Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the VH1 (T15) heavy chain
James J. Kenny,
Eric G. Derby1,
Jeffrey A. Yoder,
Shawn A. Hill1,
Randy T. Fischer,
Philip W. Tucker2,
J. Latham Claflin3 and
Dan L. Longo
National Institutes of Health, National Institute on Aging, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
1 Intramural Research Support Program/SAICFrederick, National Cancer InstituteFrederick Cancer Research and Development Center, Frederick, MD 21702, USA
2 Institute for Cellular and Molecular Biology, University of Texas at Austin, ESB-534, Austin, TX 78712, USA
3 Department of Microbiology and Immunology, University of Michigan Medical School, Building II, M6740 Medical Sciences, Ann Arbor, MI 48109, USA
Correspondence to:
J. J. Kenny
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Abstract
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Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM+ BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few VH1+ splenic B cells. These data suggest that the higher affinity T15-id+ B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the µMT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic VH1+ PC-specific B cells increased up to 5-fold and T15-id+ B cells were detectable in the spleen of T15H mice. This implies that T15-id+ PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.
Keywords: antigen binding, B lymphocytes, generation of diversity, H chain transgenic mice, idiotype, positive selection, phosphocholine
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Introduction
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The peripheral B cell repertoire has been highly selected both by clonal deletion of bone marrow (BM) cells bearing high-affinity sIgM receptors to autoantigens and by expansion of B cells with low-affinity receptors for environmental or self-antigens (reviewed in 13). Autoreactive B cells recognizing soluble self-antigens are not deleted in the BM but down-regulate their sIgM receptors before migrating into peripheral lymphoid tissues. The half-life of these anergized B cells is shortened via antigen-mediated trapping in the T cell zones of lymphoid tissues (1,4,5); thus, self-reactive B cells lose out in this competition to normal, unactivated B cells and they rapidly disappear from the peripheral B cell pool. In the absence of competition from normal B cells, autoreactive B cells exhibit a normal lifespan and some long-lived, autoreactive B cells appear to take up residence or at least become concentrated in the marginal zone of the follicles (6). High-affinity autoreactive B cells can escape clonal deletion by editing the expression of their L chain genes (7,8) and thereby differentiate into B cells expressing receptors with non-self specificities. In the presence of T cell help, low-affinity, autoreactive B cells may also be rescued from clonal deletion (9), or they may differentiate into B1 B cells (1012) or plasma cells (13) after encountering exogenous or endogenous thymus-independent antigens. Autoreactive, self-renewing B1a or B1b lymphocytes appear to preferentially home to the peritoneal cavity and up-regulate CD5 and/or CD11b (Mac-1) cell surface markers respectively (14,15). These self-reactive peritoneal B cells are responsible for the production of most of the IgM antibodies present in the serum of normal mice (16). Low-affinity autoreactive antibodies appear to play an important role as scavenger molecules which may prevent the subsequent induction of high- affinity auto-antibodies. High-affinity autoreactive antibodies produced by cells escaping to the peritoneal cavity can produce severe autoimmune hemolytic anemia (14,15).
The mechanisms and requirements for positive B cell selection are less well elucidated than those for negative selection, but recent studies indicate that signals mediated through sIgM, via the Bruton's tyrosine kinase (btk) pathway or via CD40, play an important role in the differentiation of immature B cells into long-lived, CD23+, resting B cells (17,18). Hayakawa and her collaborators (19) have also demonstrated that autoreactive B cells specific for the CD90 Thy1 glycoprotein are positively selected by self-antigen and differentiate into CD5+ B cells. Previous studies from our laboratory on mice expressing a transgene encoding the M167 H chain suggested that phosphocholine (PC)-specific B cells are positively selected into peripheral lymphoid organs of normal mice via an antigen-driven, receptor-mediated process. Thus, in mice co-expressing a normal btk and the M167 H chain transgene, we found a 100-fold higher expression of PC-specific B cells than would be expected from random H + L association. However, these same B cells were clonally deleted or failed to be selected in X-linked immune-deficient (Xid) mice, which express a mutated btk gene (20,21). We concluded that a PC-containing autologous or environmental antigen was driving the selection and expansion of these M167H:
24L PC-specific B cells in normal mice but leading to their clonal deletion in Xid mice. When the M167H:
24L anti-PC transgenes were crossed into Rag-2 knockout (KO) mice, the PC-specific B cells arrested at the transition from pre-B to immature B cells. These PC-specific B cells could be rescued from clonal deletion via co- expression of additional H and/or L chains. These data demonstrated that B cells expressing PC-specific Ig receptors are autoreactive and they use receptor dilution (decreasing the density of autoreactive binding sites) to escape tolerance.
