Ig Light Chain Precedes Heavy Chain Rearrangement during Development of B Cells in Swine

This information is current as Marek Sinkora, Jana Sinkorova and Katerina Stepanova of September 28, 2021. J Immunol published online 9 January 2017 http://www.jimmunol.org/content/early/2017/01/06/jimmun ol.1601035 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 9, 2017, doi:10.4049/jimmunol.1601035 The Journal of Immunology

Ig Light Chain Precedes Heavy Chain Gene Rearrangement during Development of B Cells in Swine

Marek Sinkora, Jana Sinkorova, and Katerina Stepanova

The current mammalian paradigm states that 1) rearrangements in the IgH locus precede those in IgL loci, 2) IgLl rearrange only when IgLk genes are consumed, and 3) the surrogate L chain is necessary for selection of productive IgH gene rearrange- ments. We show in swine that IgL rearrangements precede IgH gene rearrangements, resulting in the expression of naked IgL on a surface of precursor B cells. Findings also suggest that there is no dependency on the surrogate L chain, and thus the authentic IgL may be used for selection of the IgH repertoire. Although rearrangement starts with IgLk genes, it is rapidly replaced by IgLl rearrangement. Fast replacement is characterized by occurrence of IgLlloIgLklo dual-expressing precursors in which IgLk expression is a remnant of a previous translation. Most IgLk+ B cells are then generated later, indicating that there are two waves k l of IgL synthesis in different developmental stages with IgL gene rearrangements in between. In the absence of stromal cells, the Downloaded from stepwise order of rearrangements is blocked so that IgLl gene rearrangements predominate in early development. To our knowledge, this is the first evidence that some mammals can use an inverted order of Ig loci rearrangement. Moreover, a situation in which the generation of BCR-bearing IgLk is delayed until after IgLl becomes the dominant isotype may help explain the extreme deviations in the IgLk/IgLl ratios among mammals. The Journal of Immunology, 2017, 198: 000–000.

mmunological textbooks and reviews (1–4) describe the locus is rearranged in the surviving small preB-II cells until a http://www.jimmunol.org/ rearrangement in Ig loci as a tightly controlled sequential productive IgLk gene rearrangement creates an authentic BCR (5, I process regulated by the surrogate L chain (SLC), which is 6). The current paradigm further states that gene rearrangement in composed of l5 (CD179b) and the invariable Ig i-chain of SLC the IgLl locus only begins after all possible Vk/Jk segments have (CD179a). According to this paradigm, the first step in the rear- been exhausted and/or when Vk or the recombining element (RE) rangement leading to the formation of the BCR occurs in the IgH rearranges to the k deleting element (KDE), leading to deletion of locus of proB cells by combinatorial joining of DH to JH segments the Ck gene segment (7). on both . The resulting preB-I cells subsequently There are a few deviations from above described paradigm but rearrange certain VH segments with one of these partial VDJ gene none are substantial. For example, VH to DH rearrangement may

rearrangements for the IgH (DJH) combinations to the complete precede DH to JH rearrangement in rabbits (8). In chickens, multiple by guest on September 28, 2021 VDJ gene rearrangement for the IgH (VDJH) rearrangement, DH to DJH rearrangements may occur before subsequent rear- which is then tested for its ability to form a proper pre-BCR by rangement to the VH gene (9). Also, B cells in transgenic mice can association with SLC. There is no IgL gene rearrangement at this develop through an alternative pre-BCR–independent pathway in developmental stage. When the pre-BCR fails to fold correctly, the which the IgL genes rearrange independently of the IgH genes (10– cell has a second chance using the second . The large 14). Such findings indicate that sequential rearrangement of IgH preB-II cells die when they fail to produce a productive pre-BCR before IgL genes need not be essential. This is superimposed in whereas successful cells survive, expand, and consecutively be- birds in which IgH and IgL gene rearrangements can occur com- come small preB-II cells. petitively very early in fetal life (9, 15, 16). Currently, no homologs The importance of the SLC in selection of IgH gene rearrange- of mouse l5 have been identified in chickens or swine (17, 18), and ments is considerable because as many as 50–70% of productive IgH SLC may be lost or does not develop in other species (4). proteins do not pair with SLC and become apoptotic (4). The IgLk Our previous work disproved that the ileal Peyer’s patches are a site of primary B cell lymphogenesis in swine (19–22). Rather, Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of porcine B cells are developed throughout life in the bone marrow Sciences, 54922 Novy Hradek, Czech Republic (BM), the primary lymphoid organ also for mice and humans (23). Received for publication June 16, 2016. Accepted for publication December 6, 2016. B cell development in swine was characterized according to ex- This work was supported by the Czech Science Foundation Grant 15-02274S and by pression of MHC class II (MHC-II), CD2, CD21, CD25, CD45RC, the Institutional Research Concept of Institute of Microbiology of the Czech Academy CD172a, and IgHm Ags, and seven subpopulations developing from of Sciences RVO 61388971. subset 0 to subset 6 were identified (23). These studies also showed Address correspondence and reprint request to Dr. Marek Sinkora, Laboratory of Gno- tobiology, Institute of Microbiology of the Czech Academy of Sciences, v.v.i., Doly 183, that all seven subsets can be unambiguously identified by cell size 54922 Novy Hradek, Czech Republic. E-mail address: [email protected] and decreasing expression of CD172a on MHC-II+ BM cells as bri/hi The online version of this article contains supplemental material. B cell precursors differentiate (Fig. 1A). Thus, CD172a ex-

