies that have shown strain-dependent patterns of VH gene family usage [27, 281 with a predominant representation of the VH 5558 family in adult C57BL/6 mice (39%-49%) as compared with its utilization in adult BALB/c (13%-27%) mice (Figs. 1 and 2).

However, we also observed an enhanced expression of the VH 5558 family in adult, as compared to newborn, B6 mice (Fig. 6). These changes can be summarized as an increase in the ratio of relative expression between the 555817183 gene families, which increases from 1.4 k 0.4 in neonatal to 4.5 f 2.3 in the adult spleen. The relatively low frequencies of VH J558 (11%-27%) family in the neonatal stage appears to be accompanied by an enhanced expression of VH Q52 (16%-39%) and 7183 (10%-15%) families (Fig. 6). Surpris- ingly, however, expression of the CP12 VH gene was identical in both neontal and adult splenic B cells, in spite of the over- representation of Ly-1' B cells at birth [5 ] .

al c u m

2

CP12 44 V31 1

S107

36-60

X24

0 5 2

7183

B6NBSPL BGSPL

0 1 0 2 0 3 0 4 0 5 0 0 1 0 2 0 3 0 4 0 5 0 % of t o t a l '4, expression

% of t o t a l V, expression Figure 6 . VH gene usage in clonable splenic B cells from 2-day-old neonatal and adult C57BU6 mice. A total of 402 neonatal (three indi- vidual experiments, J558/7183p = 1.4 k 0.4) and 1253 adult (J558/7183 p = 4.55 k 2.3) spleen-derived colonies expressing detectable VH gene sequences were scored. The cloning efficiencies were 1/6 for adult spleen and 1/30 for neonatal spleen cells plated.

Figure 5. VH gene usage in FACS-purified Ly-1' and Ly-1- B cells from adult PC of C57BU6 mice. A total of 821 colonies expressing detectable VH gene sequences were scored (two experiments). The cloning efficiencies were similar (1/3) for both Ly-1' and Ly-1- B cells plated.

Eur. J. Immunol. 1989.19: 1117-1122

4 Discussion

VH gene expression in Ly-1’ B lymphocytes 1121

remarkable clonal relatedness. In the ten Ly-l+ B CH lym- phomas studied by Pennel et al. [32], all derived from old animals, the 36-60,5558, S107 and X2417183 were the only VH gene families expressed, although the three CH lymphomas producing anti-BrMRBC antibodies described as X24/7183 are identical in VH sequences to CP12 VH, and belong, therefore, to the VHll gene family [22]. In spite of the relatively small number of clones analyzed in those experiments, as compared to the present results, the data reinforce the notion that Ly-1’ B cells start from expressing a broad VH-repertoire, but, in the absence of significant turnover from uncommitted precursors, are submitted to strong selection throughout life.

Hybridomas and Abelson virus-transformed pre-B cells gener- ated from fetal liver and neonatal spleen sources enriched for Ly-1’ B cells, frequently express VH genes belonging to the 7183, Q52 and S107 VH gene family [17, 20, 341. The overre- presentation of D-proximal VH gene families [34] in early stages of ontogeny has led to the suggestion of a preferential expression of those families in Ly-1’ B cells that are produced at these developmental stages [3]. We observed the same bias for VH7183 and Q52 in splenic clonable B cells from newborn, as compared to adult mice (Fig. 6), but as shown by the analy- sis of adult Ly-1’ B cells, these preferences are not maintained in this lymphocyte compartment. Recent observations have established that preference of VH genes proximal to D has more to do with the differentiation of B cells than with cell lineage or ontogeneic stage. Thus, early B cell differentiation in adult bone marrow shows regulation of the rates of utiliza- tion of different V, gene families, followed by peripheral “normalization” of VH gene expression [36]. Since adult bone marrow is reported to be devoid of Ly-1’ B cells, these obser- vations constitute evidence against overexpression of D-proxi- ma1 families being dictated by the “Ly-1” differentiative path- way. The “normalization” process of VH gene repertoires is environmental and T cell dependent [36] and occurs through- out life, making it likely that position-dependent biases in the original Ly-l+ B cell repertoires can be “normalized” and altered for other preferences in the adult, due to the long-term persistence of this cell type.