Our laboratories (2224) as well as others (2527) have demonstrated that PC-specific antibodies are produced by variant forms of the VH1 H chain gene product in association with certain L chains. Each VH1 variant expresses a different VD junction and pairs with a different L chain to form a PC-binding antibody: the germline T15 (Asp95H) VH1 H chain associates with the
22 L chain, the M603 (Asn95H) variant associates with the
8 L chain, the D16 (Gly95H) variant associates with the
1C L chain and the M167/M511 (Ala96H) variants associate with the
24 L chain. To elucidate whether a PC antigen was responsible for amplification of the M167H:
24L B cells seen in the M167H transgenic mice, we produced additional VH1 H chain transgenic mice each expressing a different form of this VH1 gene. If a PC antigen is the positive selecting force, one should find a preferential selection and expansion of PC-specific B cells in which each H chain variant has associated with the endogenous L chain that creates a PC-specific binding site. Inasmuch as the T15H chain produces the highest affinity anti-PC antibody when associated with the
22 L chain and a very low-affinity anti-PC when associated with a
8 L chain, we also hoped to elucidate affinity limits for this apparent antigen-driven selective process. Recent work from our laboratory suggests that PC-specific antibodies are autoreactive but each family of antibody has a different affinity for PC; thus, we wanted to assess: (i) whether all the PC-specific H:L pairs would be positively selected into the peripheral repertoire and (ii) how the receptor affinity would influence this selective process.
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Methods
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Mice
Transgenic mice carrying the MOPC-167 (M167H) µ-only transgene (line 243-4, designation Tg[Igh]Bri35) (28) were obtained from Dr U. Storb (University of Chicago, Chicago, IL) through Dr R. L. Brinster (School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA). These mice have been backcrossed to C57BL/6 mice for more than 12 generations. Three additional strains of Tg[Igh] mice carrying variant forms of the V1 (T15) gene and a L chain strain (Tg[Ig
]) carrying the
22L gene (29) were produced by the Laboratory of Cell and Molecular Structure (SAIC, NCI-FCRDC, Frederick, MD). The T15µH (Asp 95H) gene (29) was mutated by site-directed PCR (30) to produce the M603H (Asn 95H) and M511H (Ala 96H) variants as described (22). Plasmid DNA for each of the V1 variant genes was cut with PvuI and XhoI, and the 17 kb fragment containing the 5'-VDJCµ-3' was isolated by agarose gel electrophoresis followed by passage over Elutip columns (Schleicher & Schuell, Keene, NH) according to the manufacturer's instructions. H chain DNA (5 ng) was injected into FVB/N oocytes and viable two-cell stage embryos transferred into the oviducts of pseudopregnant BDF1 female mice. Transgene-positive (TG+) founders were detected by PCR analysis of tail DNA. Each founder was crossed to normal FVB/N mice to determine germline transmission, and the peripheral blood of the progeny was stained with FITC-conjugated anti-VH1 antibody (hybridoma T68.3) (31) and phycoerythrin (PE)-conjugated anti-B220 to confirm B cell surface expression of the transgene-encoded H chain. L chain DNA was injected into oocytes from B6C3HF2 mice and TG+ founders were detected by PCR analysis of tail DNA using 5'V
22 and 3'J
5 primers. A single TG+ founder which expressed
22L on peripheral blood B cells was selected by staining with anti-T15-id antibody from clone T139.2 (31). This mouse was backcrossed to C57BL/6 mice to establish the
22L line.
8L transgenic mice were produced by the University of Michigan Transgenic Animal Model Core (Ann Arbor, MI) (32,33). One founder expressing four to five copies of the transgene was backcrossed to BALB/c mice. C57BL/6 µMT/µMT mice (34,35), which fail to produce B cells because they cannot insert there rearranged µH chain into the cell membrane, and T15i knockin (KI) mice (36) were obtained from Dr K. Rajewsky (University of Cologne, Cologne, Germany) through Dr C. Sidman, (University of Cincinnati, Cincinnati, OH) and Dr H. Gu (NIAID-NIH, Bethesda, MD) respectively. Each of the VH1 variant H chain transgenic mice were crossed and backcrossed to the µMT mice to obtain T15HµMT, M603HµMT, M511HµMT and M167µMT mice respectively. F1 mice expressing both the T15H and
8L or
22L were produced by crossing the respective parental lines and staining peripheral blood B cells with PEanti-VH1-id and FITCPC-dextran (PC-Dex). F1 H + L transgenic mice from M511Hx
24L matings were selected by staining with FITCanti-VH1 and the
24L-specific PEanti-M167-id (clone 28-6-20) (20).
