This information is current as
of June 16, 2017.
Single-Cell and Deep Sequencing of
IgG-Switched Macaque B Cells Reveal a
Diverse Ig Repertoire following
Immunization
Christopher Sundling, Zhenhai Zhang, Ganesh E. Phad,
Zizhang Sheng, Yimeng Wang, John R. Mascola, Yuxing Li,
Richard T. Wyatt, Lawrence Shapiro and Gunilla B.
Karlsson Hedestam
Supplementary
Material
http://www.jimmunol.org/content/suppl/2014/03/12/jimmunol.130333
4.DCSupplemental
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J Immunol published online 12 March 2014
http://www.jimmunol.org/content/early/2014/03/12/jimmun
ol.1303334
Published March 12, 2014, doi:10.4049/jimmunol.1303334
The Journal of Immunology
Single-Cell and Deep Sequencing of IgG-Switched Macaque
B Cells Reveal a Diverse Ig Repertoire following
Immunization
Christopher Sundling,* Zhenhai Zhang,†,‡ Ganesh E. Phad,* Zizhang Sheng,†
Yimeng Wang,x John R. Mascola,{ Yuxing Li,x,‖ Richard T. Wyatt,x,‖ Lawrence Shapiro,†
and Gunilla B. Karlsson Hedestam*
T
he ability of the naive B cell repertoire to recognize almost
any Ag is dependent on the process of V(D)J recombination, whereby V, D, and J gene segments generate a large
number of unique B cell clones. In addition, diversity is generated
in the recombining D-J and V-D junctions owing to trimming and
addition of nontemplated nucleotides. The resulting highly variable
domain spanning the V-D-J junction corresponds to the CDR3 of
the Ab H chains. A similar process occurs in V-J recombination
of the Ig L chain gene segments to form its CDR3. The CDR3s,
together with the V-gene–encoded CDR1 and 2 regions of both the
*Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE171 77 Stockholm, Sweden; †Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY 10032; ‡Division of Nephrology, Nanfang
Hospital, Southern Medical University, Guangzhou 510515, China; xDepartment of
Immunology and Microbial Scence, The Scripps Research Institute, La Jolla, CA
92037; { Vaccine Research Center, National Institutes of Health, Bethesda, MD
20892; ‖International AIDS Vaccine Initiative Neutralizing Antibody Center,
The Scripps Research Institute, La Jolla, CA 92037
Received for publication December 13, 2013. Accepted for publication February 16,
2014.
This work was supported by grants from Karolinska Institutet funds and foundations
(to C.S.), the Swedish Physicians against AIDS Research Foundation (to C.S.), the
Swedish International Development Agency/Department of Research Cooperation
(to G.B.K.H.), the Swedish Research Council (to G.B.K.H.), National Institutes
of Health/National Institute of Allergy and Infectious Diseases (AI102766 and
AI100663) (to Y.L. and R.T.W.), and the International AIDS Vaccine Initiative (to
R.T.W. and G.B.K.H.).
The sequences presented in this article have been submitted to GenBank (http://www.
ncbi.nlm.nih.gov/genbank/) under accession numbers KF947536-KF948098 (Envspecific) and KF948099-KF948514 (Total) and to the National Center for Biotechnology Information SRA database (http://www.ncbi.nlm.nih.gov/sra) under accession
number SRP033611.
Address correspondence and reprint requests to Dr. Christopher Sundling and
Dr. Gunilla B. Karlsson Hedestam, Department of Microbiology, Tumor and Cell
Biology, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden. E-mail addresses: [email protected] (C.S.) and [email protected]
(G.B. K.H.).
The online version of this article contains supplemental material.
Abbreviations used in this article: Env, HIV-1 envelope glycoprotein; NHP, nonhuman
primate; SHM, somatic hypermutation.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303334
H and L chains, usually constitute most Ab contacts with the Ag
(1). Additional variation is generated through random pairing of
Ig H and L chains in the developing pro–B cell. The resulting
B cell diversity is a major component of protective immunity to
pathogens following re-encounter or vaccination. Following Agspecific BCR activation of naive B cells, Ab affinity maturation
occurs through somatic hypermutation (SHM) of the Ig genes of
B cells recruited into germinal centers within B cell follicles,
eventually resulting in T cell–dependent class switching from IgM
to IgG isotype–bearing Abs (2).
