Categorical selection of the antibody repertoire in splenic B cells

Categorical selection of the antibody repertoire insplenic B cells

Robert L. Schelonka1, Jason Tanner1, Yingxin Zhuang1, G. Larry Gartland1,Michael Zemlin2 and Harry W. Schroeder Jr.1

1 Departments of Pediatrics, Microbiology, Medicine, and Genetics, University ofAlabama at Birmingham, Birmingham, USA

2 Department of Pediatrics, Philipps Universit�t Marburg, Marburg, Germany

In the bone marrow, the passage of developing B cells through critical checkpoints ofdifferentiation is associated with a reduction of specific categories of CDR3 of the Igheavy chain (CDR-H3), particularly those with excessive hydrophobic or charged aminoacids and those with a length of eight or fewer residues. To gain insight into the role ofCDR-H3 content in the development of B cells in the spleen, we compared the sequencesof VH7183DJCl transcripts from sorted transitional T1, marginal zone, and follicularB cell subsets to those expressed by immature IgM+IgD– andmature IgMloIgDhi B cells inthe bone marrow. Although differences in VH utilization were noted, the T1 CDR-H3repertoire showed extensive similarity to that of immature bone marrow B cells, and thefollicular CDR-H3 repertoiremost resembled that of mature bonemarrowB cells. Unlikethe splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginalzone B cell CDR-H3 repertoire retained both short and highly charged amino acidmotifs, including those with two arginines. Our findings suggest that antigen bindingsites containing specific categories of CDR-H3 sequence content may inhibit, permit, oreven facilitate passage of the host B cell through critical checkpoints in peripheral aswell as central development.

Supporting information for this article is available athttp://www.wiley-vch.de/contents/jc_2040/2007/36569

Introduction

Unlike H chain complementarity determining regions(CDR)1 and 2, which are entirely encoded by theVH gene segment, CDR3 of the Ig H chain (CDR-H3) iscreated de novo by VDJ rearrangement [1, 2]. Impreci-sion in gene segment joining and variation in the extentof N nucleotide insertion create a CDR-H3 repertoire

that ranges from unmodified and intact germ-line-encoded sequence to sequences where extensive nibb-ling and N addition have obscured the identity of theDH progenitor. The broad range of diversity available toCDR-H3 has functional consequences, because itslocation at the center of the antigen binding site, asclassically defined, permits this interval to often play asignificant role in antigen recognition and binding[3–5].

We previously examined the composition of theCDR-H3 repertoire in bone marrow and found that itsessential characteristics were established early in B celldevelopment, well before the expression of surface IgM[6]. Transition through bone marrow checkpoints actedto focus the repertoire by reducing or eliminatingoutliers to what appeared to be a preferred distribution

Molecular immunology

Correspondence: Robert L. Schelonka, 525 NHB, 619 19th St.South, Birmingham, AL 35233-1530, USAFax: +1-205-934-3100e-mail: [email protected]

Received 4/8/06Revised 13/12/06

Accepted 24/1/07

[DOI 10.1002/eji.200636569]

Key words:Antibodies � B cells� Repertoire devel-opment � Rodent

� Spleen

Abbreviations: CDR-H3: CDR3 of the Ig heavy chain �FO: follicular B cells � MZ: marginal zone B cells �T1: transitional phase B cells

Robert L. Schelonka et al. Eur. J. Immunol. 2007. 37: 1010–10211010

f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

of VDJ gene segment usage, DH reading frame pre-ference, amino acid composition, length, predicted baseand loop structure, and hydrophobicity. Comparisons ofthe repertoire expressed in IgM+IgD– immature B cellsand that expressed by IgMloIgDhi mature, recirculatingB cells suggested that the process of CDR-H3 focusingmight continue in the periphery [7].

To test whether the presence or absence of specificcategories of CDR-H3 hydrophobicity and length arealso associated with control of B cell development innormal spleen, we sorted wild-type BALB/cJ spleenB cells into transitional (T1), marginal zone (MZ), andfollicular (FO) populations [8]; cloned, sequenced, anddeconstructed VH7183DJCl transcripts; and comparedthese splenic CDR-H3 repertoires to those expressed inan expanded set of sequences from wild-type BALB/cbone marrow immature and mature B cells.

We show here that the splenic T1 B cell CDR-H3repertoire closely resembles that of immature bonemarrow B cells while the FO CDR-H3 repertoire mirrorsthat of mature bone marrow B cells. Splenic transitionalcell to mature FO development was characterized by thereduced representation of highly hydrophobic, highlypolar (charged), and short CDR-H3 sequences, support-ing the view that the final focusing of the repertoireoccurs in the periphery, likely as a result of exposure toantigen. Splenic MZ appeared to have a privilegedrepertoire enriched for charged and shorter CDR-H3 anddepleted of longer CDR-H3. Our findings suggest thatspecific categories of CDR-H3 length, hydrophobicity,and amino acid motifs influence peripheral B celldevelopment by facilitating or inhibiting entry intoindividual compartments in the spleen.