Flow cytometric analysis
Spleen, BM and peritoneal cells (PerC) from TG+ and TG littermates were prepared and stained as described (20,37). PE-conjugation of anti-VH1, anti-T15 (clone T139.2) (31), anti-M167-id (clone 28-6-20) (20) and anti-CD23 (38) was carried out by Molecular Probes (Eugene, OR). FITC-conjugated PC-Dex was a gift of Dr H. Dintzis (The Johns Hopkins University, Baltimore, MD) and was used at a final concentration of 2.5 µg/106 cells. The synthesis of PC-Dex was as previously described (39). Stained cells were analyzed using a FACScan equipped with Lysys II software and 5% probability contour profiles were generated using CellQuest graphics software (Becton Dickinson, San Jose, CA).
Immunization and analysis of immune response to PC
Mice were immunized i.p. with 100µg of 6-(O-phosphocholine)hydroxyhexanoate conjugated to keyhole limpet hemocyanin (PC-KLH) (40). Five days post-immunization, single spleen cell suspensions were prepared and assayed for anti-PC antibody-secreting cells (ASC) using an ELISPOT assay developed by S. Wells and A. Stall (Columbia University College of Physicians and Surgeons, New York, NY). In brief, 50 µl serial dilutions of spleen cells in RPMI 1640 medium were put into duplicate wells of flat-bottom 96-well plates which had been coated with 100 µl of PC-conjugated BSA (5µg/ml) and blocked with BSA. Plates were incubated at 37°C for 45 h in a 5% CO2 incubator, washed 5 times with PBS containing 3% BSA and 0.05% Tween 20, and developed by the addition of biotin-labeled antibodies, followed by avidinalkaline phosphatase each for 1 h at room temperature. Final color development was achieved using 5-BCIP in 2-AMP buffer (Sigma, St Louis, MO) according to the manufacturer's recommendations.
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Results
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B cell development in the BM and spleen of mice expressing variants of the VH1 H chain gene
One TG+ founder, which both transmitted and expressed the transgene-encoded product on the surface of its B220+ peripheral blood lymphocytes, was selected to establish a line for each VH1 variant. The development of B cells in the BM and spleen of each of our three H chain variants, as well as the M167 H chain line (243-4) produced by Storb et al. (28), was analyzed by flow cytometry, and the absolute number of both B cells and total lymphoid cells determined. The data in Table 1
show that the total number of BM cells in all four lines of H chain transgenic mice is not significantly reduced compared to the C57Bl/6 and FVB/N controls. On the other hand, the T15H, M603H and M511 H chain mice exhibit a significant reduction in both BM and splenic sIgM+ B cells (Table 1
, and Figs 1 and 2
). The T15H line exhibits the most profound alterations at all stages of B cell development with a >30-fold reduction in BM and splenic B cells, and an 11-fold reduction in pre-B cells (Fig. 1 A
). BM pre-B and B cell development are not significantly reduced in the M167H line (Fig. 1 D
); however, the absolute number of splenic B cells is significantly lower than that found in the C57BL/6 (TG littermate) control (Table 1
). The FACS profiles of B220:IgM stained BM cells shown in Fig. 1
(AF) indicate that not only the number of µ+ cells but the level of sIgM on the B cells from T15H, M603H and M511H mice (Fig. 1 AC
) is reduced compared to the FVB/N control. This indicates that many of the B cells are being arrested in their maturation at an early stage of BM development. The FACS profiles of VH1:IgM-stained BM B cells in column 2 of Fig. 1
(GJ) show that the VH1 variant gene product is expressed on virtually all the sIgM+ BM B cells of each transgenic strain. Since the FVB/N mouse carries the µa allotype at its endogenous H chain locus, we could not directly determine the number of B cells in the T15H, M603H or M511H mice which co-expressed endogenous sIgM. However, Fig. 1
(MP) show that very few BM B cells in these µ H chain transgenic mice express endogenously encoded sIgD. Thus, all the strains of VH1 variant mice appear to carry out H chain allelic exclusion at the level of the BM. There are no detectable PC-specific B cells present in the immature (B220lo:sIgM+) B cell population of the BM (data not shown).

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Fig. 1. FACS analysis of BM from VH1 H chain transgenic mice reveals excellent allelic exclusion, but suggests antigen-induced down-modulation of receptors. BM cells from T15H, M603H, M511H and M167H transgenic mice and normal control mice were prepared, stained and analyzed on a FACScan as described in Methods.
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Fig. 2. FACS analysis of spleen cells from VH1 H chain transgenic mice reveals poor allelic exclusion of endogenous Ig and restricted antigen-driven selection of PC-specific B cells. Spleen cells from T15H, M603H, M511H and M167H transgenic and normal control mice were prepared, stained and analyzed on a FACScan as described in Methods.