Abbreviations used in this article: BM, bone marrow; DJH, partial VDJ gene rearrange- pression is limited to early precursors until incomplete DJH rear- ment for the IgH; FCM, flow cytometry; FSC, forward side scatter; GF, germ-free; rangements, and CD172alo expression remains on the surface until KDE, k deleting element; MHC-II, MHC class II; PBS-GEL, PBS containing 0.1% 2 sodium azide and 0.2% gelatin from cold water fish skin; RAG, recombination activa- complete VDJH rearrangement(s) and CD172 cells re-present late tion gene; RE, recombining element; SJC, signal joint circle; SLC, surrogate L chain; preB-II and immature B cells (23). In this study, we characterize the VDJH, complete VDJ gene rearrangement for the IgH; VJk, VJ gene rearrangement for rearrangement and expression of IgL genes in context of IgH genes the IgL k;VJl, VJ gene rearrangement for the IgLl. and show that B cells develop in the porcine BM by a process that Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 is yet another deviation from the textbook paradigm. Analysis of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601035 2 IgL PRECEDES IgH GENE REARRANGEMENT IN SWINE developing B cells in the swine BM revealed that IgL genes rear- intracellular staining for IgH and IgL, cells that had been indirectly stained range before IgH genes and that there is no dependency on SLC, for cell surface molecules were subsequently intracellularly stained using which is consistent with the failure to recover l5 from the porcine an IntraStain kit according to a protocol recommended by the manufac- turer (DakoCytomation, Glostrup, Denmark). In experiments designated genome and the observation that the invariable Ig i-chain of the for cultures, PBS-GEL was replaced by cultivation medium and/or PBS. surrogate L chain is expressed in many nonlymphoid tissues in swine (18, 24). Whereas these initial studies suggested that ex- FCM and cell sorting pression of IgLl genes appeared before IgLk,inthisstudywe Samples were measured or sorted on standard FACSCalibur or FACSAria provide evidence that rearrangement starts with IgLk genes but then III flow cytometers, respectively (BD Immunocytometry Systems, Moun- rapidly shifts to IgLl before IgH gene rearrangements and the tain View, CA). Sorted cells were collected to 1) 1 ml of inactivated FBS formation of a BCR. Therefore, the first Ig+ cells are IgH+IgLl+ (PAA Laboratories, Pasching, Austria) if for cultivation, or 2) empty tubes + + if for PCR amplification from bulk-sorted cells. Electronic compensation whereas IgH IgLk cells are generated later. Phylogenetically it was used to eliminate residual spectral overlaps between individual appears that there are two groups of animals, one of which uses fluorochromes. Forward side scatter (FSC)–area/FSC-width parameters a pre-BCR–driven developmental pathway for B cell generation were used for elimination of doublets. The PCLysis or FACSDiva software whereas the second group uses a pre-BCR–independent pathway. (BD Immunocytometry Systems) was used for data processing. Cell cultures Materials and Methods Cell cultures of sorted cells (27) were done in RPMI 1640 medium sup- Experimental animals plemented with L-glutamine and 25 mM HEPES, 10% FBS, 100 U peni- cillin, and 0.1 mg/ml streptomycin. Final concentration of cells was always Animals used in the study were Minnesota miniature/Vietnamese–Asian– set to 1 3 106 cells/ml. Stromal cultures were established using freshly Downloaded from Malaysian crossbred pigs bred in Novy Hradek. Fetuses were obtained by isolated BM suspensions by a similar approach as described for thymic hysterectomy. Germ-free (GF) piglets were recovered from gilts by sterile stromal cells (29). After 7–14 d of incubation when the nonadherent cells hysterectomy at day of gestation 112 and were kept in isolator units under were removed during passaging, the monolayers were extensively washed GF conditions on sterile formula as previously described (25). GF piglets and then used as feeder cells. Allogeneic cultures were used in most of the were used because they have a naive immune system, in which the BM studies because the origin of stromal cells did not affect the behavior of contains a minimum of polymorphonuclear cells, and effector B cells stages examined cells in cultures. Syngeneic stromal cultures were used only for including plasma cells are missing. All animal experiments were approved testing whether allogeneic cultures produce the same effect. In that case, http://www.jimmunol.org/ by the Ethical Committee of the Institute of Microbiology, Czech Academy BM suspensions from the same donor were frozen during the time of of Science, according to guidelines in the Animal Protection Act. preparation of stromal cells. Input stromal cultures were always tested for Preparation of cell suspensions the unwanted presence of B cell lineage cells by both FCM and CDR3 spectratyping. Pure stromal cells were also involved in each experiment as Cell suspensions were prepared essentially as previously described (26). a negative control. In FCM analyses, stromal cells were gated out from all Briefly, blood was obtained by intracardial (piglets) or umbilical (fetuses) analyses by their high side scatter and low MHC-II expression. puncture. Cell suspensions from the spleen and liver were prepared in PBS by teasing apart the tissues using a forceps. Erythrocytes were removed PCR amplification and detection of transcripts from these tissues using hypotonic lysis that also destroys erythroblasts. Detection of different transcripts and gene segments was done on different BM cells were directly flushed from the tibia and/or femur, and leukocytes populations of 30,000–100,000 sorted cells that were dissolved in 5 mlof were purified using a Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) TRI Reagent per thousand cells. In a particular experiment, only the same by guest on September 28, 2021 gradient centrifugation (26). All cell suspensions were filtrated through a amount of sorted cells was used for isolation of total RNA and DNA 70-mm-mesh nylon membrane. Cell suspensions for flow cytometry (FCM) according to a protocol recommended by the manufacturer (Sigma-Aldrich). were finally washed twice in PBS containing 0.1% sodium azide and 0.2% Total cDNA was prepared using random hexamer primers. Each cDNA gelatin from cold water fish skin (PBS-GEL) whereas those for cell cul- preparation was amplified in six concurrent analyses: 1) TdT, 2) recombi- tures were transferred to cultivation medium (see below). Cell numbers nation activation gene (RAG), 3) VDJH rearrangement, 4) VJ gene rear- were determined by a hemacytometer. rangement for the IgL k (VJk rearrangement), 5) VJ gene rearrangement for Immunoreagents for FCM the IgL l (VJl rearrangement), and 6) b-actin. Each DNA preparation was amplified in four concurrent analyses: 1) signal joint circle (SJC) for DJH The following mouse anti-pig mAbs were used as primary immunoreagents: rearrangement, 2) SJC for VDJH rearrangement, 3) partial DJH rearrange- anti-IgHm (swine IgM H chain, M160 or M154, IgG1), anti–MHC-II ment, and 4) nonrearranging portion of IgM constant CH4 region. VDJH, (swine MHC-II leukocyte Ag type DR, 1038H-12-34, IgM or MSA3, VJk,andVJl rearrangements were occasionally detected at the DNA level. IgG2a), anti-CD172a (74-22-15, IgG1 or IgG2b), anti-IgLk (27.2.1, IgG1), In some cases, samples were analyzed for the presence of Vk to KDE anti-IgLl (27.7.1 or 1g6, IgG1 or K139.3E1, IgG2a), anti-CD14 (MIL-2, rearrangements and RE to KDE rearrangements in their DNA. Each analysis IgG2b), and anti-SWC8 (MIL-3, IgM). Specificities of anti-porcine IgHm consisted of two rounds of PCR. Amplification of a portion of b-actin from Abs (M160 or M154) were confirmed by comparative staining with cDNA was used as a control for sample quality and to determine the relative pan-specific mouse anti-human IgM Ab (CM7, IgG1; Sigma-Aldrich), transcript expression and efficiency whereas amplification of a Cm exon from which gave the same staining profile, and also by Western blot (see below). DNA was used as a positive control. All PCR amplifications were constantly Goat polyclonal Abs specific for mouse Ig subclasses labeled with FITC, checked on agarose gels. All primers used for amplifications are listed in R-PE, PE/Cy7 tandem complex, allophycocyanin, allophycocyanin/Cy7 Supplemental Table I, and their positions in genomic sequences are shown in tandem complex, or PE/Texas Red tandem complex were used as secondary Fig. 1B–D. The Ig gene complex in swine is simple compared with mice and immunoreagents (all secondary reagents were from SouthernBiotech, humans (reviewed in Refs. 30, 31). All porcine VH genes (IGHV) belong to Birmingham, AL). All immunoreagents were titrated for optimal signal/ the ancestral VH3 family sharing the same leader and framework sequences, noise ratios. Primary isotype-matched mouse anti-rat mAbs were used as and only one JH segment is functional (Fig. 1B). Thus, a single nondegen- negative controls. Secondary polyclonal Abs were tested for cross- erate primer set is needed to recover all VDJH rearrangements. The porcine reactivity (no primary mAbs) and also for cross-reactivity with primary IgLk and IgLl loci are also restricted and simplified. Although there are isotype-mismatched mouse anti-pig mAbs. multiple gene families for both IgL isotypes, only two IgLk families (IGKV1 and IGKV2) containing nine genes and a single Ck are functional (Fig. 1C). Staining of cells Moreover, the Jk2 segment constitutes .90% of all IgLk rearrangements k k Staining of cells for FCM was performed as described previously (25–28) because J 3–J 5 have noncanonical recombination signal sequences. Simi- l by indirect subisotype staining. Briefly, multicolor staining was done using larly, only two IgL families (IGLV3 and IGLV8) with 10 genes and two l l cells that had been incubated with a combination of three or four primary identical J -C cassettes are functional (Fig. 1D). Therefore, few primer sets mouse mAbs of different subisotypes. Cells were incubated for 15 min and are needed to recover and study all IgL rearrangements in swine. subsequently washed twice in PBS-GEL. Mixtures of goat secondary CDR3 spectratyping polyclonal Abs conjugated with different fluorochromes were then added to the cell pellets in appropriate combinations. After 15 min, cells were Electrophoretic analysis of the CDR3 regions of IgH and IgL rearrange- washed three times in PBS-GEL and analyzed by FCM. In the case of ments on polyacrylamide sequencing gels provides a clonotypic analysis of The Journal of Immunology 3 the BCR repertoire. This procedure is called CDR3 length analysis or spectratyping, and it was performed on DNA and cDNA prepared from sorted B lineage populations to show their level of BCR diversification and whether there has been selection for in-frame rearrangements (27–29). Technically, the CDR3 segments of the amplified VDJs or VJs were reamplified in the third round of PCR (see Supplemental Table I for primers) where one of the two primers was 32P labeled (28). The sequencing gels were subsequently dried and their images were obtained by a fluorescent image analyzer (FLA-7000; Fujifilm, Tokyo, Japan). Western blot Proteins from equal numbers of cells were isolated by RIPA buffer sup- plemented by the Halt protease and phosphatase single-use inhibitor cocktail according to a protocol recommended by the manufacturer (Thermo Fisher Scientific, Rockford, IL). As a control, a normal porcine adult serum was used. The samples were supplemented by 5% 2-ME and were heated at 95˚C for 5 min prior to loading onto Tris-glycine SDS 5% stacking and 8% separating polyacrylamide gels in sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 15.5 mM EDTA, 0.02% bromophenol blue) under reducing conditions. After electrophoresis, proteins were transferred to a Protran BA83 0.2-mm nitrocellulose membrane (Whatman, Dassel, Germany) by semidry blotting, and membranes were blocked for 1 h with 5% BSA in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween 20) and incubated for 1 h Downloaded from with primary anti-IgHm Abs. After washing in TBST, the blots were incu- bated for 45 min with secondary peroxidase-conjugated horse anti-mouse Abs FIGURE 1. The proposed model of B cell development in swine BM (Cell Signaling Technology, Danvers, MA). The signal was developed with an and the genomic structure of the porcine Ig loci. Depicted data for B cell ECL Plus Western blotting substrate kit (Thermo Fisher Scientific) detected development (A) summarize subset classification, cell size, and surface by a Gel Logic 2200 Pro imaging system (Carestream, Rochester, NY). In phenotype based on our previous work (23). Simplified genomic maps of some experiments, control staining of mouse IgG and IgM mAbs was used. IgH (B), IgLk (C), and IgLl (D) loci are also shown. Note that only

