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The Journal of Immunology, 2003, 171: 1192-1201.
Copyright © 2003 by The American Association of Immunologists

Antibody Repertoire and Gene Expression Profile: Implications for Different Developmental and Functional Traits of Splenic and Peritoneal B-1 Lymphocytes 1

Karsten Kretschmer2,*, Anke Jungebloud*, Jana Stopkowicz*, Britta Stoermann*, Reinhard Hoffmann3,{dagger} and Siegfried Weiss*

* Molecular Immunology, German Research Center for Biotechnology, Braunschweig, Germany; and {dagger} Basel Institute for Immunology, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In L2 mice, a high expression level of the transgenic {lambda}2315 L chain results in nearly complete exclusion of endogenous L chains and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum. The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in functional properties.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mature preimmune B cell compartment of adult mice consists of at least four subpopulations, each of which exhibits specific developmental and functional characteristics: recirculating follicular B cells (or B-2 cells) representing the major B cell subpopulation of lymphoid follicles, marginal zone (MZ)4 B cells enriched in the MZ surrounding the splenic lymphoid follicles, and B-1 lymphocytes, which can be subdivided into B-1a cells expressing CD5 and the CD5- B-1b sister population (1, 2).

Physiologically, B-1a cells are more dependent on signals via the B cell receptor (BCR) than are B-2 cells (2). In agreement, the generation and/or maintenance of B-1a cells is affected when signals via the BCR are dampened due to natural mutations or gene knockouts of BCR components, whereas inactivation of negative regulators of the BCR complex increases B-1a cell numbers. Consistently, in transgenic (Tg) mice in which B cells display a high BCR density, primarily B-1a cells are found (3, 4, 5).

The development of B-1a cells is also influenced by the specificity of their Ag receptors. Expression of a Tg BCR comprising a B-2-associated specificity results in a decrease or even loss of B-1a cells, whereas Tg expression of a B-1a-derived BCR leads to an increase of B-1a cells (2). The requirement for continuous BCR signaling and characteristic features of the Ab repertoire distinguishes B-1a cells from other B cell subsets (6, 7, 8, 9, 10). Such characteristics are paralleled by specific differences in cellular functions. B-1a cells produce the majority of natural Abs that play a nonredundant role in providing early protection against various pathogens (2, 11).

Although B-1a cells represent a minor B cell subpopulation in the spleen of normal mice, their absolute number is similar to the number of B-1a cells found in the peritoneum (12). Both B-1a compartments appear to differ in the frequencies of cells specific for bromelain-treated mouse RBCs (BrMRBCs) and ssDNA (13). The molecular basis for such differences is still unclear, because a comprehensive molecular analysis of the VHDJH repertoire of splenic B-1a cells has not been performed so far.

Except for differences in the expression levels of surface IgM and B220 (14) and differential expression of Mac-1 (12), the presently known surface marker phenotype of splenic and peritoneal B-1a populations is identical. Only a few molecular differences between these two B-1a subpopulations have been described. Peritoneal B-1a cells constitutively exhibit nuclear ptyrSTAT3, whereas splenic B-1a cells do not (15). CCR2 and CCR3 transcripts have been found in splenic, but not in peritoneal B-1a cells (16).

The acquisition of high surface IgM levels on splenic VH11V{kappa}9 Tg B-1 cells after i.p. adoptive transfer suggests that the phenotypical and physiological differences between splenic and peritoneal B-1a cells are at least partially influenced by the specific environment (14). Such differences could be due to local environmental influences on migrating B-1 cells. Alternatively, the developmental program of resident B-1 cells could determine the phenotype. In this regard, a remaining question is whether an exchange takes place between B-1a cells of the spleen and peritoneum. The potential of peritoneal B-1a cells to migrate in response to B lymphocyte chemokine (B lymphocyte chemokine/CXC chemokine ligand 13) has been demonstrated recently: B-1a cells of CXC chemokine ligand 13-deficient mice failed to home to the body cavities (17). In the same study, a significant degree of exchange of peritoneal B-1a cells between parabiosed partners was observed. Whether the peritoneal and splenic B-1a compartments are also interrelated by exchange was not addressed in this study, although a partial loss of peritoneal B-1a cells in splenectomized mice suggests a role of the spleen for maintenance of such cells (18).

We have described the mouse line L2, Tg for the {lambda}2315 L chain previously (3, 19). The peripheral B lymphocyte compartments of L2 mice are predominantly made up of IgMhighIgDlowCD5+ B-1 cells that exclusively express the Tg {lambda}2315 L chain (3). Splenic and peritoneal CD5+ B cells of such mice exhibit all characteristic markers of B-1a cells (3). Consistent with a severe block in B cell development in bone marrow (BM) of adult L2 mice, C1qRp+ (AA4.1, 493) immature B cells are below detection level in the spleen. The almost exclusive expression of a single L chain restricts the IgH chain repertoire of peritoneal B-1a lymphocytes in adult L2 mice, as well as of fetal and neonatal liver-derived B cells. This pronounced oligoclonality, characterized by a high frequency of reoccurring identical amino acid sequences, was attributed to strong positive selection mediated by (auto)Ags (19). We now extended our analysis of L2 mice by comparing the Ab repertoire and gene expression profile of B-1 cells from different anatomical locations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

L2 mice (3), backcrossed on the BALB/c background for 20–25 generations, non-Tg littermates, and IE{kappa}T mice (20) were used at 8–10 wk of age. L2 mice of the IgHb allotype were obtained by crossing L2 mice of the IgHa allotype to CB20 mice. RAG-1-deficient mice on BALB/c background were obtained from The Jackson Laboratory (Bar Harbor, ME).

Flow cytometry and cell sorting

Single-cell suspensions were obtained by flushing the peritoneal cavity and spleen with IMDM. Blood obtained by cardiac puncture was collected in PBS containing heparin (50 U/ml) followed by separation on Ficoll gradients. For each sorting experiment, pools of cells from three to five mice were used unless stated otherwise.

mAb against CD72, CD80, VCAM-1, CD1, IgMa (DS-1), IgMb (AF6-78), CD5 (53-7.3), and B220 (RA3-6B2) were obtained from BD PharMingen (San Diego, CA). The Ab to CD19 (1D3) was homemade following standard protocols. Flow cytometry was conducted using a FACSCalibur and CellQuest software (BD Biosciences, Mountain View, CA). Cell sorting was performed on a MOFLO (Cytomation, Fort Collins, CO) or a FACSVantage (BD Biosciences).