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B cell selection in the spleen of mice expressing variants of the VH1 H chain
As shown in Fig. 2
(column 1), the percent of sIgM+:B220+ B cells present in the spleens of T15H, M603H and M511H transgenic mice is reduced 2- to 4-fold compared to the FVB/N control, whereas the absolute number of B cells is reduced from 4- to 40-fold (Table 1
). On the other hand, the percent of sIgM+ B cells present in M167H transgenic mice is equivalent to that of control mice and the absolute number is only slightly reduced. When one analyzes these same B cells for the expression of the VH1 H chain (Fig. 2
, column 2) or for the expression of endogenous sIgD (Fig. 2
, column 3), one sees that the majority of splenic B cells in the T15H (Fig. 2B
) and M511H (Fig. 2
J) variant mice do not express the VH1 transgene-encoded H chain product but express endogenous sIg receptors (Fig. 2C and K
). The majority of B cells in the M603H (Fig. 2F
) and M167H (Fig. 2N
) variant mice express the VH1 transgene-encoded H chain product on their surface, but many of these cells also co-express endogenous sIg (Fig. 2G and O
). In the M167H mouse, >50% of the splenic B cells have been shown to express the endogenous µb allotype (37,41), whereas all the immature BM B cells express only the transgene-encoded µa allotype. Thus, in all lines of VH1 variant mice, there appears to be positive selection for splenic B cells expressing endogenous sIg. When the spleen cells from these same four strains of VH1 variant mice were analyzed for their ability to bind PC (Fig. 2
, column 4), elevated numbers of PC-specific B cells were present in the M603H (5%, Fig. 2H
) and M167H (6%, Fig. 2P
) mice but not in the T15H (Fig. 2D
) or M511H (Fig. 2L
) mice. The lack of PC-specific B cells in the T15H spleen is not surprising because few B cells expressing the VH1 gene product have been selected into the periphery of these mice. The lack of PC-specific B cells in the M511H spleen may be because they have such low affinity for PC that they are not detectable by FACS (see below). The fact that large numbers of PC-specific B cells are present in the spleen but not in the BM of M603H and M167H mice indicates that these B cells have been positively selected into and expanded within the splenic population. Previous data from our laboratory has shown that this is accomplished via an antigen-driven, receptor-mediated process (20) and J. Kearney has recently shown that the M167H:
24 PC-specific cells that develop in M167H transgenic mice are expanded after reaching the spleen and develop into B cells which reside in the marginal zone of splenic follicles (J. F. Kearney and F. Martin, unpublished abstract).
Expansion of T15-id+, PC-binding B cells in the peritoneal cavity of T15H mice
In normal mice, T15-id+ B cells reside mainly in the B1a or CD5+ B cell subset (42), which is found primarily in the peritoneal cavity. Reduced numbers of CD5+ VH1+ B cells are present in the peritoneal cavity of gene targeted T15i KI mice (36), but the number of T15-id+ PC-binding B cells within this B1 subset was not determined. Many of the receptor specificities expressed preferentially within the B1 subset are thought to be autoreactive (16,43) and self-reactive B cells have been shown to escape clonal deletion by residing in the peritoneal cavity (14,15). Autoreactive PC-specific M167-id+ B cells also escape clonal deletion in the peritoneal cavity of Rag-2 mice expressing M167H:
24L transgenes (J. J. Kenny et al., submitted). We therefore analyzed the peritoneal cells from T15H mice to determine if these potentially autoreactive T15+, PC-specific B cells were sequestered in this anatomical location. Approximately 5% of the peritoneal VH1+ cells were T15-id+ and bound PC-Dex but such cells were absent in the spleen (Fig. 3
); however, these cells also lacked both CD5 and CD23 (data not shown), and thus may represent B1b cells or marginal zone cells. When T15i KI mice are crossed to
22 transgenic mice, the resulting T15-id+, PC-binding B cells look like splenic marginal zone cells rather than B1 B cells; thus, they were long-lived CD23CD21hiCD38+ cells (6) (J. J. Kenny, unpublished observation). We have also recently shown that PC-specific B cells in M167H:
24L Rag-2 KO transgenic mice arrest their development at the transition from pre-B to immature B cells (J. J. Kenny et al., submitted). This is the point in development where autoreactive anti-DNA, anti-H-2b and anti-HEL B cells (1,5,4448) arrest and undergo receptor editing (7,8,4951). In Rag-2 KO mice, receptor editing is not possible, and the PC-specific B cells undergo clonal deletion in the BM and spleen, but they escape deletion in the peritoneal cavity where they develop into B1 B cells (J. J. Kenny et al., submitted).

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Fig. 3. FACS analysis of spleen and peritoneal cells from T15H transgenic mice shows that PC-specific B cells are present in the peritoneum but not the spleen. Spleen and PerC from T15H transgenic mice were prepared, stained and analyzed on a FACScan as described in Methods.