Results

Composition of spleen and bone marrow B cellCDR-H3 sequences

We cloned 397 V7183DJCl sequences from spleen, ofwhich 364 were in-frame, open, and unique (SeeSupporting Information for all of the unique CDR-H3

sequences reported in this manuscript). Of these,102 were derived from sorted splenic T1(CD19+sIgMhiCD21lo) B cells, 151 from MZ(CD19+CD21hiCD23lo), and 111 from FO(CD19+CD21+CD23+) [8] (Fig. 1). We compared thesesets of sequences to a compendium of 255 unique,translatable sequences from bone marrow CD19+sIgM-hisIgD– fraction E and 254 sequences from CD19+sIgMlo-

sIgDhi fraction F, including 57 fraction E and60 fraction F sequences newly obtained for this work[6, 7, 9, 10].

Immature and T1 CDR-H3 lengths lie betweenMZand FO mature, recirculating CDR-H3

Between fraction E and fraction F, the average length ofCDR-H3 increased by 1.5 bases, or a half-codon(p=0.02, Student's t-test, two-tailed; Fig. 2). Theaverage length of T1 CDR-H3 matched bone marrowfraction E, whereas the average length of FO CDR-H3matched bone marrow fraction F. MZ CDR-H3 were onehalf-codon shorter on average than of fractions E andT1, placing them a full codon shorter than CDR-H3 fromFO (p�0.05) and bone marrowmature fraction F B cells(p�0.005).

The variance in CDR-H3 length narrowed during theprogression from fraction E to T1 to FO to fraction F(p<0.04; Fig. 3). This reflected, in part, elimination ofCDR-H3 consisting of eight or fewer codons, which arehereafter defined as 'short'. Short CDR-H3 comprised11% of fraction E and T1, but only 4% of FO andfraction F sequences (p�0.001, fraction E vs.fraction F). Fraction E cells had a higher prevalenceof CDR-H3 in which extensive exonucleolytic nibblingprecluded identification of the DH (21% fraction E vs.13% fraction F or FO, p<0.03).

The prevalence of short CDR-H3 in themarginal zone(13%) was not different from fraction E and T1, butmore than three times that in the follicle or fraction F(p�0.01; Fig. 3). When compared to splenic FO andbone marrow mature fraction F CDR-H3, marginal zonesequences were also depleted of CDR-H3 of 15 or morecodons (19% vs. 12%, p�0.05). Thus, the shorter lengthof MZ CDR-H3 reflected both enrichment for shortsequences and depletion of longer ones. This shiftresulted in a distribution of CDR-H3 lengths of equalvariance between splenic FO and bone marrow maturefraction F CDR-H3 vs. MZ CDR-H3, even though themean was one codon shorter.

To further assess the relative contribution of VDJgerm-line sequence, nibbling, and N addition to theseshifts in CDR-H3 length, we deconstructed CDR-H3 thatcontained identifiable DH gene segments (Fig. 4). FOand mature fraction F CDR-H3 contained a greaternumber of N nucleotides between V and D than among

Figure 1. Representative gates used to identify and sort spleenB lineage cell populations.

Eur. J. Immunol. 2007. 37: 1010–1021 Molecular immunology 1011


Figure 2. Average CDR-H3 length and hydrophobicity of VH7183DJCl transcripts isolated from sorted spleen and bone marrowB cells. (A) Average CDR-H3 codon lengths; *p�0.05 compared to fraction F, ***p�0.005. (B) Average CDR-H3 hydrophobicities asassessed by a normalized Kyte–Doolittle scale [14, 15] from bonemarrow fraction E (immature), spleen transitional T1, spleen FO,bone marrow fraction F (mature, recirculating), and spleen MZ CDR-H3. Error bars depict the standard error of each mean. Thesolid line depicts the accepted relationship between these developing B cell subsets. The dotted line depicts the potentialrelationship between T1 and MZ. *p�0.05 compared to fraction E.

Figure 3. Distribution of CDR-H3 lengths and average hydrophobicity of VH7183DJCl transcripts isolated from sorted spleen andbonemarrow B cells. The tissue source of the cells is given in the leftmost column, followed by the B cell subset and the number ofunique sequences from each subset. The distribution of CDR-H3 lengths in codons is shown in the penultimate middle column,and the distribution of average CDR-H3 hydrophobicities is given in the rightmost column. Prevalence is reported as the percent ofthe sequenced population of unique, in-frame, open transcripts from each B cell subset. To facilitate visualization of the change invariance of the distribution, the lines mark the preferred range of lengths in bone marrow fraction F.



those from fraction E and transitional T1 (p�0.05). MZexhibited a decrease in the contribution of JH sequenceto CDR-H3 (p�0.05), which opposes the patternobserved during development of fraction F B cells inthe bone marrow [6].