Phosphatidylcholine (PtC) liposomes were prepared using a protocol essentially identical with methods described previously (8). PtC liposome specificity was confirmed by IgH chain sequence analysis of sorted PtC+ peritoneal B-1a cells from normal BALB/c mice (8).

Amplification of VHDJHµ chain transcripts, sequencing, and sequence analysis

Amplification, cloning, and sequencing of IgH µ chain transcripts from bulk sorted IgM+CD5+ cells was done as described previously (19). For single-cell RT-PCR, the Qiagen (Valencia, CA) OneStep RT-PCR kit was used. B cells were first bulk sorted and then sorted again to deposit single cells into strips of eight PCR tubes containing 15 µl of RNase-free water plus the supplied buffer and 5 U of RNAsin. This two-step sorting process ensures deposition of single cells by exclusion of doublets. As a negative control, one of the eight tubes did not receive a cell but was otherwise processed like tubes containing single cells. Samples were freeze-thawed three times (-20°C/65°C) and put on ice, and a mix containing RNase-free water, dNTPs at 400 µM each, 1.7 µl of Qiagen OneStep RT-PCR enzyme mix, the oligonucleotides VHcons and Cµ1 (VHcons, 5'-GAGGTGCAGCTGCAGGAGTCTGG-3'; Cµ1, 5'-ATGGCCACCAGATTCTTATCAGA-3') (both at 0.4 µM), and the supplied buffer were added to a final volume of 30 µl. Reverse transcription was performed at 50°C for 45 min followed by 95°C for 15 min. PCR conditions were as follows: 94°C for 15 s, 50°C for 1 min, 68°C for 1 min for 10 cycles, followed by 94°C for 15 s, 50°C for 1 min, 72°C for 30 s for 15 cycles, with final extension at 72°C for 15 min.

For the second amplification, 1 µl of first-round RT-PCR product was added to a 20-µl mix containing each dNTP at 200 µM, 1.5 U of HotStarTaq (Qiagen), the supplied buffer, and the VHcons oligonucleotide in combination with an internal constant region primer (Cµ2, 5'-CATTTGGGAAGGACTGA-3') each at 0.2 µM. Conditions for the second round of amplification were as follows: 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s. PCR was conducted for 35 cycles followed by incubation at 72°C for 10 min. PCR products were gel purified and then sequenced directly using the oligonucleotide Cµ2.

Generation of cRNA and microarray hybridization

All of the experiments were performed in triplicate. For each replicate experiment, cells were isolated from different pools of mice, with the exception of B-2 cells, which were isolated from the same pool of mice. A total of 150,000 cells of each B cell subpopulation was sorted directly into TRIzol. RNA amplification and hybridization of Affymetrix GeneChip Probe arrays were done as previously described (21). Affymetrix U74Av2 high-density oligonucleotide arrays, interrogating 12,488 transcripts, were hybridized, washed, stained, and scanned according to the manufacturer’s specifications. Scanned raw data images were processed with Affymetrix GeneChip, version 4.0, software. Arrays were normalized to correct for variations in fluorescence intensities using average difference values and the DNA chip analyzer software (dChip) according to the author’s instructions (22). Then, the hybridization background was calculated, and all gene expression values below 61 were set to 61 to eliminate low-level signals. Assessment of differentially expressed genes was done by a nonparametric, permutation-based method using the program significance analysis of microarrays (SAM) (23). To control for multiple testing, a false discovery rate (24) was calculated as the percentage of false-positive genes among all genes detected as differentially expressed. The q value used by SAM is similar to the familiar p value, but adapted to the analysis of a large number of genes, and represents the lowest false discovery rate at which the gene is called significant. Genes were considered to be differentially expressed if they were detected at a false discovery rate of 0.15%, which was the highest false discovery rate by which the number of falsely called genes was <1. Additionally, only transcripts that were scored as positive (perfect match intensity > mismatch intensity), showing a change of at least 2-fold and a difference of at least 200 average difference units, were considered to be differentially expressed. Cluster analysis of either all transcripts present on the Affymetrix U74Av2 high-density oligonucleotide arrays or exclusively differentially expressed genes was performed according to Eisen et al. (25).

The following oligonucleotides were used for the validation of the array-based expression of some transcripts in cluster 6 by RT-PCR: SpiC, forward, 5'-CAGAGGCAACGCTAACTACTATGG-3', and SpiC, reverse, 5'-CAGCTTTCTCCGGATTTTTATGAT-3'; NAD, forward, 5'-GCAGGCGTGAACAATGAGAAAAAC-3', and NAD, reverse, 5'-CACAAAGCCTGGGCAAATGACA-3'; CCR3, forward, 5'-CTCGCTATCCAGAAGGTGAAGAAG-3', and CCR3, reverse, 5'-GTGGAAAAAGAGCCGAAGGTGTT-3'; CD206, forward, 5'-TTTTACGAGAAGTTGGGGTCAGG-3', and CD206, reverse, 5'-TTTGGTCAGCTTTGGTTGTAATGG-3'; VCAM-1, forward, 5'-TCCCTAATGTGTATCCCTTTGACC-3', and VCAM-1, reverse, 5'-AGGCTGCAGTTCCCCATTATTTAG-3'; LPL, forward, 5'-CGAGAGGATCCGAGTGAAAG-3', and LPL, reverse, 5'-CTAATGCTGGAAGACCTGCTATG-3'; CD5, forward, 5'-AGAAAAAGCAGCGTCAGTGGATTG-3', CD5, reverse, 5'-TCAGGATGGAGGGGATGC-3'; and HPRT, forward, 5'-GCTGGTGAAAAGGACCTCTCG-3', and HPRT, reverse, 5'-CTTGCGCTCATCTTAGGCTTTG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The predominance of B-1a cells exclusively expressing the Tg {lambda}2315 L chain in the peripheral B lymphocyte compartments of L2 mice facilitates the comparison of B-1 subpopulations (3). In the peritoneum, IgMlowIgDhighCD5-Mac-I- B-2 cells are completely absent, whereas the few CD5- B cells are IgMhighIgDlowCD5-Mac-I+, thus showing the B-1b surface marker phenotype (supplementary Fig. 1 and data not shown). Similarly, B-1a cells represent the major B cell population in the spleen. The remaining minor population of IgM+CD5- B cells exhibit the CD23low/-CD21high MZ surface marker phenotype. 5