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B cell development in the BM and spleen of µ MT mice expressing variants of the VH1 H chain
Autoreactive B cells are filtered out of the peripheral repertoire in the T cell regions of lymphoid tissues (4) and thus fail to compete with non-autoreactive B cells. Surface IgM interactions with autoantigens can also induce receptor editing at the L chain locus (7,8) and induce rearrangements at the H chain locus in transgenic mice (J. J. Kenny et al., submitted). To elucidate whether the expression of endogenous Ig H chains altered the development and selection of PC-specific B cells in our H chain transgenic mice, we backcrossed these strains expressing VH1 variant genes into the C57BL/6 µMT/µMT background, a mouse that cannot produce endogenous sIgM due to a deletion in the transmembrane region of the endogenous µ gene (34,35). The spleen and BM of backcross (BC1) mice were then analyzed by flow cytometry to determine the number of B cells expressing the VH1 transgene-encoded H chain. BC1 mice which expressed a functional endogenous Ig locus (µMT/+) as determined by the presence of sIgD (VH1+:
+) were compared to homozygous µMT/µMT (VH1+:
) littermates. As seen in Table 2
, the total number of spleen cells is lower in all the µMT/µMT lines except the T15H µMT mice, whereas the sIgM+ B cell number is significantly reduced in all the µMT/µMT lines (P < 0.01) except the M167H µMT mice (P = 0.37). Figure 4
shows that all the B cells in these µMT mice express the VH1 transgene-encoded µ H chain (Fig. 4AD
) and lack endogenous sIgD as expected (Fig. 4EH
). Less than 15% VH1+ B cells are present in the spleens of T15H, M603H and M511H µMT/µMT mice, whereas the M167 µMT/µMT mice have normal numbers of B cells in their spleen. Thus, going from the founder's FVB/N genetic background to a predominantly C57BL/6 background did not normalize the number of B cells expressing these three VH1 H chain variants, suggesting that the regulation of VH1+ B- cell numbers is not controlled by unknown, non-Ig background genes. This has remained true through six backcross generations. The absolute number of VH1+ B cells present in each of the VH1 variant strains is also not controlled by the presence of B cells expressing endogenous sIg. The M603 µMT mice were the only strain showing a significant increase (P = 0.03) in VH1+ splenic B cell numbers when compared to their µMT/+ littermates (Table 2
); thus, B cell competition for limited space or follicular-derived survival factors are not controlling the homeostasis of VH1+ B cell numbers. Elimination of B cells expressing endogenous sIg, however, has a profound effect on the number of PC- specific B cells seen in some of these VH1 H chain variant mice (Fig. 5
). PC-specific B cells were undetectable in the T15H µMT/+ mice (Fig. 5A
), whereas 10% of the VH1+ B cells in the T15H µMT/µMT mice bound PC-Dex (Fig. 5K
) and expressed the
22L-specific combinatorial idiotope detected by anti-T15-id (clone T139.2) (data not shown). The PC-specific B cells in the M603H µMT/µMT mice increased 5-fold over that in the M603H mice (Fig. 5L
versus B) and the M167H µMT/µMT mice (Fig. 5N
) had 9% PC-specific B cells compared to 6% in their µMT/+ littermates (Fig. 5D
). There were no detectable PC-specific B cells in the M511H mice either in the presence or absence of endogenous sIg (Fig. 5C, H and M
). Since this H chain variant makes a low-affinity anti-PC antibody when combined with the
24 L chain (22), it was possible that its affinity for PC was below the level needed for positive selection, or that the number and/or affinity of these receptors did not allow detection of binding by FACS analysis. To test these possibilities, we crossed M511H mice with the
24 L chain transgenic mouse (strain 194-2) (28), and analyzed the spleens of the F1 male progeny for PC-binding and M167-id+ B cells. The H + L double-positive F1 mice expressed both the VH1 and M167 idiotopes, but they failed to bind detectable amounts of PC-Dex (data not shown). However, upon immunization of M511H µMT/µMT mice with PC-KLH, one can detect a low-level M167-id+, anti-PC response (Table 3
) indicating that some M511VH1:
24 PC-binding B cells are present but not detectable by FACS.

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Fig. 4. FACS analysis of spleen cells from VH1 µMT H chain transgenic mice shows increased positive selection of PC-specific B cells in the absence of B cells expressing endogenous H chains. Spleen cells from T15H, M603H, M511H and M167H µMT transgenic mice were prepared, stained and analyzed on a FACScan as described in Methods.
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Fig. 5. FACS analysis of spleen cells from VH1 µMT H chain transgenic mice demonstrates expression of the transgene-encoded VH1 H chain on all B cells in the absence of endogenous H chains. Spleen cells from T15H, M603H, M511H and M167H µMT transgenic mice were prepared, stained and analyzed on a FACScan as described in Methods.