MZ CDR-H3 included fewer total N nucleotides thanthe FO and fraction F subsets (5.9�0.4 vs. 7.4�0.3,p<0.003). This analysis combines the extent ofN addition between V and D and between D and J.The relative paucity of N nucleotides in MZ reflected ahigher prevalence for CDR-H3 that lacked N nucleotidesentirely. Less than 2% of fraction E, T1, FO, andfraction F CDR-H3 were N addition-free, while 8% ofMZ contained only germ-line sequence (p=0.004).

More extensive exonuclease nibbling of germ-linesequence also contributed to the decrease in lengthobserved inMZ CDR-H3. MZ germ-line nucleotide lossesexceeded that of T1 by 1.9 nucleotides, FO by1.5 nucleotides, fraction E by 1.5 nucleotides, andfraction F by 1.4 nucleotides, (p=0.01, p=0.03,p=0.01, and p=0.02, respectively).

Altered usage of DH and JH in MZ

Usage of DH DFL family members was enhanced in MZwhen compared to fraction E, T1, and FO (p�0.03;

Fig. 5A). There were no differences in reading framepreference for any of the cell populations (Fig. 5B).Usage of JH2 was also increased in the MZ (p�0.01 vs.fractions E and F; Fig. 5C). Among the MZ CDR-H3 thatcontained identifiable DH, increased use of JH2, theshortest of the four JH gene segments, was a majorcontributor to the decrease in germ-line JH sequenceamong MZ CDR-H3. MZ CDR-H3 using JH2 were1.2 codons shorter than mature recirculating B cellbone marrow transcripts using the same JH (p=0.006).This again reflected both an increase in nibbling and adecrease in N addition (data not shown). A decrease inthe use of JH4, the longest JH, was a second JH factor inthe overall decrease in MZ CDR-H3 length (Fig. 5C).

Usage of VH81X (VH7183.1) is uncommon in allsplenic subsets, including the marginal zone

Usage of VH81X (VH7183.1) decreased from 7% offraction E to only 2% of fraction F (p=0.02; Fig. 5D).VH81X is the most frequently rearranged member of theVH7183 family [11]. In our studies, VH81X contributed toone-third of the VH7183 transcripts from the earliestB cell progenitors analyzed [6]. As these progenitorssuccessfully navigate through sequential developmentalcheckpoints in the bone marrow, VH81X usage declines.

Figure 4. Deconstruction of the components contributing to CDR-H3 length in sequences containing identifiable DH genesegments. The potential contribution of the germ-line sequence of the VH gene segment, P junctions, N region addition, theDH gene segment, and the JH gene segment to CDR-H3 length is illustrated. The length of DFL16.2 is identical to that of DSP familymembers. The B cell subset is identified in the left column, followed by the number of sequences analyzed in the flanking columnto the right. All components are shown to scale. All comparisons in length were made to the referent population, bone marrowfraction F; *p�0.05, **p<0.01, ***p�0.005.



Usage of VH81X in T1 B cells (2%) matched thatobserved in fraction F. This suggests that selectionagainst VH81X either disfavored release from the bonemarrow or reduced entry into the spleen.

Although B cells in a VH81X H chain-transgenicmouse tended to accumulate in theMZ [12], in these un-manipulated mice with access to a broad, diverserepertoire accumulation of VH81X sequences was notobserved in the marginal zone. To the contrary, only oneof 151 MZ sequences bore VH81X (<1%).

Usage of other VH7183 gene segments also differedbetween bone marrow fraction E and spleen. Taking thespleen as awhole by combining T1,MZ, and FO, usage ofVH7183.18 (p=0.02) and VH7183.13 (p=0.02) wasincreased, whereas usage of VH7183.10 (p<0.05) andVH7183.1 (VH81X) (p=0.003) was diminished (Fig. 5D).A shift in the use of VH7183.18 from fraction E toT1 wassignificant at p=0.01. A trend towards increased use ofVH7183.12 and VH7183.13, and towards a decrease inthe use of VH7183.10 and VH7183.1 (VH81X), is alsoapparent upon inspection (Fig. 5D), although the

numbers of sequences compared were insufficient toachieve statistical significance. These shifts in VH usageaway from that observed in fraction E appeared tointensify in the MZ compartment, where the increaseduse of VH7183.12 and VH7183.13 approached orachieved significance (p=0.06 and p<0.01, respec-tively).

VH usage in FO was very similar to that observed inbone marrow mature fraction F (Fig. 5D). This wouldsupport the hypothesis that FO and the recirculatingfraction of mature B cells in the bone marrow are theproducts of a similar range of selection pressures.