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  		FIGURE 1. BCR specificities overlapping between splenic and peritoneal B-1 cells. IgM+CD5+ B-1a cells from spleen and peritoneum and IgM+CD5- B-1b cells from peritoneum were isolated from a single L2 mouse by sorting and used for the amplification of IgH chain transcripts. B-1b cells could be identified as IgM+CD5-, because B-2 cells are completely absent (supplementary Fig. 1). Reanalysis revealed a purity of cells of >94%. The CDR3 nucleotide sequence and the corresponding amino acid sequence of IgH chains completely identical in amino acid sequence but recovered from different B-1 compartments are displayed. Many additional sequences differed only in a single CDR3 amino acid (data not shown). The frequency of plasmids (n), the VH gene family, the D element, if possible, the JH element, and the loss of JH nucleotides is indicated (VH, D, JH, and -JH). N/P indicates the addition of N and/or P nucleotides. Boxes indicate triplets that differ in their nucleotide sequence but encode an identical amino acid sequence. n.f., Not found. The complete sequence data are available from GenBank under accession numbers AY172346–AY172404 (peritoneal B-1a), AY172405–AY172437 (peritoneal B-1b), and AY172455–AY172509 (splenic B-1a).

 
The same Ab specificities can be selected in the splenic and peritoneal B-1a compartment

Earlier studies have shown that peritoneal B-1a cells from adult L2 mice exhibit a high frequency of reoccurring identical VHDJH amino acid sequences (19). We now performed a comparative analysis of splenic and peritoneal B-1 cells, isolated from a single L2 mouse. Fifty-two splenic B-1a, 48 peritoneal B-1a, and 26 peritoneal B-1b sequences were established, representing unique rearrangements that were distinguishable by their nucleotide sequences. Some of these IgH chains displayed identical amino acid sequences, although they were encoded by variant nucleotide sequences (Fig. 1, groups A and C). Thus, such B-1 cells were descendants of different clones. Some identical VH sequences are shared among the three B-1 populations. In total, 17.3% (9 of 52) of splenic B-1a-derived sequences were identical with IgH chains recovered from the peritoneum of the same mouse. Six of these splenic IgH chain sequences were found in the peritoneal B-1a compartment (Fig. 1, groups A–F), whereas three were identical with IgH chains found among peritoneal B-1b cells (Fig. 1, groups G–I). Such high frequency of overlapping sequences between both anatomical locations could be the result of independent identical VHDJH rearrangements of different B-1 cell precursors, possibly selected by the same (auto)Ag. Consistently, six overlapping IgH chains did not exhibit complementarity determining region 3 (CDR3) diversification (Fig. 1, groups A, B, D—F, and I), thus increasing the chance of independent identical rearrangement events. Alternatively, the single overlapping sequence from the splenic and the peritoneal B-1a compartment displaying JH nibbling (Fig. 1, group C) might be indicative of migration between the two locations, because the introduction of CDR3 diversity limits the probability of independent identical rearrangements.

Two identical IgH chain amino acid sequences were shared between splenic and peritoneal B-1a and peritoneal B-1b cells (Fig. 1, groups A and C) indicating that some Ab specificities can be selected into all three B-1 compartments analyzed, but cross-contaminations when isolating B-1 lymphocytes cannot be totally excluded.

Increased junctional diversity of splenic B-1a cells

Despite the reoccurrence of identical sequences between splenic and peritoneal B-1 cells, the CDR3 diversity of peritoneal B-1a cells was more restricted, because 45.9% did not exhibit nontemplated (N) nucleotides in their VH-D and D-JH junctions compared with splenic B-1a (23.1%) and peritoneal B-1b cells (11.5%) (Table I). A similar increase of CDR3 diversity was observed in normal mice: 45.8% of IgH chains from peritoneal B-1a cells (n = 24) did not exhibit N nucleotides in their VH-D and D-JH junctions, whereas only 18.8% of splenic B-1a cells exhibited no N nucleotides (Table I).


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  		Table I. Comparison of N nucleotide insertions

 
To allow conclusions about the frequency of individual clones, we amplified IgH chains of individual IgM+CD5+ B cells from spleen, peritoneum, and blood by single-cell RT-PCR (Fig. 2). No striking differences in the pattern of VH, D, and JH gene segments engaged in these rearrangements could be detected (data not shown). However, peritoneal B-1a cells from L2 mice showed a lower frequency of N nucleotides compared with splenic and blood-derived B-1a cells (Table I). The slightly lower frequency of N nucleotides in the splenic and peritoneal B-1a compartment of L2 mice revealed by the bulk sorting compared with the single-cell RT-PCR experiment was most likely due to an underestimation, because in the case of bulk sorting, repeatedly isolated identical transcripts were counted only once.



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  		FIGURE 2. Comparative IgH chain repertoire analysis of B-1a cells from L2 mice derived from different anatomical locations. A, For single-cell RT-PCR, splenic, peritoneal, and blood-derived IgM+CD5+ B-1a cells were isolated by two subsequent rounds of cell sorting. After the first round of sorting, B-1a cells were >98% pure. IgH chains of B-1a cells from the different anatomical locations were grouped according to amino acid homology and identity. The frequency of cells exhibiting the same VHDJH nucleotide sequence is given (Freq). See Fig. 1 for further explanations. B, IgH chains with identical amino acid sequences can be recovered from spleen, peritoneum, and blood (S, P, and B). C, Analysis of the peritoneum and spleen for the presence of PtC-specific B-1a cells. The complete sequence data are available from GenBank under accession numbers AF546708–AF546754 (splenic B-1a), AF537038–AF537090 (peritoneal B-1a), and AF547095 (blood-derived B-1a).

 
Consistent with such restricted CDR3 diversity, 44.4% of IgH chains analyzed from peritoneal B-1a cells could be summarized into homology groups with identical or very similar amino acid sequences (Fig. 2A). In contrast, from spleen and blood, only 18.8 and 15.0% of IgH chains, respectively, could be grouped in such a way. Thus, the strongly restricted IgH chain repertoire that is consistently found in peritoneal B-1a cells of L2 mice is less pronounced in spleen and blood.