In that case, mouse IgG was detected by the same secondary peroxidase- functional genes are shown; no functional gene is inverted, and genes are http://www.jimmunol.org/ conjugated horse anti-mouse Abs only, whereas mouse IgM was detected not scaled. Positions of all used primers are shown and their numbering by primary goat anti-mouse IgM followed by secondary peroxidase-conjugated corresponds to numbers in Supplemental Table I. Gene nomenclature mouse anti-goat Abs. follows the international ImMunoGeneTics information system.

Results were detected both in DNA and cDNA levels (Fig. 3C). At this stage IgL is present on the surface of early precursors in the absence of development, the level of IgLk rearrangements was always higher of IgH than for IgLl, and in some experiments only IgLk rearrangement The earliest precursors of B cells in swine are characterized by the was detected. Subsets 1a and 1b showed also both types of IgL high expression of CD172a on large MHC-II+ BM cells (23) (Fig. 1). rearrangements as well as transcripts for RAG and, additionally, by guest on September 28, 2021 The present studies were initiated after finding that FCM surface evidence of partial DJH rearrangements (Fig. 3C, DJH), including staining cannot detect IgH on these large precursors (Fig. 2A) SJC for these partial DJH rearrangements (Fig. 3C, SJC DJH). In whereas IgL can be detected (Fig. 2B). Screening of fetal liver and subsets 2a and 2b, the first signs of IgH gene rearrangements were BM during ontogeny revealed similar precursors, and these were not identified along with SJC (Fig. 3C, SJC VDJH). From subset 3 and found in circulation or in secondary lymphoid tissues (Supplemental onward, IgH and IgL gene rearrangements and transcripts were Fig. 1). Intracellular staining for IgH and IgL confirmed that early routinely detected. Interestingly, no apparent downregulation of precursors do not contain IgH (Fig. 2C) but contain IgL (Fig. 2D). RAG or TdT expression between preB-I (subsets 2–3) and preB-II However, immature B cells express IgH (Fig. 2E) together with IgL cells (subsets 4–5) was seen as has been reported in mice (4). (Fig. 2F). Intracellular staining showed that some portion of the Cultivation of sorted BM subsets provided insight into CD172alo precursors still contains IgL (Fig. 2H) without IgH (Fig. developmental pathways, changes in expression of IgLk and 2G). The existence of IgL+IgH2 precursors in the BM (Fig. 2I) but IgLl transcripts, and identification of a checkpoint in B cell not in the periphery (Fig. 2J) was also confirmed by simultaneous development IgL/IgH staining. Western blot analysis further proved that sorted IgL+IgH2 precursors do not contain IgH (Fig. 2K, black arrow) Our finding of preferential IgL rearrangements (Figs. 2, 3) prompted whereas sorted IgL+IgH+ immature B cells contain IgH (Fig. 2K, in vitro cultivation of cells at different stages of development. In- open arrow). Western blot analysis was also used to show that the dividual subsets were sorted by FCM under aseptic conditions, as anti-IgHm Abs are IgHm-specific, and that the secondary Abs did not were those used for PCR studies (Fig. 3B). The sorted cell pop- recognize swine Igs (Fig. 2K, rest of the gel). ulations were then cultivated for 4 d at 4˚C or at 37˚C in medium only or at 37˚C in medium on a bed of BM stromal cells (Fig. 4). The order of Ig loci recombination is reversed compared with mice Stromal cells were included because previous findings revealed Previous studies characterized B cell development in swine and that development of subset 3 is not supported in medium alone identified seven subpopulations developing from subset 0 to subset (23). Stromal cells were always checked for their phenotype 6 (23). These subsets can be distinguished by cell size and ex- (large MHC-II2/lo cells) and an absence of MHC-IIhi B cell lineage pression of CD172a on MHC-II+ BM cells (Fig. 1A). Because of cells (data not shown). As might be expected, cultivation at 4˚C did the detection of free IgL proteins on the surface of subsets 1 and 2, not lead to any phenotypic changes (Fig. 4, all dot plots for 4˚C) we have subdivided these subsets and designated them 1a, 1b, 2a, whereas cultivation at 37˚C caused a variable decrease in expres- and 2b in this study (Fig. 3A). FCM sorting of individual subsets sion of CD172a (Fig. 4B–E). This is consistent with the develop- (Fig. 3B) followed by PCR amplification of DNA and cDNA mental pathway for porcine B cells (23). However, cultivation of revealed that precursor subset 0 does not contain any rearranged IgH subsets 0–2b did not result in complete loss of CD172bri cells genes, but transcripts for RAG and both types of IgL rearrangements (Fig. 4A–D). Rather, even cells expressing CD172hi/lo after sorting 4 IgL PRECEDES IgH GENE REARRANGEMENT IN SWINE Downloaded from