Altered use of glycine and proline in marginalzone CDR-H3

A general bias for tyrosine and glycine and against use ofhighly charged or highly hydrophobic amino acids in theCDR-H3 loops is established in early B cell development[6]. The same general pattern of amino acid usage wasapparent in the spleen. However, subtle modifications

Figure 5. DH, DH reading frame, JH and VH usage of VH7183DJCl transcripts isolated from sorted spleen and bone marrow B cells.VH, DH, and JH usage is reported as the percent of the sequenced population of unique, in-frame, open transcripts from bonemarrow fraction E (immature), spleen transitional T1, spleen FO, bone marrow fraction F (mature, recirculating), and spleen MZCDR-H3. The number of sequences analyzed is given in the bottom right. Usage is reported as the percent of the sequencedpopulation of DFL- and DSP-containing transcripts from each B lineage population. Arrows point to features of particular interest.(A) DH family usage. (B) DH reading frame usage among CDR-H3-containing members of DFL or DSP DH families. (C) JH genesegment usage. (D) VH7183 family gene segment usage. The VH segments are arranged in germ-line order with the most DH

proximal sequences to the right.



were observed in marginal zone. When compared tobone marrow immature fraction E, usage of proline inMZ was significantly reduced (p=0.004) and usage ofglycine was enhanced (p<0.02) (data not shown).Differences in amino acid usage were observed for otheramino acids, but did not achieve statistical significance.

Altered range of predicted CDR-H3 loop structuresin the marginal zone

The “H3-rules” of Shirai et al. [13] can be used to predictCDR-H3 structure. First, the base of the loop can bepredicted to form either a kinked, extra-kinked, orextended structure. Second, among those sequenceswhere kinked or extra-kinked CDR-H3 bases are likely,the structure of subsets of the loops can be predicted toform either intact hydrogen bond ladders or deformed(broken) hairpins.

At the base of CDR-H3, bone marrow immaturefraction E sequences were less likely to contain a kinkedstructure (81%) than MZ (93%) or FO (95%) popula-tions (p<0.05), and more likely to contain extra-kinkedbase structures (11%) than MZ (2%) or FO (5%)(p<0.05) (Fig. 6). Bone marrow immature fraction E(6%) and T1 (8%) CDR-H3 were also predicted to

contain more extended base structures than FO (1%)CDR-H3. Kinked, extra-kinked, and extended basestructures in bone marrow immature fraction E andmature fraction F were predicted to be of similarprevalence.

In the CDR-H3 loop itself, deformed hairpins werepredicted to be less frequent in MZ (6%) than bonemarrow immature fraction E (19%) (p<0.05). Intacthydrogen bond ladders were predicted with similarfrequency in all five of the subpopulations studied.

Loss of charged and hydrophobic CDR-H3 loops inconventional mature B cell repertoires

We have previously shown that as B cells mature in thebone marrow, these developing cells express fewercharged or hydrophobic CDR-H3 loops [6]. We definecharged loops as those whose average normalizedKyte–Doolittle hydrophobicity [14, 15] is lessthan –0.700, whereas hydrophobic CDR-H3 exhibit anaverage hydrophobicity that is greater than +0.600.

The process of reduction in the prevalence of chargedand hydrophobic CDR-H3 that first began in developingpre-B cell populations continues in the spleen. Whencompared to bone marrow mature fraction F, splenic

Figure 6.Distribution of the predicted structures ofthe base and the loop of CDR-H3 from VH7183DJCltranscripts isolated from sorted spleen and bonemarrow B cells. Structural properties were pre-dicted according to Shirai's [13] “H3-rules” de-duced from the analysis of well-determinedCDR-H3 crystal structures. These rules allow toprediction of structural features from the primaryCDR-H3 sequence based upon the location andhydrophobicity of amino acids, and size of the sidechain. The structure of the CDR-H3 base termedkinked, extra-kinked, or extended can be predictedfor sequences that contain a minimum of fiveamino acid residues at IMGT positions 105–118(Kabat positions 93–103). For asmany as 30% of thesequences with a kinked or extra-kinked CDR-H3base, the H3-rules can predict whether an intacthydrogen bond ladder may be formed within theloop of the CDR-H3 region or whether the hydro-gen bond ladder is likely to be broken. Frequenciesare reported as percentage of all sequencesanalyzed. (A) Frequency of kinked (k–), extra-kinked (k+) and extended (E) CDR-H3 bases. (B)Frequency of broken and intact hydrogen bondladderswithin the CDR-H3 loop for those H chainsthat contain kinked or extra-kinked bases; *p<0.05using v2-test or Fisher's exact test as appropriate.



transitional T1 B cells expressed greater numbers of bothhydrophobic (p=0.008) and charged (p=0.03) CDR-H3loops (Fig. 3, Table 1, 2). Hydrophobic sequencesproved uncommon in splenic MZ and FO, as well asin bone marrow mature fraction F. Their absencecontributed to a progressive decline in average CDR-H3loop hydrophobicity for each of these populations whencompared to T1 (Fig. 2).