The peritoneal data set was dominated by three different nucleotide sequences representing 14.8%, all of them encoding the same amino acid sequence (Fig. 2, A and B, group E). The same IgH chain amino acid sequence also dominated the data set from spleen (10.4%) and could be recovered from blood as well (5.0%) (Fig. 2B, group E). Remarkably, both nucleotide sequence variants recovered from spleen and peritoneum show no CDR3 diversification, just like the overlapping sequences identified by the bulk sorting experiment. Of note, the same VHQ52-DFL16.2-J4H-encoded amino acid sequence dominating the peritoneal repertoire at a similar frequency (14.4%; 17 of 118 sequences) was previously described by ourselves, using hybridoma techniques (19). Four additional IgH chain sequences present in blood could also be detected either in peritoneum or spleen (Fig. 2B, groups A–D).

The frequency of PtC-specific B-1a cells differs between the peritoneum and spleen of L2 mice

Despite the fact that some identical IgH chains can be readily detected in the spleen, peritoneum, and blood, certain B-1a specificities prevalent in the peritoneal repertoire (VHQ52-DSP2.7/DST-JH1: 13.0%, 7 of 54 sequences; VHQ52-DSP2.10-JH4: 7.4%, 4 of 54 sequences) (Fig. 2A) were found neither in spleen nor blood. Because one could argue that the absence of such sequences is due to the limited sample size, we analyzed L2 mice for the presence of PtC-specific B cells. In normal mice, this specificity is only found among B-1a cells expressing IgH and IgL chains that are restricted to a few IgH chains in combination with certain {kappa}L chains (8). Surprisingly, despite the virtually exclusive expression of the Tg {lambda}2 L chain, PtC-specific B-1a cells are present in the peritoneum of L2 mice in substantial numbers (Fig. 2C). Parallel analysis of IE{kappa}T mice that only produce {lambda} L chains, due to a deletion of the Ig{kappa} chain intron enhancer (20), showed that PtC-specific B-1a cells can be formed in the absence of {kappa} L chains (Fig. 2C). The important role of anti-PtC Abs in immunity (26) might explain the compensation of PtC specificity by {lambda} L chains even in the absence of {kappa} L chains.

Although the splenic B cells compartment of L2 mice is dominated by B-1a cells, PtC binders were below detection (at least 60- to 70-fold lower frequency) (Fig. 2C). Consistently, the splenic B-1a compartment of normal mice contains a lower frequency of B-1a cells specific for BrMRBC than the peritoneum (13). The epitope recognized on BrMRBCs has been identified as PtC (27).

Evidence of previous Ag encounter

Despite considerable differences in their Ab repertoire, freshly isolated splenic and peritoneal B-1 cells from L2 mice show evidence of previous antigenic experience. The level of the costimulatory molecule CD80 is low on B-2 cells of normal mice, whereas splenic and peritoneal B-1 cells of L2 and control mice show constitutive expression of CD80 (Fig. 3). Similarly, surface levels of CD86 on splenic and peritoneal B-1 cells of L2 mice were slightly increased compared with B-2 cells from normal mice (data not shown).



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  		FIGURE 3. High constitutive expression levels of CD80 on splenic and peritoneal B-1 cells of L2 and non-Tg littermates. Consistent with the complete absence of B-2 cells, all IgM+ cells in the peritoneum of L2 mice are CD80+, whereas IgMlow B-2 cells in the peritoneum of control mice are CD80-.

 
Limited capacity of peritoneal B-1a cells to enter the splenic B-1a compartment

In L2 mice, certain B-1a specificities show a clearcut segregation between the splenic and peritoneal B-1a compartment. Therefore, the oligoclonal B-1a repertoire in the peritoneum of L2 mice cannot be extrapolated to the splenic compartment of such mice. However, recovery of blood-derived B-1a IgH chain sequences from splenic and peritoneal B-1a cells from the same pool of mice underlines the capacity of B-1a cells to circulate through the body. It does not necessarily indicate an exchange between peritoneum and spleen, especially because all overlapping IgH chain sequences of splenic B-1a cells (6 of 48 (12.5%) from the bulk sorting and 5 of 52 (9.6%) from the single-cell RT-PCR experiment) did not exhibit N and palindromic (P) nucleotides. Hence, they might be derived from independent rearrangement events.

Intraperitoneal transfer of L2-derived peritoneal exudates into RAG1-/- mice revealed that peritoneal B-1a cells have the capacity to enter the spleen in substantial numbers under such conditions (Fig. 4A). To analyze the extent of exchange between spleen and peritoneum under more physiological conditions, reconstitution experiments were performed using peritoneal B-1 cells of L2 mice and a congenic IgH allotype system (Fig. 4B). Although a substantial population of donor-derived B-1a cells (5–10%) could be detected in the peritoneum of recipients after i.p. transfer, such cells were below the detection limit in the spleen, even 6 wk after transfer. As expected, after i.v. injection, donor-derived B-1a cells could be detected in both spleen and peritoneum (Fig. 4B). Transfer of host syngeneic BM into irradiated recipients revealed that adult BM of L2 mice does not result in the production of CD5+ B-1 cells (3), suggesting that both splenic and peritoneal B-1a cells from L2 mice originate from early mouse development.



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  		FIGURE 4. Limited capacity of peritoneal B-1a cells to enter the splenic B-1a compartment of normal L2 mice. Peritoneal B-1a cells (1–1.5 x 107) obtained by peritoneal lavage of L2 mice congenic for the IgHb allotype were transferred into RAG1-/- or L2 mice displaying the IgHa allotype. A, B-1a cells can be identified in the spleen of RAG1-/- mice 4 wk after i.p. transfer. B, Representative analysis of IgMb+ donor-derived B cells after i.p. or i.v. transfer. Donor-derived B cells were below detection level even 6 wk after i.p. transfer and at any other time analyzed (2 days to 6 wk after transfer). Similar results were obtained by transferring IgHa B cells into IgHb recipients. As expected, i.v. transfer results in the presence of donor-derived B-1a cells in peritoneum and spleen at similar frequencies at any time analyzed (2, 4, and 8 days after transfer). Cells were gated on CD19+ cells.

 
Gene expression profile of splenic and peritoneal B-1 cells from L2 mice

To further characterize B-1 cells of L2 mice from different anatomical locations, we studied their gene expression profile. To this end, total RNA was extracted from 1.5 x 105 sorted B lymphocytes (Fig. 5A) and used for mRNA amplification. The samples were then hybridized to high-density oligonucleotide arrays (Affymetrix).