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PC-specific B cells expressing the T15-idiotope are not dominantly selected into the peripheral B cell pool
T15-id+ (T15H:
22L) B cells dominate the PC-specific immune response of virtually all normal mice. The T15 H chain can also produce a low-affinity anti-PC specific antibody when it associates with a
8 L chain (22,52). In ELISA assays, this T15H:
8 antibody exhibits a 40-fold lower binding avidity for PC-BSA than the T15H:
22L antibody (22). Neither high-affinity T15H:
22L nor low-affinity T15H:
8L PC-Dex-binding B cells appeared to be positively selected into the spleens of our T15H transgenic mice (Figs 2 and 5
), but these mice expressed <1% VH1+ B cells in their spleen. We therefore analyzed the spleens of another T15H mouse, the gene-targeted T15i KI mouse developed by Taki et al. (36). Large numbers of VH1+ B cells can be seen in the spleens of both heterozygous and homozygous T15i KI (36) mice, and ~10% VH1+ PC-binding cells are detectable in these mice (Fig. 6A and B
). B cells bearing the T15-id represent <20% of this PC-specific subset of VH1+ cells in homozygous T15i mice (Fig. 6C
) and the L chain(s) expressed in the other 80% of PC-binding cells has not yet been determined since a combinatorial anti-id specific for
8-containing antibodies does not yet exist.
To show that large numbers of T15-id+ B cells are permitted to exist in the spleens of T15i mice, we crossed them to
22L transgenic mice and analyzed the spleen cells of H + L double-positive F1 progeny for B cells expressing T15-id and for PC-Dex binding cells (Fig. 6E and F
). These data show that virtually all the VH1+ splenic B cells bound PC-Dex (Fig. 6E
) and the same number (20%) were T15-id+ (Fig. 6F
). The majority of these VH1+ B cells are neither mature, resting CD23+ B cells nor CD5+ B cells (Fig. 6G and H
). Their T15-id+ B cells appear to be CD21hi CD38+ marginal zone cells (unpublished data). These data demonstrate that T15-id+ B cells can persist in the spleen in the absence of B cells expressing other receptor specificities. However, the B cell subset into which these T15-id+ B cells have differentiated does not appear to be either the traditional B1 or B2 subsets (16) and appears to represent marginal zone B cells (6). These B cells also co-express endogenous H and/or L chains in addition to the transgene-encoded T15 H + L. We have hypothesized that they escape clonal deletion via co-expression of multiple receptors (receptor dilution) (J. J. Kenny et al., submitted). T15Hx
22L transgenic mice look exactly like the T15ix
22L transgenic mice except that they do not express the low levels of sIgD seen on the surface of the latter strain (J. J. Kenny, unpublished data). These T15-id+ B cells also fail to develop in T15H:
22L Xid transgenic mice (J. J. Kenny, unpublished data), which further indicates the autoreactive nature of anti-PC-specific B cells and their failure to be positively selected in the absence of receptor-mediated signals transmitted via the btk signaling pathway which is defective in Xid mice (21,39).
Immune response to PC-KLH in VH1 µ MT/ mu; T mice
We have demonstrated that transgenic mice expressing the M167 µ
anti-PC transgenes are capable of responding to the thymus-dependent antigen PC-KLH both in vivo (53) and in vitro (54). To determine whether the B cells present in our VH1+ µMT/µMT transgenic mice were capable of responding in vivo to PC-KLH, five to 10 mice of each variant H chain line, as well as C57BL/6 and M167 µ
207-4 control mice, were immunized with 100 µg of PC-KLH (40). Five days after immunization, the spleens were analyzed by ELISPOT for the number of B cells secreting antibodies specific for PC. The data in Table 3
show that after immunization every strain of VH1 µMT mice, including the M511H, exhibited at least a 10-fold increase in PC-specific ASC over the background level present in unimmunized mice. The T15H µMT produced the highest response in terms of both total number of ASC per spleen and number of ASC per 106 B cells. These T15H µMT mice also exhibited the greatest change (240-fold in VH1 ASC per spleen) between unimmunized and immunized spleens, whereas the M167µ
207-4 and the M167H µMT mice, which have the most PC-binding B cells per spleen, produced only a 2- to 10- fold increase respectively (Table 3
). The M511H and M603H µMT mice also showed marked increases in PC-specific ASC following immunization. Note, however, that the magnitude of the ASC response is not directly proportional to the number of antigen-binding cells present in the spleen before immunization. The magnitude of the response appears to be related more to the relative affinity for PC of the receptor expressed on the B cell (T15 > M603 ~ M167 > M511) (55).