Although the MZ population was depleted ofhydrophobic CDR-H3, enrichment for charged CDR-H3loops was observed (p<0.01; Table 2, Fig. 2, 3). Amongthe various charged amino acids, attention has beendrawn to arginine due to its association with anti-DNAreactivities [16]. Charged sequences with two CDR-H3arginine residues were detected in bone marrowimmature fraction E, in splenic transitional T1 cells,and in MZ. Sequences containing two or more arginineswere not detected in either the splenic FO or bonemarrow mature fraction F subsets.

Discussion

Our work suggests that the sequence content andpredicted structure of CDR-H3 may shape the fate of theB cell in peripheral lymphoid tissues. We have previouslyshown that selection for or against certain categories ofCDR-H3 sequence occurs during the B cell developmentin the bone marrow. The most apparent categoriessubject to selection include CDR-H3 with excessive useof non-polar or highly polar (charged) amino acids, andthose with lengths of eight or fewer amino acids. Weshow here that this process of CDR-H3 selectioncontinues in the spleen and that B cells enriched forspecific CDR-H3 sequence categories appear to eithergain ready access to or are excluded from individualsplenic B cell compartments. Our findings also confirmand extend previous observations that selection ofB cells into the various compartments of the spleen canbe influenced by V usage [17–19]. Collectively, theseobservations support the hypothesis that entry to thevarious peripheral B cell compartments is influenced byligand selection [17, 18, 20, 21] and further suggest thatselective pressure may be exerted at the level ofcategories of antigenic epitopes.

Table 1.CDR-H3 loop and base sequences of spleen andbonemarrowB cell transcriptswithKyte–Doolittle hydrophobicity value ofgreater than 0.600a)

Tissue n (%) p VH DH JH Base Loop Base Charge

BM E 5 (2) 0.21 5 DSP2.9 2 AR SMMVT IDY 0.606

9 DQ52 2 AE LG QDY 0.715

3 DSP2.2 4 AR ILMITYA MDY 0.861

ND DFL16.1 1 TR LLFT ADV 0.958

16 DSP2.2 1 AS L LLF 1.400

Spleen T1 5 (5) 0.008 18 DFL16.1 2 AR SITTV VDY 0.612

9T DFL16.1 3 AR HLITTVVAP FAY 0.628

12 DSP2.2 4 AR GPTMIIA MDY 0.651

16 DQ52 3 AR LG GAY 0.715

6 No D 2 AS V GDY 1.600

Spleen FO 2 (2) 0.22 9T DSP2.9 4 AR LGLLRA MDY 0.617

10 DFL16.1 4 AR LITTVVATRA MDY 0.633

BM F 1 (<1) 14 DFL16.1 2 AS LITTV VDY 0.912

Spleen MZ 1 (1) 0.51 13 DFL16.2 2 AS LITTA LDY 0.746

a) Hydrophobic CDR-H3 loop amino acid residues are bold and italicized. Proportion of highly hydrophobic sequences for eachpopulation was compared to bone marrow fraction F (BM F).



VH usage in the bone marrow mature B cell subsetwas highly similar to that observed in progenitorimmature B cells, but differs from that observed in allof the splenic subsets that we examined, including thetransitional T1 population. This was true in particularfor the use of the VH7183.10 gene segment, whichdominates bone marrow repertoires. These findingssuggest that VH content may not only influence entryinto specific splenic B cell subsets, but into the spleenitself. The fate of B cells bearing VH7183.10 remainsundetermined, but could reflect selection into otherB cell niches, such as themucosa or the peritoneal cavity.

We have previously shown that developmentalprogression from the immature B cell stage (fraction E)to the recirculating, mature B cell population(fraction F) is associated with the loss of highly non-

polar or charged CDR-H3, as well as with reducedrepresentation of short intervals [6] The similarity inCDR-H3 content between bone marrow fraction E andthe splenic transitional T1 population indicates that theuse of non-polar, charged, or short CDR-H3 does notprevent B cells from leaving the bone marrow andentering a primary lymphoid organ. However, once inspleen, the fate of B cells using these categories ofCDR-H3 subsequently appears to diverge.

The prevalence of highly non-polar CDR-H3 mark-edly drops in all B cell subsets after the transitional T1stage of development. It remains unclear whether theexcess presence of hydrophobic CDR-H3 in T1 reflects along-lasting accumulation of these cells within thistransitional population, or the relative failure of cellsbearing these CDR-H3 to progress to the next stage of

Table 2.CDR-H3 loop and base sequences of spleen andbonemarrowB cell transcriptswithKyte–Doolittle hydrophobicity value ofless than –0.700a)