A

FIGURE 2. Analysis of IgL expression on different subpopulations of porcine cells. Fresh BM cells isolated from newborn pigs were gated for large ( – http://www.jimmunol.org/ D) and small (E–H) MHC-II+ cells only, and their representative surface expression of CD179a and either IgHm or IgLl detected by FCM on the surface (A, B, E, and F) or intracellularly (C, D, G, and H) are shown. Staining for IgLk (data not shown) gave a similar result as for IgLl. Filled arrows indicate large MHC-II+CD179ahiIgLl+IgH2 early precursors whereas open arrows indicate small MHC-II+CD179a2IgLl+IgH+ immature B cells. Results are repre- sentative of nine independent experiments using fetal, young, and adult pigs. Comparison of IgHm and IgLl surface expression on BM (I) and spleen (J) cells is also shown. Western blot under reducing conditions (K, left) demonstrates that IgH is absent in sorted large MHC-II+CD179ahiIgLl+IgH2 early precursors (filled arrow) whereas it is present in small MHC-II+CD179a2IgLl+IgH+ immature B cells (open arrow). Shown is also a confirmation that anti- porcine IgHm mAbs (M145 and M160) recognize IgHm from porcine serum and cells (∼75 kDa), which is different from other Ig classes (∼50–60 kDa). Secondary peroxidase-conjugated horse anti-mouse Abs do not recognize swine Igs. Note that mouse IgG (moIgG) and IgM (moIgM) were used for comparison, and moIgM was detected differently by GoaMo-IgM followed by MoaGo-HRP Abs (indicated by asterisk). The dashed vertical lines between gel strips indicate where parts of the images were jointed from different gels. A loading control stained by Coomassie blue is also by guest on September 28, 2021 shown (K, right). generated a portion of CD172bri cells (Fig. 4B–D). These obser- and 4 plus 47% with stromal cells) whereas IgLk expression is vations correspond with our earlier findings that a portion of early mostly lost (2 plus 2% in medium alone and 2 plus 3% with stromal B cell lineage cells can revert to myeloid cells (23). A third ob- cells). These results indicate that IgLl+ cells accumulate during servation was that cultivation of subsets 0–2b generated few early phases of B cell development whereas IgLk+ cells disappear. CD1722IgL+ immature B cells (Fig. 4A–D). This is also the case Finally, subset 4 (Fig. 4F) generated significantly more IgLk+ im- for subset 3, but only when cultivated with stromal cells (Fig. 4E). mature B cells (18% in medium alone and 21% with stromal cells) This indicates that some of the early subsets can generate immature than IgLl+ immature B cells (2% in medium alone and 5% with B cells directly but that subset 3 cannot develop further without stromal cells). In contrast, subset 5 (Fig. 4G) generated significantly help from stromal cells. This finding confirms the presence of a more IgLl+ immature B cells (10% in medium alone and 20% with developmental checkpoint associated with the subset 3 described stromal cells) than IgLk+ immature B cells (4% in medium alone earlier (23). The developmental block cannot be reached by all and 13% with stromal cells). These results indicate that IgLk+ upstream subsets after 4 d. This concerns mainly very early pop- immature B cells are preferentially generated from subset 4 whereas ulations (0–1a) that develop downstream but not into population 3 IgLl+ immature B cells can be also generated from subset 5. (Fig. 4A, 4B). Further developmental stages (populations 1b, 2a, and 2b) develop partially into subset 3 but do not develop further In the absence of IgH gene rearrangement, early precursors k (Fig. 4C, 4D). More time is probably needed for the earliest subsets display gradual restriction of the IgL repertoire whereas the l to develop into subset 3. Unfortunately, prolonged cultivation could IgL repertoire diversified not be done because of the excessive dying of cells (data not Sorted and thereafter cultivated subsets (Fig. 4) were also subse- shown). With regard to the stromal cells, there was no apparent quently examined for their CDR3 spectratypes to characterize the effect on the number of CD1722IgL+ immature B cells generated rearrangements (Fig. 5). This length analysis offers 1-bp resolu- by cultivation of subsets 0–2b (Fig. 4A–D). Alternatively, stromal tion and allowed determination of whether a particular subset had cells seem to have a positive effect on the genesis of CD1722IgL+ undergone selection for productive rearrangement, indicated by immature B cells in subsets 3–5 (Fig. 4E–G). Fourth, cultivation of almost exclusive presence of in-frame bands, or whether no se- subsets 0–2a with stromal cells generated significant amounts of lection had occurred such that many out-of-frame bands would be CD172+IgLl+ cells but almost no CD172+IgLk+ cells (Fig. 4A–C). present. Moreover, results can also show whether the rearranged This preferential generation of IgLl+ cells was especially apparent repertoire is expanded, resulting in a diversified distribution of for subsets 1b plus 2a (Fig. 4C). Thus, subsets 1b plus 2a remain bands, or whether diversification is restricted as represented by a generally IgLl+ during cultivation (5 plus 44% in medium alone scattered distribution of bands. We choose to analyze transcripts The Journal of Immunology 5

rather than DNA because: 1) both productive and nonproductive rearrangements are transcribed (33), 2) the yield of PCR ampli- fication is much higher for cDNA so that smaller amount of cells can be used for population studies, and 3) the analysis of cDNA reflects the actual transcription activity. In any case, comparison of DNA and cDNA amplifications did not reveal considerable dif- ferences (Supplemental Fig. 2). Staining, sorting, and cultivation were carried out by the same procedure as described in Fig. 4. CDR3 spectratypic analyses con- firmed that IgH transcripts were absent in sorted subset 1a–1b and infrequent in subsets 2a–2b when cells were cultivated at 4˚C, that is, the equivalent of freshly sorted cells (Fig. 5C, 5F, strip 4). However, both IgLk (Fig. 5A, 5D, strip 4) and IgLl (Fig. 5B, 5E, strip 4) transcripts were recovered from these early subsets. A comparison of the IgLk and IgLl transcripts in early subset 1a showed that the initial IgLk repertoire was more diverse and contained many out-of- frame transcripts (Fig. 5A, 5D, strip 4). The initial IgLl repertoire in subset 1a also showed prominent out-of-frame transcripts but the

repertoire was more restricted (Fig. 5B, 5E. strip 4). Further data Downloaded from showed that the CDR3 diversity of IgLk transcripts decreases as the cells develop to subsets 1b–3 (Fig. 5A, 5D, strip 4), whereas during the same transition, the diversity of IgLl transcripts increases (Fig. 5B, 5E, strip 4). These results show that in an absence of IgH gene rearrangement, the initially diversified IgLk repertoire becomes

restricted as B lineage cells develop whereas the initially restricted http://www.jimmunol.org/ IgLl repertoire is becoming diversified. This trend becomes more prominent when sorted subsets 1a–2b were cultivated in medium alone (compare Fig. 5A and 5D, strip M for IgLk spectratyping, with Fig. 5B and 5E, strip M for IgLl spectratyping, respectively). Repertoire diversity of IgL and IgH gene rearrangement is inhibited during the developmental checkpoint in subset 3 Apparently, there is little transcription of IgLk (Fig. 5A, 5D, stars),