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Discussion
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The accumulation of B lymphocytes in peripheral lymphoid tissues is not a random event determined by the stochastic rearrangement of Ig H and L chain V region genes in the BM. The development and homeostatic maintenance of the peripheral B cell repertoire is a dynamic process involving both the negative and positive selection of B cells. The studies presented in this manuscript were undertaken to elucidate how minor changes in the CDR3 region of the VH1 H chain gene influence both V region homeostasis and PC-specific B cell selection. To progress toward this goal, it is necessary to develop a working hypothesis that can explain our observations that: (i) H chain allelic exclusion was highly efficient in the BM, yet many B cells expressing endogenously encoded sIg-receptors appeared in the periphery (Figs 1 and 2
); (ii) the number of immature B cells emerging from the pre-B cell pool was greatly reduced in all the VH1 variants, and the amount of sIgM receptor appeared to be down-regulated on T15H, M603H and M511H BM B cells (Fig. 1
and Table 1
); (iii) the number of PC-specific B cells present in the spleens of T15H, M603H and M167H mice increased substantially when competition from B cells expressing endogenous sIgM was prevented (Fig. 5
)nevertheless, the absolute number of VH1+ B cells present in the spleens of three out of four of the VH1 variant mice was not significantly changed (Table 2
); (iv) M511H mice do not have detectable numbers of PC-specific B cells (Fig. 5
), but can produce id+, PC-specific antibodies following immunization (Table 3
); (v) T15-id cells dominated the PC-binding cells of gene-targeted T15i KI mice (Fig. 6
), yet splenic B cells expressing T15H:
22L receptors could exist in large numbers in T15ix
22L transgenic mice (Fig. 6
) and T15-id+ B cells could dominate the PC-specific B cells in the spleens of T15H µMT mice (Fig. 5
); and (vi) PC-specific B cells failed to develop in the spleens of VH1 H or H + L transgenic mice co-expressing the X-linked immune deficiency gene, xid (20,21,39).
PC-specific B cell development follows the pattern of autoreactive lymphocytes
The above observations may be explained by our working hypothesis: an endogenous PC-containing antigen, or a self-antigen that cross-reacts with PC, induces a receptor-mediated activation that will lead to either clonal deletion or anergy in the absence of T cell help. Many of these autoreactive, PC-specific B cells can escape from clonal deletion by co-expressing endogenous H and/or L chains or by differentiating into B1 B cells and taking up residence in the peritoneal cavity. T15-id+ B cells were detected only in the peritoneal cavity of T15H mice until endogenous Ig expression was eliminated (Fig. 5
). B cells expressing T15H:
22L receptors have the highest affinity for PC (~5x105), M603H:
8 and M167H:
24 receptors have a slightly lower affinity (55), and those expressing M511H:
24 receptors have at least a 40-fold lower avidity (22). Thus, according to our hypothesis, when the T15-id+, M603-id+ and M167-id+ B cells encounter endogenous PC antigen, they will be partially activated and undergo clonal deletion at check point #1 in the BM or at check point #2 in the T cell zones (PALS) of the peripheral lymphoid tissues (1). By reactivating their Rag genes in order to express endogenous H and/or L chain genes, these B cells would lower the number of functional PC-specific receptors below the threshold needed to obtain sufficient receptor cross-linking to induce apoptosis in the BM. These dual receptor-expressing B cells would then migrate into the periphery, where the continued ligation of their receptors by autoantigen would result in their selective expansion. Alternatively, H chain transgene expression would be suppressed and a B cell expressing only the endogenous H chain produced. Both developmental schemes would account for the fact that most of the B cells in the periphery of all the VH1 variant mice expressed only endogenous sIg or both endogenous and transgene- encoded sIg. The double-expressing PC-specific B cells in the periphery would still bind self-antigen and their migration to B cell follicles would be delayed or prevented. Thus, these PC-specific B cells would be out-competed by B cells expressing only endogenous sIg receptors or by VH1+ B cells with low or no affinity for self-antigens. That this second check point in the PALS plays a major role in controlling the level of PC-specific B cells in most of our VH1 H chain variant mice was demonstrated by the dramatic increase in PC-specific cells seen in all VH1 H chain µMT mice except the M511 µMT where the receptor affinity for PC is apparently too low to induce positive selection. Thus, PC-specific B cells went from undetectable in the T15H mice to 10% of VH1+ B cells in the T15H µMT mice and M603H PC-Dex binding cells went from 5 to 30% of the splenic B cells in the absence of endogenous H chain expression. This increase in positive selection for PC-specific B cells occurred without an increase in the absolute number of VH1+ B cells. In T15H mice, only those T15-id+, PC-specific B cells that differentiated into B1 B cells and homed to the peritoneal cavity following receptor cross-linking survived in the presence of large numbers of normal B cells. Thus, T15-id+ B cells compete poorly with endogenous B cells in the peripheral lymphoid tissues. Our working hypothesis is further supported by the observation that peritoneal B cells in several lines of transgenic mice co-express both endogenous and transgene-encoded H chains (56,57), and by the fact that high-affinity autoreactive B cells have been shown to escape clonal deletion by homing to the peritoneal cavity (14,15). Hayakawa et al. (19) have also shown that autoreactive B cells specific for the Thy1 CD90 glycoprotein get positively selected and amplified in the peritoneal cavity of transgenic mice expressing a VH3609 H chain. In this situation, it is possible that the T cells supply co-stimulatory signals or activation factors to the B cells binding to their surface.