The aim of this investigation was to compare the antibody repertoires of the two murine B cell subsets defined by the differential expression of the Ly-1 marker in terms of VH gene family distribution. While the patterns of expression of VH gene families do not give any indication as to their antibody reactivities, they may provide a simple and direct way to dis- close similarities or differences between antibody repertoires, and it is to date the only method that considers the totality of repertoires. The present findings were obtained by studying VH gene expression in single cells, by in situ hybridization, or in CFU-B colonies by RNA colony blot assay. Although these two methods show somewhat different patterns of VH gene family representation in BALB/c mice, which may be related to the different culture conditions employed (Andrade et al., in preparation), we can pinpoint concordant findings in both techniques that allow differentiation of Ly-1’ from conven- tional B cell repertoires in the PC of adult BALB/c mice, in terms of V H family usage.

Based on the overall VH gene family representations, we con- clude from the present investigation that the most significant difference between murine Ly-1’ and Ly-1- B cell subsets is related to the utilization of CP12 genes. This VH segment that encodes anti-bromelinized mouse red blood cell autoanti- bodies (anti-BrMRBC) defines a recently described VH gene family (VHll), which appears to contain one to three members in the various laboratory mouse strains [22].

Unlike adult peritoneal Ly-1’ B cells, a preferential utilization of CP12 VH genes was not observed in the analysis of the newborn spleen, another tissue enriched in Ly-1’ B cells. This would suggest that the overexpression of CP12 VH genes in adult Ly-1’ B cells is generated by a selective process during murine post-natal development. This hypothesis is supported by previous observations on the development of BrMRBC- specific B lymphocytes. In fact, it has been established that the large majority of anti-BrMRBC antibodies are produced by Ly-1’ B cells [7, 291, and most of them appear to be encoded by CP12 genes (unpublished observations). Nevertheless, it has been established that the absolute frequencies of BrMRBC-specific B cells reach adult levels only at 3 weeks of age in the PC, and by 6-10 weeks of age in the spleen [30]. This process of selection is also shown to be T cell-dependent [9] and to continue throughout life, reaching very high fre- quencies in aged animals (Weksler et al., submitted). Since the production of Ly-1’ B cells from uncommitted precursors appears to take place only in the perinatal period [3], it is plausible that this whole set of results can be explained by the positive selection and expansion of a rather infrequent CP12 VH gene-encoded specificity necessary for anti-BrMRBC reac- tivity. This process, which occurs in the particular environ- mental conditions of the peritoneum, requires time and results in persistence of the selected clones in the PC.

Our data add to previous findings showing VH gene restriction among Ly-1’ B cells of older animals [3, 31-33]. Extreme selection for a given set of specificities seems to be reinforced with age, as shown by structural work [31-331 and the analysis of antibody reactivities (Weksler et al., submitted). Forster et al. [31] have shown that 12 out of 14 Ly-1’ B cell hybridomas, obtained from transfers of adult CB20 peritoneal cells into newborn BALB/c mice and derived many months after selec- tion in the host, expressed VH genes of the 5558 family and

In conclusion, the present study indicates that the peritoneal Ly-1’ B subset in young adults uses all ten murine VH families, and shows a preferential utilization of the CP12 VH genes, most likely as a result of the local environmental selection during life.

We are grateful to Drs. C. Paige and M. Brahic for introducing us to the RNA colony blot assay and in situ hybridization techniques. We thank Drs. U. Krawinkel, J . Kearney, F. Ah, S. Riley and L. Reininger for VHprobes, Dr. H . Sauter for helpful suggestions and comments, M.-P. Lembezat for the expert technical assistance and J . Badella for secreta- rial expertise.

Received March 10. 1989.

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Note added in proof: We have recently confimred by in situ hybridiza- tion that the expression of CP12 V, genes is up to 40% of sorted Ly-l+ peritoneal B cells in adult B6 mice.

Received May 19, 1989.