IgLl (Fig. 5B, 5E, stars), and IgH rearrangements (Fig. 5C, 5F, by guest on September 28, 2021 stars) when subset 3 is cultivated in medium alone. However, this apparent transcriptional block is overcome when subset 3 was cul- tivated together with stromal cells (Fig. 5, S strips). These results agree with the FCM analyses (Fig. 4) and demonstrate that subset 3 has a transcriptional block in the absence of stromal cells. These findings also correlate with the earlier described developmental checkpoint in subset 3 (23). Interestingly, cultivation of subset 3 in medium alone also resulted in considerable differences in IgH spectratypic profiles between cDNA and DNA that were not ob- served in any other samples (Supplemental Fig. 2). The apparent lack of IgH transcripts was accompanied by an entirely unselected IgH repertoire in DNA. The finding that in-frame IgH rearrangements had the same intensity as out-of-frame rearrangements indicates a loss of selection for productive rearrangements and subsequent transcription blockade in the absence of stromal cells. IgL repertoire in late developmental stages is selected in IgL+ cells but unselected in IgL2 cells The analyses of later developmental stages in subsets 4–6 reveal FIGURE 3. Detection of rearrangement-specific products and transcripts that they contain a selected IgH repertoire (Fig. 5C, 5F, strip 4) from sorted BM cells. According to expression of CD172a and either IgLl (A) or IgLk (B) on large and small MHC-II+ BM cells, individual subsets and that selection for in-frame rearrangements was similar when 0–6 were sorted by FCM as pooled cells. Polymorphonuclear cells (PMN) cells were cultivated in medium alone (Fig. 5C, 5F, strip M) or were included as non–B lineage cells and sorted according to expression of with stromal cells (Fig. 5C, 5F, strip S). These findings indicate CD14 and SWC8 (23). Each sorted subset was thereafter examined (C) for that subsets 4–6 went through selection for productive IgH gene the presence of rearrangement-specific products by DNA amplification and rearrangements, which is consistent with earlier findings (23). also transcripts by cDNA amplification (cDNA) using primer sets noted in Supplemental Table I. The results are representative of five independent experiments done for IgLl (A) and IgLk (B) sorting with the same output. PCR cycles was used. The dashed vertical line in SJC DJH amplification indicates In a particular experiment, only the same amount of sorted cells was used where parts of the image were jointed from different gels due to a different for isolation of total RNA and DNA and the same number of amplification arrangement of samples. Cm, nonrearranging portion of IgM C region. 6 IgL PRECEDES IgH GENE REARRANGEMENT IN SWINE Downloaded from http://www.jimmunol.org/

FIGURE 4. FCM analysis of IgL expression on sorted and thereafter cultivated BM cell subpopulations. Individual BM subsets 0–6 (A–H) were aseptically sorted by FCM as pooled cells (for sorting strategy, see Fig. 3) and analyzed in individual sets of three dot plots in line for each sort. Dot plots are representative of nine independent experiments and show surface expression of CD172a and IgLl on different subsets after cultivation of sorted cells in RPMI 1640 medium for 4 d at 4˚C (first dot plots in sets), at 37˚C (second dot plots in sets), and at 37˚C in presence of BM stromal cells (third dot plots in sets). Note that all sorted subsets were restained for the same molecules after cultivation. Cells cultivated at 4˚C had the same phenotype as freshly sorted cells, and subset numbers are given below these dot plots. Only staining for IgLl is shown because staining for IgLk gave similar results. However, the by guest on September 28, 2021 proportions of individual populations differed in some cases for IgLl and IgLk sorting and staining. For this reason, numbers under dot plots indicate the proportions of individual cell populations for IgLl and IgLk sorting separated by a slash. Statistically significant differences (p , 0.01) between IgLl and IgLk staining are indicated by underlined values.