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  		FIGURE 5. Gene expression profiles of splenic and peritoneal B-1 cells. A, Purification of splenic and peritoneal B-1 cells from L2 mice by FACS sorting. B, Identification of gene expression patterns by self-organizing maps. Expression levels of each gene were normalized so that the mean = 0, and SD = 1. Black and red lines indicate expression level means and SDs, respectively. C, Hierarchical cluster analysis of 262 genes and 156 expressed sequence tags with a change of at least 2-fold. Shown are normalized expression levels below (green), near to (black), and above (red) the mean. D, Hierarchical cluster analysis of ~700 transcripts differentially expressed between splenic and peritoneal B-1 cells from L2 mice and splenic B-2 cells from normal mice with a change of at least 2-fold. E, Analysis of gene expression by RT-PCR. B-1a cells from spleen and peritoneum of L2 mice were sorted to high purity (>98.5%) and used for RT-PCR. LPL, Lipoprotein lipase; NAD, hydroxyprostaglandindehydrogenase 15. F, The expression levels of CD72, CD1, and VCAM-1 on mRNA level revealed by gene expression profiling correlate with differential surface expression.

 
Comparison of gene expression profiles by cluster analysis indicates a close correlation between the corresponding B-1a populations from Tg and normal mice (supplementary Fig. 2). Between splenic B-1a cells from L2 and normal mice, only 60 transcripts were found to be at least 5-fold regulated, while 25 were regulated at least 5-fold between the peritoneal B-1a cells. Approximately one-third of such genes are {kappa} L chain transcripts up-regulated in the populations of normal mice (data not shown). The few differentially expressed transcripts might reflect the transcriptional regulation of the Tg and/or the higher homogeneity of B-1a cells in L2 mice. Thus, the B-1a cells in Tg L2 mice reflect the normal situation to a large extent.

Between splenic and peritoneal B-1 cells from L2 mice, a total of 522 genes were identified as differentially expressed at a false discovery rate of 0.15%. Four hundred and eighteen genes were regulated by a factor of 2 or more: 262 known genes and 156 expressed sequence tags (Fig. 5C). Six different patterns of differentially expressed genes could be identified by grouping the 262 known genes using self-organizing maps with a 1 x 6 geometry (Fig. 5B). Fig. 6 summarizes 50 transcripts of known genes present in clusters 5 and 6 that are at least 5-fold regulated.



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  		FIGURE 6. Differentially expressed genes among B-1 subsets from L2 mice. The unique Affymetrix probe set identifier (Affy_id), the mean average difference value, and the SD of the three replicate experiments, as well as a description of the particular gene are shown. SD was calculated as implemented in Excel (Microsoft, Redmond, WA). Only at least 5-fold-regulated genes, comparing the populations expressing the lowest and highest levels, are displayed. Clusters 1–6 (C1–C6) relate to the groups displayed in Fig. 5B. The two right columns show the mean average difference values and SDs of the corresponding transcripts of splenic CD5-IgM+ B-2 cells from normal mice.

 
The comparison of the ~12,000 expression values by cluster and dot-plot analysis revealed good correlation between replicate experiments (supplementary Figs. 2 and 3). To further validate the array-based gene expression, some transcripts present in cluster 6 (Figs. 5B and 6) were selected according to the putative absence in peritoneal B-1a cells but expression in splenic B-1a cells and validated by RT-PCR. Fig. 5E shows that the detection of these transcripts by RT-PCR was in concordance with the array results.

The analysis for marker genes with a known expression pattern revealed that our expression data for CCR3 (Figs. 5E and 6, cluster 6) are concordant with previous observations, whereas CCR2 (cluster 2) does not follow the expression pattern described previously (Fig. 6) (16). CCR2 might indicate a difference between Tg and normal splenic B-1a cells. Although the array-based analysis indicated an ~5-fold regulation of CD5 between splenic and peritoneal B-1a cells (cluster 6), RT-PCR (Fig. 5E) and FACS analysis (data not shown) revealed similar expression levels in both B-1a subsets.

Detailed analysis of the six different gene expression patterns of B-1 cells from L2 mice (Figs. 5B and 6) demonstrated that only one gene present in cluster 1 shows >5-fold increase in the peritoneal B-1a cells. In contrast, cluster 2 contains 12 genes that show an increase of at least 5-fold in the peritoneal B-1 compartment. Of particular interest are the following: the chemokine receptor CCR2; Mac-2, a lectin implicated in cell adhesion; S100A4, which is involved in cell cycle regulation and intercellular adhesion; and Annexin A1, which mediates anti-inflammatory responses and inhibits T cell proliferation (supplementary Fig. 4).

The number of 128 genes following the expression pattern of clusters 3 and 4 indicates a high degree of similarity between splenic B-1a and peritoneal B-1b cells. Genes present in clusters 3 and 4 are associated with intracellular vesicular trafficking, protein trafficking, general metabolism, cytoskeleton, proteolysis/peptidolysis, transcriptional regulation, and signal transduction (supplementary Fig. 4). Interestingly, splenic B-1a and peritoneal B-1b cells express H2-M transcripts (cluster 4; two hits), a molecule involved in MHC class II presentation, at higher levels than peritoneal B-1a cells. H2-O also belongs to this cluster (data not shown). Only the arbitrarily set cutoff of at least 5.0-fold regulation excluded this 4.8-fold-regulated transcript.

Genes potentially involved in mediating specialized functional characteristics of splenic B-1a cells

Clusters 5 and 6 contain genes that show increased mRNA abundance only in splenic B-1a cells compared with peritoneal B-1 cells (Fig. 5B). Some of these molecules are involved in mediating chemotaxis, e.g. CCR3, the chemokine orphan receptor, the high- and low-affinity receptors for bacterial N-formyl-methionyl peptides (FPR1 and FPR2), and calgranulin A (S100 calcium-binding protein 8), a chemotaxin to neutrophils. The mRNA expression levels of molecules involved in Ag uptake and presentation like the mannose receptor (CD206), CD1, and CD68 are also enhanced. CD206 is a pattern recognition receptor that is involved in TAP-independent presentation of lipoglycan Ags to T cells via CD1 (28). The high expression levels of CD1 on splenic B-1a cells is comparable to MZ B cells (data not shown) and might improve the ability to present such nonclassical Ags. CD68 plays a role in phagocytic activities, intracellular lysosomal metabolism, and extracellular cell-cell and cell-pathogen interactions.