Tissue n (%) p VH DH JH Base Loop Base Charge

BM E 8 (3) 0.04 6 No D 3 AR HD RAY –0.955

10 No D 3 AR H AAY –0.910

10 No D 4 AR H MDY –0.910

10 No D 3 AR QRDG LAY –0.818

9 DSP2.3 2 AR QRGYDERHY FDY –0.780

10 No D 2 AR QRG LDY –0.757

10 DQ52 3 AR HENWEP FAY –0.737

10 DSP2.5 3 AR QDGNRG FAY –0.707

Spleen T1 4 (4) 0.03 18 DQ52 2 AR N WDY –1.000

2 No D 4 AR NRS MDY –0.800

13 DSP2.2 3 AR RGD YDD –0.757

18 DSP2.11 2 AR RDYRSY FDY –0.707

Spleen FO 1 (1) 0.51 9T No D 3 AR N GAY –1.000

BM F 1 (<1) referent 9 DSP2.2 4 AR RDDD LDY –1.075

Spleen MZ 8 (5) <0.01 6 No D 4 AR RR GDY –1.300

9T No D 2 AY D FDY –1.000

12 No D 4 AR E EDY –1.000

10 No D 2 AR HRP FDY –0.860

2 DSP2.02 3 AR ENDYDEG FAY –0.749

6 DSP2.02 2 AR KDYDY FDY –0.728

9 DSP2.11 2 AR HEDYRYY FDY –0.717

a) Charged CDR-H3 loop amino acid residues are bold and italicized and arginine residues are underscored. Proportion of highlycharged sequences for each population was compared to bone marrow fraction F (BM F).



development. Unlike T cell receptors, which by virtue ofMHC peptide presentation can potentially access allsubfragments of a protein molecule irrespective ofhydrophilicity, Ig respond to epitopes found on thesurface of soluble antigens which of necessity tend to behydrophilic. We and others have speculated that highlyhydrophobic CDR-H3 are thus less likely to createoptimal paratopes [22, 23] If so, the decline inhydrophobic CDR-H3 may reflect death from neglectrather than active negative selection per se. Resolution ofthis issue will require manipulation of the hydrophobi-city characteristics of the repertoire as a whole, such asthat which we have previously achieved by altering thesequence of the DH [7], combined with kinetic analysis.

Unlike the T1 repertoire, which appears to reflect thecomposition of newly formed bone marrow immigrants,the CDR-H3 repertoires of MZ and FO differ not onlyfrom bone marrow fraction E and splenic T1 cells, theyalso differ from each other. B cells bearing short orcharged CDR-H3 appear to be acceptable or even toaccumulate in the marginal zone, whereas the FOpopulation is depleted of both and bears extensivelysimilarity to recirculating bone marrow fraction F. Thisprovides further support for the FO fraction serving asthe major contributor to the pool of recirculatingconventional B cells. In our studies of mice forced toexpress highly charged CDR-H3, we have observeddepletion of splenic FO and bone marrow recirculatingB cells coupled with enhanced numbers of MZ [7]. Thesestudies would support the view that expression of Igwith highly polar or charged antigen binding sites eitherfacilitates entry into the MZ compartment or is activelyselected against in the FO and recirculating B cell pools.

A previously recognized feature of MZ is enrichmentfor short CDR-H3 [18, 19]. Deconstruction of thesequences we obtained in this study revealed thatshorter MZ CDR-H3 length cannot be attributed solely toan increased prevalence of sequences bearing thehallmarks of neonatal origin [24]. Only 8% of the MZsequences lacked any N addition and, given that 2% ofthe sequences from the adult, immature B cells also lackN addition, even those MZ sequences which lackedN nucleotides could also have been drawn from adultprogenitors. Moreover, the shorter lengths of these MZCDR-H3 also reflect increased exonucleolytic nibblingand altered use of shorter JH gene segments. Although itis possible that these are features of an as yetunrecognized bone marrow progenitor pool, there is areasonable likelihood that they are a manifestation ofligand selection for a specific set of antigen binding siteparatopes, and that this selection process could serve asa motive force for entry into the marginal zone [17, 18,20, 21].

B cells within the FO and MZ spleen compartmentsalso differ in their functional characteristics. The FO

subset contains the resting precursors of cells most likelyto engage in immune responses toT-dependent, typicallyprotein-based, antigens. Upon stimulation by antigen,FO can give rise to both primary antibody-forming cellsandmemory B cells of high affinity. In contrast, based onsurface phenotypic criteria, the MZ subset appears to beenriched for cells that exist in a semi-activated state.Although given the proper stimuli MZ can engage inT-dependent responses, a large proportion of thispopulation includes cells that appear primed to respondtoT cell-independent challenges, such as those providedby polysaccharides on the surface of encapsulatedbacteria [21, 25, 26]. The question then arises whethercorrelations can be made between the enrichment forshort, polar CDR-H3 in the marginal zone and either thelikelihood of pre-activation, or an altered range ofpreferred epitopes, or both.