However, analyses of IgL transcripts disclose that selection for in- (Fig. 6A), and the same strategy was done for IgLk sorting (Fig. 6B). frame rearrangement is dependent on the type of IgL for which As is evident from CDR3 analyses of sorted cells, IgLlo populations cells were sorted. Specifically, Fig. 5A shows that CDR3 spec- always contained selected in-frame transcripts for the same type tratypes for IgLk transcripts were always selected in subsets 4 and of IgL (Fig. 6D, 6E, populations I and III) whereas depleted IgL2 5 but unselected in subset 6 for IgLl sorting (Fig. 5A, arrow). The populations always contained a selected CDR3 repertoire for the same is true for IgLl transcripts in a case of IgLk sorting (Fig. 5E, second type of IgL (Fig. 6C, 6F, populations II and IV). This is a arrow). Oppositely, Fig. 5B shows that IgLl transcripts were al- clear demonstration that the surface staining discriminates two ways selected in subset 6 but unselected in subsets 4 and 5 for Igl types of IgL in an allelic-exclusive manner. Thus, IgLl+IgLk2 cells sorting (Fig. 5B, arrows). A similar situation was found for IgLk have productive IgLl gene rearrangements in IgLl+ and IgLk2 transcripts in the case of IgLk sorting (Fig. 5D, arrows). These fractions but nonproductive gene rearrangements in IgLl2 and results indicate that only productive IgL gene rearrangements can IgLk+ fractions. Similarly, IgLl2IgLk+ cells have productive IgLk be expressed on the surface of cells. Furthermore, both IgLl+ and gene rearrangements in IgLl2 and IgLk+ fractions but nonpro- IgLk+ late B lineage cells with IgH gene rearrangements also ductive gene rearrangements in IgLl+ and IgLk2 fractions. Im- contain IgL transcripts for the second type of IgL that is mostly portantly, these results also indicate that IgLk+ as well as IgLl+ nonproductive. To further elucidate these conclusions we have B cells may contain rearrangements for the second type of IgL, performed a more detailed sorting described below. which is different from the mouse/human paradigm that IgLl genes rearrange only when IgLk genes are consumed (5, 6). Late IgLl+ precursors contain nonproductive IgLk transcripts and IgLk+ precursors contain nonproductive IgLl transcripts IgLk precedes IgLl gene rearrangement, but many IgLl+ To investigate late B lineage precursors with IgH gene rear- B cells are generated early in B cell development whereas most k+ rangement, more stringent sorting conditions for subsets 4 and 5 IgL cells are generated later were established. Importantly, note that all earlier sorting proce- Detection of IgLl and IgLk on the surface of precursors in the dures were set to acquire strictly IgL2 cells for subsets 4 and 5. absence of IgH prompted us to sort MHC-II+ BM cells by gating However, subsets 4 and 5 also contain IgLlo cells. For this reason, on those bearing IgLl and/or IgLk (Fig. 7A), and then to analyze we have set up a new sorting strategy (Fig. 6) to sort IgLl2 and these for size and CD172a expression (Fig. 7B–F). As shown ear- IgLllo cells for large and small MHC-II+CD172a2 BM cells lier, CD172a expression decreases as B cell lineage cells develop The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. CDR3 spectratypic analyses of sorted and thereafter cultivated BM cell subpopulations. According to expression of CD172a and either IgLl (A–C) or IgLk (D–F) on large and small MHC-II+ BM cells, individual subsets (depicted as “sub.” above gel strips) 1–6 were sorted by FCM as pooled cells and cultivated in RPMI 1640 medium for 4 d at 4˚C (depicted as “4” above gel strips), at 37˚C in medium only (depicted as “M” above gel strips), and at 37˚C in presence of BM stromal cells (depicted as “S” above gel strips). The same amount of sorted and thereafter cultivated cells for each subset was used for cDNA preparation and subsequent CDR3 analysis of VJk (first row of gels), VJl (second row of gels), and VDJH rearrangements (third row of gels). Lines in gels represent position of in-frame rearrangements (3 bp difference), which was previously demonstrated by sequence analyses (32). The asterisks by guest on September 28, 2021 indicate a developmental stop in subset 3 for cultivated cells in medium only. The arrows indicate an unselected IgL repertoire in dependence on which IgL subsets were cell sorted (see text). Results are representative of four independent experiments. further (23). Unexpectedly, we found three populations of IgLlo BM IgLk2 cells in ∼3d,IgLlloIgLklo precursors do so in ,1d.These cells that were composed of IgLl2IgLklo (R1), IgLlloIgLklo (R2), results indicate that the loss of surface IgLk expression in IgLlloIgLklo and IgLlloIgLk2 (R3) subsets (Fig. 7A). Analysis of the sorted cells after IgLl gene rearrangement occurs in hours. IgLl2IgLklo subset (Fig. 7A, R1) showed that they were composed In accordance with cultivation studies (Fig. 7M), the freshly almost exclusively of large CD172abri/hi early precursors (Fig. 7B) sorted IgLlloIgLk2 subset (Fig. 7A, R3) did not contain IgLk that contained only IgLk and RAG transcripts (Fig. 7H). transcript but did contain IgLl, and also contained partial DJH and lo lo The sorted IgLl IgLk subset (Fig. 7A, R2) also contained full VDJH rearrangements as well as Vk–KDE and RE–KDE large CD172ahi precursors, but CD172abri cells were almost rearrangements (Fig. 7J). FCM analysis showed that these IgLllo missing (Fig. 7C). Interestingly, cells with surface IgLl and IgLk IgLk2 cells consisted of some large CD172ahi precursors but proteins lacked detectable IgLk transcripts but contained IgLl mainly of their progenies, including small and large CD172alo and transcripts and carried Vk to KDE and RE to KDE rearrangements CD172a2 cells (Fig. 7D). (Fig. 7I). An absence of IgLk transcripts in cells with Vk–KDE Finally, we analyzed the sorted IgLl2IgLkhi (Fig. 7A, R4) and and RE–KDE rearrangements indicates that IgLlloIgLklo cells had IgLlhiIgLk2 (Fig. 7A, R5) BM cells and found that they represent undergone IgLk gene rearrangement and the Ck gene segment had exclusively CD172a2 immature B cells (Fig. 7E and 7F, respec- been deleted. Therefore, the presence of IgLk proteins on the tively). Both populations contained IgH and IgL transcripts and surface of IgLlloIgLklo precursors is a remnant of a previous other products of rearrangements (Fig. 7K, 7L). Combined with translation event. We interpret these findings as evidence that IgLk other data presented above, these studies indicate a developmental precedes IgLl gene rearrangement as has been described in mice transition from IgLk to IgLl transcription that is followed by the (5–7), but the initial IgLk locus activity is quickly replaced by second wave of IgLk gene rearrangement. rearrangement in the IgLl locus prior to the onset of IgH gene rearrangement. Neither IgLl2IgLklo (Fig. 7H) nor IgLlloIgLklo (Fig. 7I) precursors carry partial DJH or full VDJH rearrangements. Discussion To further investigate the loss of IgLk expression, IgLl2IgLklo Our findings indicate that in swine the order of IgH and IgL loci and IgLlloIgLklo cells were sorted and subsequently cultivated for recombination is reversed from that in mice and as described in 1–5 d (Fig. 7M). Both populations were shown to lose IgLk expression textbooks (3, 4). This results in the expression of IgL without IgH in and differentiate into IgLlloIgLk2 cells. Kinetic studies demonstrated early B cell precursors (subsets 0–1). The observation that IgL gene that whereas IgLl2IgLklo precursors develop into .90% IgLllo rearrangement can be initiated before IgH gene rearrangement and 8 IgL PRECEDES IgH GENE REARRANGEMENT IN SWINE

FIGURE 6. CDR3 spectratypic analyses of late CD172a2 B lineage precursors. According to expression of CD172a and either IgLl (A) or IgLk (B) on large and small MHC-II+ BM cells, individual subsets I–IV (depicted in dot plots) were sorted by FCM as pooled cells. The same amount of sorted cells for each subset was used for cDNA preparation and subsequent CDR3 analysis of VJk (C and E) and VJl (D and F) rearrangements in IgLl (C and D) or IgLk (E and F) sorted cells. Subset numbers are depicted above each gel strip. Re- sults are representative of three independent experiments. Lines in gels represent position of in-frame rearrangements. Arrows indicate mostly unselected CDR3 repertoire. Downloaded from

pre-BCR expression in nonengineered swine supports the conclu- The reverse order of IgH and IgL gene rearrangements brings sion that IgH and IgL gene rearrangements are independent. The into question the concept of allelic exclusion because IgL gene same was predicted from studies done using IgH-deficient mice (2, rearrangement is not controlled by productive IgH gene rear-