VCAM-1 (CD106) (Figs. 5, E and F, and 6; two hits) mediates adhesion of leukocytes but is also supposed to be a costimulatory molecule for T cells (29). VCAM-1 expression has been described for BM stromal cells, follicular dendritic cells, and stimulated endothelial cells (30). Additional molecules mediating adhesive and cell-cell communication functions that were found to be up-regulated in splenic B-1a cells are procollagen XIV, Ig superfamily 4, and ficolin.

CD82 (31) and CD72 (32) are two up-regulated molecules that have established negative regulatory functions. CD82 may also play an important role in MHC class II maturation. Furthermore, the gene knockout suggests that Ly-6E has an inhibitory role in regulation of T cell activation, whereas cross-linking of the Ly-6E molecule on B cells provides a signal for proliferation (33).

Four (Deltex homolog 1, pim-2, NF-{kappa}BI{epsilon}, NFATc1) and five (c-mer; nuclear receptor subfamily 1, group H, member 3; cellular repressor of E1A-stimulated genes; calcyclin; Spi-C) molecules involved in regulation of transcription and signal transduction are present in cluster 5 and 6, respectively. Deltex homolog 1 is a regulator of Notch signaling, and NF-{kappa}BI{epsilon} might be involved in the regulation of cell adhesion, immune and inflammatory responses, apoptosis, differentiation, and growth. Pim-2 is expressed in hemopoietic tissues, and leukemia and lymphoma cell lines, and is potentially involved in proliferation. NFATc1 has been described to regulate activation, proliferation, and differentiation of T and B lymphocytes. The protooncogene mer, which is involved in the regulation of cell growth and maintenance, has been described in numerous neoplastic B and T cell lines, but not in normal B cells. Calcyclin expression is induced by growth factors and overexpressed in myeloid leukemias. The array-based strong up-regulation of the Ig J chain in splenic B-1a cells is consistent with the ~40-fold higher frequency of IgM-secreting splenic B-1a cells compared with peritoneal B-1a cells (13).

An additional comparison of the expression data from Tg B-1 cell with splenic B-2 cells of normal mice revealed ~700 at least 2-fold-regulated transcripts. Approximately 90 {kappa} L chains transcripts were detected only in the B-2 population from normal mice (Fig. 5D). Similarly, CD9 was found to be up-regulated in B-1 cells and thus was detected at the predicted levels (1). A more extensive comparison of B-1 and B-2 cells is displayed in Fig. 6.

Taken together, many of the molecules that are specifically up-regulated in the splenic B-1 subpopulation compared with peritoneal B-1 and splenic B-2 cells might facilitate the function of splenic B-1a cells as a first line of defense and link them to cognate T cell-mediated immune responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent data on the functional abilities of peritoneal B-1a and splenic MZ B cells indicate that they provide a first line of defense against pathogens to ensure rapid development of short-term responses to a limited number of conserved Ags (1, 2). Our data suggest that splenic B-1a cells, like peritoneal B-1a and MZ B cells, represent a distinct B-1 cell subpopulation with unique functional characteristics.

In contrast to conventional Ig and IgH chain Tg mice that have widely been used previously to study various aspects of B-1 cell development, the restriction of the Ab repertoire by Tg L chain allows the analysis of IgH chain repertoire generation (19). The combined analysis of the Ab repertoire, gene expression profile, and homing properties of B-1 cells from spleen and peritoneum was facilitated by the several characteristics of the Tg mouse line used in this study. First, the almost exclusive expression of only a Tg L chain in L2 mice restricts the IgH chain repertoire to an analyzable degree. Second, the predominance of B-1a cells in L2 mice facilitates the isolation of appropriate cell numbers, which especially holds true for the splenic B-1a compartment. Third, L2 mice allow the analysis of the full potential of B-1 cells in the absence of B-2 cells.

The IgH chain repertoire analysis was now extended to additional anatomical locations. Despite a comparable pattern of VH, D, and JH gene segments engaged in functional IgH chains of B-1a cells from spleen, peritoneum, and blood of L2 mice, the lower CDR3 diversity in peritoneal B-1a cells is paralleled by a pronounced oligoclonality. A similar difference in CDR3 diversity by N nucleotides was found in normal mice. The difference in N nucleotides has implications in both the development and function of such cells. The most reliable indicator for the origin of a CD5+ B cell from fetal sources is the lack of N nucleotides at both VH-D and D-JH junctions due to the absence of TdT activity during this period (34). Transfer experiments have revealed that adult BM of L2 mice is not capable of producing CD5+ B cells (3). Therefore, the differences of CDR3 diversity between splenic and peritoneal B-1a cells must be due to differences in the generation of these cells early in mouse development. Thus, peritoneal and splenic B-1a cells might be generated at different ontogenetic time points, e.g. before and after initiation of TdT expression.

Given that splenic and peritoneal B-1a cells exhibit a similar pattern of VH genes, the differences in CDR3 diversity might explain to some extent the differences in frequencies of certain specificities in both B-1a compartments (Fig. 2C and Ref. 13). Hence, this appears to be consistent with the reduction of phosphorylcholine-specific B-1a cells in mice Tg for TdT (35).

The skewed IgH chain repertoire of splenic compared with peritoneal B-1a cells could reflect selection of a more diverse splenic B-1a repertoire, recruited from the same pool of mature B-1a cells from which peritoneal B-1a cells are also derived. Differences in the selection and maintenance of both B-1a subsets might be indicated by the higher expression level of H2-M and H2-O transcripts in splenic compared with peritoneal B-1a cells of L2 mice. Recent evidence indicates that the ratio of H2-O and H2-M differs in dependence on the B cell differentiation stage and influences the levels of MHC II-peptide complexes (36). In addition, H2-O appears to modify peptide loading of MHC II molecules and drive Ag presentation toward proteins that have been internalized by surface Ig (37). Therefore, splenic and peritoneal B-1a cells might have different Ag-presenting capabilities, which could influence the interaction with T cells and change selective processes shaping the Ab repertoire of such B-1 cell subsets.

The Ab repertoire clearly demonstrates the heterogeneity between splenic and peritoneal B-1 cells. Restriction of B-1a specificities to peritoneal cavity and isolation of some oligoclonal IgH chains at high frequencies from the peritoneum but not spleen is found on the one hand. In contrast, identical IgH chains from both spleen and peritoneum were isolated, clearly indicating that the same Ag specificities can be selected into both B-1a compartments.