Studies from a number of investigators indicate thatthe marginal zone in both mice and rats, is oftenenriched for self- or poly-reactive B cells [12, 27–29].Although without direct analysis of the bindingcharacteristics of these marginal zone Ig these datamust be viewed as circumstantial, there is evidence inthe literature of an association between short, N region-depleted CDR-H3 and self- and poly-reactivity, andbetween enrichment for charged CDR-H3 amino acidsand pathogenic self-reactivity. For example, short, N-lessCDR-H3 are a marker of the neonatal CDR-H3 repertoirein both mouse and human, which is itself enriched forself- and poly-reactivity [30, 31] An excess of chargedamino acids in CDR-H3 has been associated withpathogenic self-reactivity, especially to DNA [16,32–34]. In this light, it should be noted that B cellsexpressing anti-dsDNA reactivity have been shown to beexcluded from the follicles [35]. It is possible that use ofshort, polar CDR-H3 sequences facilitates self- and poly-reactivity, which in turn contributes to the pre-activationthat marks the MZ subset.

The more complex issue is whether differences inCDR-H3 structure and amino composition could yieldsets of paratopes that would interact preferentially withepitopes that are associated with either T-dependent orT-independent responses. The complexity of CDR-H3has made such comparisons problematic, but insight hasbeen gained for CDR–H1, CDR-H2, CDR-L1, andCDR-L2, which are encoded entirely by their V genesegments and thus begin in strict germ-line conformity.Each of these V-encoded CDR has been shown to possessone of a small set of main chain conformations termedcanonical structures [36–39]. Each canonical structureis determined by the loop size and by the presence ofcertain residues at key positions in both the loop andframework regions.

Surveys have shown that the lengths of CDR-H2 andCDR–L1 appear to correlate with the type of recognized



antigen. Antibodies with short loops in CDR-H2 andCDR-L1 appear to be preferentially specific for largeantigens, whereas antibodies with long loops in CDR–H2and CDR–L1 appear to be preferentially specific forsmall molecules, including haptens [40]. It is possiblethat the length, structure, and amino acid compositionof CDR-H3 may also correlate with epitope structuresthat are likely to be found in either T-dependent orT-independent antigens and thus lead to differentialselection of the CDR-H3 repertoire. At present, however,the database of sequences is too small to enable testingof this provocative hypothesis.

At first glance, the interplay between developingB cells and the diversity of the Ig they express may beviewed as a matter of gradation. Under this view,selection of individual B cells might be viewed as theconsequence of a gradient in B cell signaling. Ouranalysis of CDR-H3 composition suggests that differ-ences between B cell subsets reflect more than a fine-gradation in the repertoire. In both mouse and humanwe have observed categorical changes in the repertoire,wherein CDR-H3 can be grouped into sequencecategories defined by hydrophobicity, amino acidcontent, and length. We suggest that these categoriescould prove helpful in understanding the developmentalfate and functional behavior of B cells.

Further studies are required to test whether thenumber and type of categories observed in this work aresufficient to characterize B cell subsets from otherorgans and immunological reactions and whether thesecategories can be associated with and perhaps evenpredict distinct functional properties for the B cells thatexpress them. Such an approach may facilitate betterunderstanding of the biologic role of the variousB lymphocyte subpopulations, provide a frameworkfor further dissection of the mechanisms that underliepassage through both central and peripheral develop-mental checkpoints, and yield insight into the patho-genic mechanisms that contribute to the development ofdiseases of immune function.

Materials and methods

Mice

We isolated three different splenic B cell fractions from fourseparate mice on two separate occasions. The mice analyzedrepresent the progeny of a mixed 129/C57BL6 forebearer thathad been backcrossed for ten generations onto BALB/cJ (StockNo. 000651; Jackson Laboratories, Bar Harbor, ME.) [6]. Ourinitial analysis of bone marrow CDR-H3 repertoire develop-ment had been performed on four separate mice derived fromthe same breeding pool [6]. Additional samples wereindependently obtained from five individual 8–10-wk-oldBALB/cJ mice bred in our own mouse colony [7, 10]. To

increase the power of our comparisons for this work, we sortedbone marrow cells from two additional, individual 8-wk-oldinternally bred BALB/cJ mice. In total, we were able tocompare CDR-H3 repertoires expressed in immature andmature bone marrow B cells from 11 IgMa BALB/c mice fromnine different sorts to CDR-H3 repertoires expressed in splenicB cells from four IgMa BALB/c mice from two different sorts.All studies were performed in accordance with the Universityof Alabama Institutional Animal Care and Use Committeeregulations.

Flow cytometry and cell sorting

Single-cell suspensions were prepared by passing wholespleens though 70-lm filters and flushing with ice-cold FACSbuffer (1� PBS + 2% heat-inactivated fetal calf serum).Single-cell suspensions were independently prepared fromthe bone marrow of two femurs as previously described [6].Red blood cells were lysed in 5 mL erythrocyte lysing buffer(0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA; Cat. No. ACK;Comprehensive Cancer Center Media Preparation) for 5 minon ice. Cells were washed and resuspended in an appropriatevolume of FACS buffer for counting and staining. Total spleencells andmononuclear cells from bonemarrow from individualmice were incubated in 100 lL of fluorescently labeledantibodies in FACS buffer. Sorting was performed on a MoFloinstrument (Cytomation, Ft. Collins, CO).