10, 13, 34) and humans (35). rangement. However, an authentic IgL gene rearrangement has http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Detailed analyses of IgLl and/or IgLk expression during B cell development. Fresh BM cells were stained by anti–MHC-II, anti-CD172a, anti-IgLl, and anti-IgLk Abs, gated for MHC-II+ cells only, and individual IgLllo/hi and/or IgLklo/hi subsets were sorted by FCM as pooled cells R1–R5 (A). Representative analysis of cell size (FSC) and expression of CD172a in sorted cells is shown (B–F). Each sorted subset was thereafter examined for the presence of rearrangement-specific transcripts and products by PCR amplification (H–L) using primer sets noted in Supplemental Table I. The results are representative of three independent experiments and they are shown under each dot plot (B–F). The arrangement of shown PCR products is the same as depicted in the lower left outline (G). In a particular experiment, only the same amount of sorted cells was used for isolation of total RNA or DNA and the same number of amplification PCR cycles was used. Note that porcine KDE has two substitutions in the nanomer (underlined nucleotides in the sequence) that, however, does not prevent its function. Sorted subsets R1 and R2 were also cultivated in RPMI 1640 medium for 1–5 d (M). Representative dot plots before and after 3 d culture are shown, and the decline in the frequency of sorted IgLkloIgLl2 (R1, solid line) and IgLkloIgLllo cells (R2, dashed line) during 5 d of culture is depicted in a graph. Note that all sorted subsets were restained for the same molecules after cultivation. The Journal of Immunology 9 been shown to serve in selection of the IgH repertoire when SLC is rearrangement in mice is controlled by productive IgH gene deleted from the genome (14, 36). Presumably, this occurs natu- rearrangement (35, 36), whereas our findings in this study thus rally in swine that lack a SLC (18). Only targeted disruption of the indicate that productive IgL are able to escape from the endoplasmatic membrane exon of IgH genes causes allelic inclusion (34). Studies reticulum without chaperoning by IgH. A similar phenomenon in chickens demonstrate that allelic exclusion occurs when IgL wasobservedinhumanswhereIgLproducedinexcessofIgH and IgH genes are competitively rearranged (15, 16, 37). Fur- are secreted as free IgL (42) and can then associate with the outer thermore, sharks display allelic exclusion even though they have membrane of cells via interaction with phospholipids such as many functional IgL genes that are prerearranged in the germline sphingomyelin A (43). Moreover, free IgL associate only on the and subsequently expressed (38). Thus, allelic exclusion appears surface of cells that produce these IgL (43). Our results also to be independent of SLC and occurs irrespective of the IgH/IgL exclude the possibility that free IgL on a surface can be adven- recombination order. titiously acquired from other sources because intracellular stain- Although IgL precedes IgH gene rearrangement in the pig, IgL ing showed the same IgL isotype inside of cells, and sorting gene rearrangements occur in the same stepwise order as in humans revealed rearranged IgL in DNA and in transcripts of the same IgL and mice, starting with IgLk genes and proceeding to IgLl genes isotype. In any case, further analyses are needed to explain the (5–7). However, initial IgLk gene rearrangements in swine are attachment of free IgL on the cell surface and their possible role in rapidly replaced by IgLl gene rearrangement before recombina- B cell selection. tion of IgH genes, giving the impression that IgLl precedes IgLk Our data indicate that swine do not use an invariable SLC but rearrangement in swine (18). The first substantial IgH gene rear- rather authentic IgL. This could be an advantage because each type 2 rangement occurs in subset 3, and these IgLlloIgLk precursors of IgL could serve as a different type of SLC without excessive Downloaded from can generate only IgLl+ B cells. Most IgLk+ B cells are generated skewing of IgH repertoire (36). Usage of authentic IgL instead of subsequently from subsets 4 and 5 (Fig. 4). This indicates that SLC for selection of the IgH repertoire may not be limited to IgLk gene rearrangement occurs in two successive waves in dif- swine because inactivation of SLC in mice does not prevent an ferent developmental stages with IgLl gene rearrangement in initiation of IgL gene rearrangement or development of mature between. Thus, most IgLl+ B cells are generated earlier whereas B cells, and it causes only a temporal decrease in a number of + most IgLk B cells arise later. Moreover, the second wave of IgLk B cells, which is compensated for latter in life (14, 36). Further- http://www.jimmunol.org/ gene rearrangement occurs in the presence of IgLl gene rear- more, mice deficient in SLC also have normal IgM serum levels rangement(s). This differs from the mouse-based paradigm in (14) and immune responses (36). One may speculate that SLC is which IgLl genes rearrange only when IgLk genes are consumed, only important for species in which IgH precede IgL gene rear- which results in .90% of IgLk+ B cells (6). rangement. Another group of species may lose (or did not de- The present study confirmed the developmental checkpoint in velop) l5 gene (37), including swine (18, 24). It remains to be subset 3 when the selection for a productive IgH gene rear- investigated whether a pre-BCR–independent developmental rangement occurs (23). These small MHC-II+CD172alo precursors pathway is evolutionarily more ancient than the pre-BCR–driven cannot develop further without BM stromal cells whereas it is pathway. Our current belief is that IgH before IgL gene rear- possible for subsequent developmental stages (Figs. 4, 5). This rangement is the most ancestral because this is used in amphibians by guest on September 28, 2021 closely resembles mouse preB-II cells that do not need the BM (44). In any case, one group of animals might increase an effi- and/or cytokines for expansion once they express functional pre- ciency of B cell generation by employing components of SLC BCR (1). FCM showed that subset 3 almost exclusively expresses whereas others might invert the order of IgH and IgL gene IgLl proteins. The lack of stromal cells (or BM environment) rearrangement or employ yet other mechanisms such as gene should therefore lead to accumulation of IgLl+ cells. This is ex- conversion in chickens (37) or usage of prerearranged IgL genes actly what we described in vivo during early ontogeny (18) when in sharks (38). BM is not functional (32), which led us to the incorrect conclusion that IgLl may precede IgLk gene rearrangement in swine (18). Acknowledgments The