The recovery from blood of some splenic and peritoneal IgH chain sequences underlines the migratory capacity of B-1a cells (17). The intraperitoneal reconstitution of RAG-1-/- mice revealed that peritoneal B-1a cells are in principle capable of entering the spleen. Similarly, i.v. injection of peritoneal B-1a cells results into the homing of such cells into the splenic compartment of L2 mice. However, i.p. reconstitution experiments indicate that exchange between peritoneum and spleen of L2 mice appears to be very limited even 6 wk after transfer. In agreement, splenic B cells of VH11V{kappa}9 Tg mice, adoptively transferred into the peritoneal cavity of normal mice, were not detectable in the spleen after 72 h (14). Therefore, the overlapping identical IgH chains appear to be unlikely due to a migratory exchange of dominant B-1a clones, but might well be derived from different rearrangement events. Consistently, all splenic IgH chain sequences that are also found in peritoneum do not exhibit N and P nucleotide insertions despite the fact that only 23.1% (bulk sorting) and 31.3% (single-cell RT-PCR) of splenic sequences have no N nucleotides. Therefore, we also argue that the reduction of B-1a cells in splenectomized mice observed by Wardemann et al. (18) is not due to the requirement of B-1a cells to continuously circulate through the spleen, but rather to a loss of a survival factor generated in the spleen.

Splenic B-1a and peritoneal B-1b cells show comparably high levels of CDR3 diversity and striking similarities in gene expression profiles. One hundred and nineteen of 262 differentially expressed transcripts of known genes show comparable expression levels in both B-1 subsets (clusters 1, 3, and 4). Interestingly, all three overlaps of identical IgH chains between splenic B-1a and peritoneal B-1b cells show substantial CDR3 diversification by the addition of N and P nucleotides and/or JH nucleotide loss. This finding might suggest a migratory exchange between these B-1 subpopulations in L2 mice, obscured in reconstitution experiments due to the low frequency of peritoneal B-1b cells in L2 mice.

The diverse Ab repertoire and characteristic gene expression profile might qualify splenic B-1a cells for mediating specialized functions in first-line-of-defense responses at their particular location. The expression of FPR1 and FPR2 may help B-1a cells to survey the extracellular environment for the presence of bacteria. Such chemotactic responses might be followed by the uptake of polymannose ligands mediated by CD206 which then could lead to Ag presentation via CD1 (38). An additional potential linkage between innate and adaptive immune responses might be represented by the VCAM-1 expression on splenic B-1a cells, supposedly a costimulatory molecule for T cells. The high expression levels of the Ig J chain in splenic B-1a cells could be the result of persistent activation and the high levels of CD72 and CD82 might have important regulatory functions in limiting the response to continuous antigenic stimulation.

In summary, our combined analysis of the Ab repertoire and gene expression profile of splenic and peritoneal B-1 cells indicates that differences in gene expression appear to be due to a different developmental program rather than local environmental influences on migrating B-1 cells. Splenic and peritoneal B-1a cells constitute unique populations of B-1 cells, each with different developmental and functional features.


   Acknowledgments
 
The expert technical assistance of Susanne zur Lage, Regina Lesch, Maria Höxter, Lothar Gröbe, Hubertus Kohler, Tracy Hayden, and Stephanie Thies is gratefully acknowledged. We also thank Thomas Jacobs for advice on liposome preparation. We also gratefully acknowledge Ulrich Certa at F. Hoffmann-LaRoche, Ltd., for providing the facility for gene expression analysis.


   Footnotes
 
1 This work was partly supported by grants from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and niedersächsisches Ministerium für Wissenschaft und Kultur. Part of the expression analysis was performed at the Basel Institute for Immunology, which was founded and supported by F. Hoffmann-LaRoche, Ltd. Back

2 Address correspondence and reprint requests to Dr. Karsten Kretschmer, Molecular Immunology, German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail address: kkr{at}gbf.de Back

3 Current address: Department of Bacteriology, Max-von-Pettenkofer-Institut, Pettenkoferstrasse 9a, D-80336 Munich, Germany. Back

4 Abbreviations used in this paper: MZ, marginal zone; BCR, B cell receptor; Tg, transgene/transgenic; BrMRBC, bromelain-treated mouse RBC; BM, bone marrow; CDR3, complementarity determining region 3; PtC, phosphatidylcholine; N, nontemplated; P, palindromic. Back

5 K. Kretschmer, A. Jungebloud, J. Stopkowicz, T. Kleinke, R. Hoffman, and S. Weiss. The selection of marginal zone B cells differs from that of B-1a cells. Submitted for publication. Back