Testing autoreactivity in the Rag KO mouse
The µ
anti-TNP transgenic mice developed by Rusconi and Kohler (58), whose transgene-encoded receptors cross-react with DNA, cannot develop B cells in Rag-deficient animals where endogenous L chains cannot be rearranged (13). B cells expressing anti-PC, M167H:
24L receptors also undergo clonal deletion in the BM of Rag-2 KO mice (J. J. Kenny et al., submitted). PC-specific CD5+ B cells develop in the peritoneal cavity of these mice but these cells appear to be anergic in that there is no circulating anti-PC antibody present in their serum. Based on our working hypothesis, we would also predict that the reason homozygous T15i KI mice, which express few or no endogenous VH regions, do not have large numbers of T15-id+, PC-specific B cells in their spleens is because the binding of endogenous PC antigen by T15H:
22L receptors reactivates the Rag genes, which in turn causes the recombinase-mediated destruction of both JH locus inserted T15V genes. Since the T15V region in our T15H transgenic mice is not located at the H chain locus, it cannot be readily targeted by the recombinase for V gene replacement. Thus, in the T15H µMT mice, 10% of the B cells express T15H:
22L receptors; however, note that PC-Dex binding is low (Fig. 5
). Thus, these cells probably co-express a second
L chain which would lower the functional receptor number and consequently raise the deletional threshold following binding to autoantigen. Preliminary PCR data indicate that several other L chains in addition to the
22L are expressed in these T15-id+, PC-binding B cells (L. Rezanka and J. J. Kenny, unpublished data). In (T15ix
22L)F1 mice, where all the VH1+ B cells are T15-id+ and PC specific, we have observed that most of the B cells co-express endogenous H chains (J. J. Kenny et al., submitted). Thus, this rearrangement of endogenous H chain may be raising the threshold for clonal deletion by creating mixed H chain molecules and hence lowering the number of functional PC-specific receptors.
In the absence of competition from B cells expressing endogenous sIg receptors, one would expect transgenic mice expressing low-affinity autoreactive B cells to have normal numbers of VH1+ B cells in their spleens since these cells would no longer be deleted at check point #2. However, all the VH1 variant µMT mice expressed approximately the same number of VH1+ B cells as their normal VH1+ µMT/+ littermates. Thus, the homeostatic level of total B cells in each VH1 variant appeared to be regulated by the form of the VH1 gene being expressed; we cannot rule out the possibility that the integration site of the Ig transgene is also having an effect. It is clear, however, that the genetic background in which the transgene is being expressed does not alter the level of expression of each VH1 variant. We are currently trying to determine whether the number of endogenous L chains that can pair with each of the VH1 variant H chains might be limited. If the association of H and L chains is also restricted, the potential diversity of the B cell repertoire could be highly restricted. The restriction in H:L association may account for the finding that very few B cells appear to emerge from the pre-B cell pool in the T15H, M603H and M511H mice. This restricted development could also be due to the possibility that most of the H + L combinatorial associations formed with these VH1 variants may be highly autoreactive and deleted at check point #1 in the BM. Preliminary data from 15 HxL Rag-2 KO transgenic strains indicates that approximately two-thirds of these H:L combinations give rise to autoreactive specificities (J. J. Kenny, unpublished data). Inasmuch as the pre-B cell pool was significantly reduced only in our T15H mice, we do not feel that the lack of VH1+ BM B cells is due to a failure of these H chains to associate with and signal through the surrogate L chain receptor complex.
Overall, the results presented in this study suggest that the functional B cell repertoire may be highly restricted in usage of H + L chain combinations and that this loss of receptor heterogeneity is primarily due to autoimmunity. Low-affinity autoreactive B cells may avoid negative selection by rearranging additional H and L chains. This dual expression of H or L chains lowers the number of functional receptors and thus raises the threshold for negative selection. The autoreactive B cell can then be positively selected and expanded by the autoantigen. After positive selection, the homeostatic level of autoreactive B cells is controlled by competition with other B cells.
 |
Acknowledgments
|
---|
This work was supported in part by ACS grant no. IM 664 to J. L. C. and NIH grant AI-18016 to P. W. T.
 |
Abbreviations
|
---|
ASC antibody-secreting cells |
BC backcross |
BM bone marrow |
btk Bruton's tyrosine kinase |
KO knockout |
KI knockin |
PE phycoerythrin |
PC phosphocholine |
PC-Dex PC-conjugated dextran |
PC-KLH 6-(O-phosphocholine)hydroxyhexanoate-conjugated keyhole limpet hemocyanin |
PerC peritoneal cells |
TG transgene |
 |
Notes
|
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Transmitting editor: J. J. Kenny
Received 21 January 2000,
accepted 21 February 2000.
 |
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