The scheme of Loder et al. [8] was used to isolate individualsplenic B cell subpopulations (Fig. 1): anti-CD21-PE (gift of Dr.John Kearney, Birmingham, AL), anti-CD23-FITC (BD Phar-Mingen, San Diego, CA), anti-IgM-Cy5 (Jackson ImmunoR-esearch, West Grove, PA) and anti-CD19-spectral red (South-ern Biotechnology, Birmingham, AL). Following the scheme ofHardy [9], B lineage cells from bone marrow were enrichedusing anti-CD19 magnetic beads and AutoMACSJ (MiltenyiBiotech, Auburn, CA). CD19+ cells were incubated on ice withthe following: polyclonal FITC-anti-IgM (Southern Biotech-nology), monoclonal PE-anti-IgD (11-26; Southern Biotech-nology) and spectral red-anti-CD19 (Southern Biotechnology).Cells within the lymphocyte gate were sorted as fraction E(CD19+IgM+IgD–) and fraction F (CD19+IgMloIgDhi) lympho-cytes as previously reported [6].

Sorting, RNA preparation, RT-PCR and sequencing

For each splenic B cell population, 5�104 cells were sorteddirectly into RLT lysis buffer (RNeasy mini-kit; Qiagen,Valencia, CA). For each bone marrow population,1�104–2�104 cells of fraction E and fraction F cells weresimilarly sorted into the same RLT lysis buffer. This procedureprecludes reanalysis of the lysed cells. Therefore, for qualitycontrol, we routinely performed a reanalysis sort session fromcompanion, unlysed, sorted cells. The “purity” of thepopulations, based on “tight gaiting” for surface expressionof IgM, CD21, and CD23, was typically 96% for the FOpopulation and 99% for the MZ population (data not shown).

RNA isolation, RT-PCR amplification, and sequencing wasperformed as previously described [6]. All unique sequenceshave been placed in the GenBank database (DQ439001-



DQ439323, DQ439420-DQ439537 and DQ845299-DQ845339).

Sequence analysis

CDR-H3 was identified as the region between (but notincluding) the 30 VH-encoded conserved Cys (TGT) at Kabat[3] position 92 (IMGT 104) [41] and the 50 JH-encodedconserved Trp (TGG) at Kabat position 103 (IMGT 118).CDR-H3 was separated into two components, the base [Kabatamino acids 93 and 94 (IMGT 105 and 106; typically alanineand arginine) and Kabat amino acids 100–102(IMGT 115–117; typically phenylalanine, aspartic acid, andtyrosine] and the loop (the intervening amino acids).

The “H3-rules” published by Shirai [13, 42] were firstdeduced from the analysis of well-determined CDR-H3 crystalstructures [42] and then confirmed and refined as moreCDR-H3 crystal structures became available [13]. With theserules, one can use the location and characteristics of the aminoacids in the primary sequence of CDR-H3, including hydro-phobicity and the size of the side chain, as a basis for theprediction of structural features in the CDR-H3 loop and base.The structure of the CDR-H3 base (termed kinked, extra-kinked, or extended) can be predicted in sequences thatcontain a minimum of five amino acid residues, includingIMGT positions 105–118 (Kabat positions 93–103).

In approximately 25–30% of the sequences with a kinked orextra-kinked CDR-H3 base, the H3-rules can predict whetheran intact hydrogen bond ladder may be formed within the loopof the CDR-H3 region or whether the hydrogen bond ladder islikely to be broken. For example, proline residues tend toinhibit formation of a stable hydrogen bond ladder, thepresence of a VH-encoded arginine at the amino terminus ofCDR-H3 in conjunction with a JH-encoded aspartic acid at theC terminus permits formation of a salt bridge that stabilizes thebase, and glycine residues permit greater flexibility [13].Identification of the DH required a minimum of fiveconsecutive nucleotide matches with germ-line DH sequence.

Statistical analysis

Differences between populations were assessed where appro-priate by Student's t-test, two tailed; Fisher's exact test, twotailed; v2-test; and either Levene's or O'Brien's tests for thehomogeneity of variance. Analysis was performedwith JMP INversion 5.1 (SAS Institute, Inc., Cary, NC). Means are reportedwith the standard error of the mean.

Acknowledgements: The authors wish to thankT. Carvalho and J. Kearney for their invaluable adviceand support. Thisworkwas supported byNIHHD043327(R.L.S.), NIH AI07051 (J.T.), DFG TR22/TP17 (M.Z.), NIHAI42732 (H.W.S.), and NIH AI48115 (H.W.S.).

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Categorical selection of the antibody repertoire in splenic B cells