differences in the ability of fetal liver and BM to support We thank Lucie Poulova, Mirka Kratochvilova, Sarka Pfeiferova, and B cell development (4) appear to be related to the developmental Blanka Dusankova for excellent technical assistance. Our warm gratitude checkpoint in subset 3 in which the IgLl+ B cell accumulates. also goes to John E. Butler and Nancy Wertz (University of Iowa, Iowa City, Differences in this checkpoint or its timing might explain why IA) for discussions and critical reading of the manuscript. certain species express .90% of IgLl proteins such as in horses, cows, sheep, cats, or dogs (39) rather than being simply attributed Disclosures to a disproportionate repertoire of germline IgLk versus IgLl The authors have no financial conflicts of interest. genes (30, 31). Thus, it would be interesting to further study these species to establish the order of IgL and IgH gene rearrangement and whether they possess a consensus SLC and the IgLl/IgLk References 1. Rolink, A. G., T. Winkler, F. Melchers, and J. Andersson. 2000. Precursor B cell ratio throughout B cell development. Studies in transgenic mice receptor-dependent B cell proliferation and differentiation does not require the that have prolonged time for successive IgL gene rearrangement bone marrow or fetal liver environment. J. Exp. 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H. Potockova, K. Karova, and J. Sinkorova. 2011. Ileal Peyer’s patches are not Immature surface Ig B cells can continue to rearrange k and l L chain gene by guest on September 28, 2021 necessary for systemic B cell development and maintenance and do not con- loci. J. Exp. Med. 178: 1263–1270. tribute significantly to the overall B cell pool in swine. J. Immunol. 187: 5150– 41. Shaffer, A. L., and M. S. Schlissel. 1997. A truncated heavy chain re- 5161. lieves the requirement for surrogate light chains in early B cell development. 21. Butler, J. E., and M. Sinkora. 2013. The enigma of the lower gut-associated J. Immunol. 159: 1265–1275. lymphoid tissue (GALT). J. Leukoc. Biol. 94: 259–270. 42. Hopper, J. E., and E. Papagiannes. 1986. Evidence by radioimmunoassay that 22. Potockova, H., J. Sinkorova, K. Karova, and M. Sinkora. 2015. The distribution mitogen-activated human blood mononuclear cells secrete significant amounts of of lymphoid cells in the small intestine of germ-free and conventional piglets. light chain Ig unassociated with heavy chain. Cell. Immunol. 101: 122–131. Dev. Comp. Immunol. 51: 99–107. 43. Hutchinson, A. T., P. A. Ramsland, D. R. Jones, M. Agostino, M. E. Lund, 23. Sinkora, M., and J. Sinkorova. 2014. B cell lymphogenesis in swine is located in C. V. Jennings, V. Bockhorni, E. Yuriev, A. B. Edmundson, and R. L. Raison. the bone marrow. J. Immunol. 193: 5023–5032. 2010. Free Ig light chains interact with sphingomyelin and are found on the 24. Wertz, N., J. Vazquez, K. Wells, J. Sun, and J. E. Butler. 2013. Antibody rep- surface of myeloma plasma cells in an aggregated form. J. Immunol. 185: ertoire development in fetal and neonatal piglets. XII. Three IGLV genes com- 4179–4188. prise 70% of the pre-immune repertoire and there is little junctional diversity. 44. Mussmann, R., M. Courtet, and L. Du Pasquier. 1998. Development of the early Mol. Immunol. 55: 319–328. B cell population in Xenopus. Eur. J. Immunol. 28: 2947–2959. Supplemental Table I: List of primers used in this study: Amplification Template # 1st Round Sense # 1st Round Antisense Tm # 2nd Round Sense # 2nd Round Antisense Tm (CDR3 spectratyping) (CDR3 spectratyping) b-actin cDNA ATTGTCATGGACTCTGGGGA GATCCACACGGAGTACTT 58°C TCATGAAGATCCTCACGGAG GATCCACACGGAGTACTT 58°C TdT cDNA GAGCTCAGTGATTCTGTC GAACTTTCTCCATCTTCAA 53°C GTCTCCTGGCTGATAGAA GAACTTTCTCCATCTTCAA 53°C RAG cDNA GAGAATGAATGGCAACTT GACTGCCTGGCATTCATT 55°C CATGAAGCTCTGAGGGAA GACTGCCTGGCATTCATT 55°C SJC D-JH DNA S4 GGTTTTTGCCACCGGGTCTG S5 TGGGCCGCCCTCCAAGGA 58°C S4 GGTTTTTGATGGGCGCTGTG S6 GACAAGGGCTTCTTGGCCTG 58°C SJC V-DJH DNA S1 GGTTTGTGTCTGGGCTCAGA S2 CGGTCCCGGGAGGACAGA 58°C S1 GGTTTGTGTCTGGGCTCAGA S3 CCCACACACTTTGGGGAAAAT 58°C DJH DNA TGCCTGTGCCCTAGACCAT TGAGGACACGACGACTTCAA 56°C CGGCCGATGACGTACT TGAGGACACGACGACTTCAA 56°C VDJH DNA H1 GAGGAGAAGCTGGTGGAGT H4 TGAGGACACGACGACTTCAA 58°C H2 CTCCTGTGTCGGCTCTGGA H4 TGAGGACACGACGACTTCAA 58°C H3 (GTTTCTTTGAGAACCGAAGACACGGC) (55°C) VDJH cDNA H1 GAGGAGAAGCTGGTGGAGT H5 CGGGAAGTCCTGGATGTT 58°C H2 CTCCTGTGTCGGCTCTGGA H4 TGAGGACACGACGACTTCAA 58°C H3 (GTTTCTTTGAGAACCGAAGACACGGC) (55°C) VJk DNA K1 CTCCTGGGGCTCCTCCTGC K4 CCGTTTGAGCTCCAGCTT 58°C K2 CCATYGTGYTGACCCAGWCTCCAC K4 CCGTTTGAGCTCCAGCTT 58°C K3 (TGGATCAGGSACAGATTTCACCCT) (55°C) VJk cDNA K1 CTCCTGGGGCTCCTCCTGC K5 CACACTCGTTCCTGTTGAA 58°C K2 CCATYGTGYTGACCCAGWCTCCAC K5 CACACTCGTTCCTGTTGAA 58°C K3 (TGGATCAGGSACAGATTTCACCCT) (55°C) VJl DNA L1 GGTGCTTCTGATCGGGCT L5 GAGGACGGTCAGATGGGTCC 58°C L3 TGATCCAGGAGCCGGCGA L6 AGATGGGTCCCACCGCCGAA 58°C L2 CCCTCTCCTGCYCCCCC L4 (CTGAGGACGAGGCCGACTA) (55°C) VJl cDNA L1 GGTGCTTCTGATCGGGCT L7 CTGGCCGCGTACTTCTT 58°C L3 TGATCCAGGAGCCGGCGA L6 AGATGGGTCCCACCGCCGAA 58°C L2 CCCTCTCCTGCYCCCCC L4 (CTGAGGACGAGGCCGACTA) (55°C) Cm exon DNA C1 GCACATGCCGTCCGTGTA C3 CCACGGTCCTCTCGGTCA 58°C C2 GCGGACGTG TTCGTGCAG C3 CCACGGTCCTCTCGGTCA 58°C Vk to KDE DNA L1 CTCCTGGGGCTCCTCCTGC R2 TCCAGACGTATACCTTGAGAATGAG 58°C L1 CTCCTGGGGCTCCTCCTGC R4 AGGCAGATGATCTTCTGTGG 54°C RE to KDE DNA R1 TCGCGATTGAATGACCTTGG R2 TCCAGACGTATACCTTGAGAATGAG 58°C R3 AACCCCGCGACCGCCCGTTCA R4 AGGCAGATGATCTTCTGTGG 58°C

Supplemental Figure 1: FCM analysis of IgL expression during ontogeny. The cells isolated from different tissues from DG35 (a-b) and DG55 fetuses (c-f), newborn piglet (g) and 3yr old sow (h) were stained for CD172a, MHC-II and IgLl. Representative dotplots show the expression of CD172a and IgLl on gated large and small MHC-II+ cells. Note that spleen and bone marrow at DG35 are rudimental and not populated by cells so they cannot be analyzed and shown. Arrows indicate large CD172a+ precursors of B cells expressing free IgLl on the surface. The results are representative of three independent experiments.

Supplemental Figure 2: Comparison of CDR3 spectratypic analyses done using cDNA and DNA isolated from sorted and thereafter cultivated subsets from bone marrow. Staining, sorting and cultivation were done by the same experimental design as described in Figure 4. cultivation in medium alone cultivation with stromal cells cDNA DNA cDNA DNA 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6

nt 3

R <24>

D <21> C -

k <18> L g

I <15>

1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6

nt

3 <30> R

D <27> C -

l <24> L g

I <21>

1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 1a 1b 2a 2b 3 4 5 6 nt

<69>

<60>

<51> 3 R

D <42> C - H g I <33>

<24>

<15>