Received for publication December 23, 2002. Accepted for publication June 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Martin, F., J. F. Kearney. 2001. B1 cells: similarities and differences with other B cell subsets. Curr. Opin. Immunol. 13:195.[Medline]
  2. Berland, R., H. H. Wortis. 2002. Origins and functions of B-1 cells with notes on the role of CD5. Annu. Rev. Immunol. 20:253.[Medline]
  3. Engel, H., B. Bogen, U. Muller, J. Andersson, A. Rolink, S. Weiss. 1998. Expression level of a transgenic {lambda}2 chain results in isotype exclusion and commitment to B1 cells. Eur. J. Immunol. 28:2289.[Medline]
  4. Lam, K. P., K. Rajewsky. 1999. B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development. J. Exp. Med. 190:471.[Abstract/Free Full Text]
  5. Watanabe, N., S. Nisitani, K. Ikuta, M. Suzuki, T. Chiba, T. Honjo. 1999. Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment. J. Exp. Med. 190:461.[Abstract/Free Full Text]
  6. Tornberg, U. C., D. Holmberg. 1995. B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures. EMBO J. 14:1680.[Medline]
  7. Kantor, A. B., C. E. Merrill, L. A. Herzenberg, J. L. Hillson. 1997. An unbiased analysis of VHD-JH sequences from B-1a, B-1b, and conventional B cells. J. Immunol. 158:1175.[Abstract]
  8. Seidl, K. J., J. D. MacKenzie, D. Wang, A. B. Kantor, E. A. Kabat, L. A. Herzenberg, L. A. Herzenberg. 1997. Frequent occurrence of identical heavy and light chain Ig rearrangements. Int. Immunol. 9:689.[Abstract/Free Full Text]
  9. Herzenberg, L. A., N. Baumgarth, J. A. Wilshire. 2000. B-1 cell origins and VH repertoire determination. Curr. Top. Microbiol. Immunol. 252:3.[Medline]
  10. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, D. Allman, C. L. Stewart, J. Silver, R. R. Hardy. 1999. Positive selection of natural autoreactive B cells. Science 285:113.[Abstract/Free Full Text]
  11. Herzenberg, L. A.. 2000. B-1 cells: the lineage question revisited. Immunol. Rev. 175:9.[Medline]
  12. Kantor, A. B., A. M. Stall, S. Adams, L. A. Herzenberg, L. A. Herzenberg. 1992. Differential development of progenitor activity for three B-cell lineages. Proc. Natl. Acad. Sci. USA 89:3320.[Abstract/Free Full Text]
  13. Klinman, D. M., K. L. Holmes. 1990. Differences in the repertoire expressed by peritoneal and splenic Ly-1 (CD5)+ B cells. J. Immunol. 144:4520.[Abstract]
  14. Chumley, M. J., J. M. Dal Porto, J. C. Cambier. 2002. The unique antigen receptor signaling phenotype of B-1 cells is influenced by locale but induced by antigen. J. Immunol. 169:1735.[Abstract/Free Full Text]
  15. Fischer, G. M., L. A. Solt, W. D. Hastings, K. Yang, R. M. Gerstein, B. S. Nikolajczyk, S. H. Clarke, T. L. Rothstein. 2001. Splenic and peritoneal B-1 cells differ in terms of transcriptional and proliferative features that separate peritoneal B-1 from splenic B-2 cells. Cell. Immunol. 213:62.[Medline]
  16. Rothstein, T. L., G. M. Fischer, D. A. Tanguay, S. Pavlovic, T. P. Colarusso, R. M. Gerstein, S. H. Clarke, T. C. Chiles. 2000. STAT3 activation, chemokine receptor expression, and cyclin-Cdk function in B-1 cells. Curr. Top. Microbiol. Immunol. 252:121.[Medline]
  17. Ansel, K. M., R. B. Harris, J. G. Cyster. 2002. CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity 16:67.[Medline]
  18. Wardemann, H., T. Boehm, N. Dear, R. Carsetti. 2002. B-1a B cells that link the innate and adaptive immune responses are lacking in the absence of the spleen. J. Exp. Med. 195:771.[Abstract/Free Full Text]
  19. Kretschmer, K., H. Engel, S. Weiss. 2002. Strong antigenic selection shaping the immunoglobulin heavy chain repertoire of B-1a lymphocytes in {lambda}2(315) transgenic mice. Eur. J. Immunol. 32:2317.[Medline]
  20. Takeda, S., Y. R. Zou, H. Bluethmann, D. Kitamura, U. Muller, K. Rajewsky. 1993. Deletion of the immunoglobulin {kappa} chain intron enhancer abolishes {kappa} chain gene rearrangement in cis but not {lambda} chain gene rearrangement in trans. EMBO J. 12:2329.[Medline]
  21. Hoffmann, R., T. Seidl, M. Neeb, A. Rolink, F. Melchers. 2002. Changes in gene expression profiles in developing B cells of murine bone marrow. Genome Res. 12:98.[Abstract/Free Full Text]
  22. Li, C., W. H. Wong. 2001. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl. Acad. Sci. USA 98:31.[Abstract/Free Full Text]
  23. Tusher, V. G., R. Tibshirani, G. Chu. 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98:5116.[Abstract/Free Full Text]
  24. Benjamini, Y., Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57:289.
  25. Eisen, M. B., P. T. Spellman, P. O. Brown, D. Botstein. 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95:14863.[Abstract/Free Full Text]
  26. Boes, M., A. P. Prodeus, T. Schmidt, M. C. Carroll, J. Chen. 1998. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188:2381.[Abstract/Free Full Text]
  27. Arnold, L. W., G. Haughton. 1992. Autoantibodies to phosphatidylcholine: the murine antibromelain RBC response. Ann. NY Acad. Sci. 651:354.[Medline]
  28. Prigozy, T. I., P. A. Sieling, D. Clemens, P. L. Stewart, S. M. Behar, S. A. Porcelli, M. B. Brenner, R. L. Modlin, M. Kronenberg. 1997. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6:187.[Medline]
  29. Burkly, L. C., A. Jakubowski, B. M. Newman, M. D. Rosa, G. Chi-Rosso, R. R. Lobb. 1991. Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation. Eur. J. Immunol. 21:2871.[Medline]
  30. Koni, P. A., S. K. Joshi, U. A. Temann, D. Olson, L. Burkly, R. A. Flavell. 2001. Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow. J. Exp. Med. 193:741.[Abstract/Free Full Text]
  31. Hammond, C., L. K. Denzin, M. Pan, J. M. Griffith, H. J. Geuze, P. Cresswell. 1998. The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules. J. Immunol. 161:3282.[Abstract/Free Full Text]
  32. Pan, C., N. Baumgarth, J. R. Parnes. 1999. CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation. Immunity 11:495.[Medline]
  33. Snapper, C. M., H. Yamada, J. J. Mond, C. H. June. 1991. Cross-linkage of Ly-6A/E induces Ca2+ translocation in the absence of phosphatidylinositol turnover and mediates proliferation of normal murine B lymphocytes. J. Immunol. 147:1171.[Abstract]
  34. Gregoire, K. E., I. Goldschneider, R. W. Barton, F. J. Bollum. 1979. Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse. J. Immunol. 123:1347.[Abstract/Free Full Text]
  35. Benedict, C. L., J. F. Kearney. 1999. Increased junctional diversity in fetal B cells results in a loss of protective anti-phosphorylcholine antibodies in adult mice. Immunity 10:607.[Medline]
  36. Glazier, K. S., S. B. Hake, H. M. Tobin, A. Chadburn, E. J. Schattner, L. K. Denzin. 2002. Germinal center B cells regulate their capability to present antigen by modulation of HLA-DO. J. Exp. Med. 195:1063.[Abstract/Free Full Text]
  37. Liljedahl, M., O. Winqvist, C. D. Surh, P. Wong, K. Ngo, L. Teyton, P. A. Peterson, A. Brunmark, A. Y. Rudensky, W. P. Fung-Leung, L. Karlsson. 1998. Altered antigen presentation in mice lacking H2-O. Immunity 8:233.[Medline]
  38. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro, T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, P. J. Brennan, et al 1995. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 269:227.[Abstract/Free Full Text]



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