In any given individual, at any given moment, the circulating
B cell repertoire comprises naive B cells poised to respond to new
Ags and IgG-switched memory B cells generated from prior exposure to pathogens, environmental Ags, or vaccine Ags (3). Agspecific memory B cells have the capacity to rapidly differentiate
into Ab-producing cells upon Ag re-encounter (4, 5). Examination of Ag-specific memory B cell repertoires therefore comprehensively surveys the B cell clones engaged by a specific Ag
following infection or immunization. Single-cell sorting of HIV-1
envelope glycoprotein (Env)–specific memory B cells from chronically HIV-1–infected individuals indicates a limited-memory
B cell repertoire size of ∼50 clonotypes, with a bias toward
the use of IGHV1 family gene segments (6). Vaccination with
tetanus toxoid, in contrast, was shown to yield a repertoire size
of ∼100 clonotypes, which was not diversified further by boosting (7, 8). So far, little is known about V-gene segment usage and
clonality of B cell responses elicited by other vaccine Ags. Yet,
interest has increased in understanding germline VH gene activation and Ab maturation in response to immunization, not the
least in the HIV-1 vaccine field because it is known that several
highly potent, broadly neutralizing Abs against the Env from
HIV-1–infected individuals use the same IGHV1 family gene segment (9, 10).
To establish a baseline of VH usage in rhesus macaques, we
investigated the contribution of individual Ab H chain V-gene
segments in total IgG-switched rhesus macaque B cells. Next,
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The nonhuman primate model is important for preclinical evaluation of prophylactic and therapeutic intervention strategies. The
recent description of the rhesus macaque germline Ig loci and establishment of a database of germline gene segments offer improved
opportunities to delineate Ig gene usage in the overall B cell repertoire as well as in response to vaccination. We applied 454-pyrosequencing and single-cell RT-PCR of bulk and sorted memory B cells, respectively, to investigate IGHV gene segment expression
in rhesus macaques. The two methods gave remarkably concordant results and identified groups of gene segments that are
frequently or rarely used. We further examined the VH repertoire of Ag-specific memory B cells induced by immunization with
recombinant HIV-1 envelope glycoproteins, an important vaccine component. We demonstrate that HIV-1 envelope glycoprotein
immunization activates a highly polyclonal response composed of most of the expressed VH gene segments, illustrating the
considerable genetic diversity of responding B cells following vaccination. The Journal of Immunology, 2014, 192: 000–000.
2
Materials and Methods
Samples and ethics statement
Immunizations and blood samples from the rhesus macaques used in the
current study are described elsewhere (12). Briefly, Chinese rhesus macaques were immunized five times in a monthly interval with recombinant
gp140-F trimers based on the HIV-1 YU2 isolate (13) in AbISCO-100 and
CpG-C adjuvant. Peripheral bleeds were taken while the macaques were
under ketamine sedation, 1 and 2 wk after each immunization, and the
PBMCs were isolated via density-gradient centrifugation with FicollHypaque. After extensive washing in PBS, the cells were frozen in FCS
supplemented with 10% DMSO. During the study the macaques were
housed at the Astrid Fagraeus Laboratory animal facility at Karolinska
Institutet (Stockholm, Sweden). All procedures were approved by the
Local Ethical Committee on Animal Experiments. Some Ig sequences
from macaques F125, F126, F128, F129, and F130 were described previously (11, 14), whereas new memory B cell isolations were performed
from frozen PBMCs for macaque F124, following the fourth immunization, and macaques F125 and F128, following the second and fifth
immunizations.
Cell preparation and single-cell flow cytometric sorting
Frozen PBMCs were thawed and resuspended in 10 ml RPMI 1640 media
supplemented with 10% FBS and 10,000 U/ml DNase I. After washing, the
pellet was resuspended in 50 ml PBS and 5 ml Aqua Dead Cell Stain and
incubated for 20 min at 4˚C. The cells were then stained essentially
as previously described (11), using a mixture of Abs for human CD3
(allophycocyanin-Cy7; SP34-2), CD8 (Pacific blue; RPA-T8), CD14 (Qdot
605; M5E2), CD20 (PE-Alexa Fluor 700; 2H7), CD27 (PE-Cy7; M-T271),
IgG (FITC; G18-145), and IgM (PE-Cy5; G20-12) in 100 ml for 1 h at 4˚C.
To allow sorting of B cells expressing an Env-specific BCR, gp140-F–
biotin preconjugated to streptavidin-allophycocyanin was included at
4 mg/ml. Following staining, the cells were washed in prechilled PBS,
resuspended in 500 ml, and passed through a 70-mm nylon cell mesh.
Total (CD20+CD27+IgG+) and Env-specific memory B cells (CD20+
CD27+IgG+gp140-F+) were sorted at single-cell density into 96-well
PCR plates containing 20 ml lysis buffer using a three-laser FACSAria cell
sorter. All sorted cells were negative for Aqua Dead Stain mix, CD14,
CD3, CD8, and IgM. The lysis buffer was composed of 6.25 mM DTT,
20 U RNase inhibitor, 5 ml 53 First Strand cDNA Synthesis Buffer (Life
Technologies), and 0.0625 ml Nonidet P-40. In sorts performed for F124,
an additional 10 mg/ml carrier RNA (poly-A; QIAGEN) was included in
the lysis buffer.
Single-cell RT-PCR
The sorted plates were reverse transcribed and the Ab genes amplified as
previously described (14). Briefly, the RNA was reverse transcribed to
cDNA following the addition of 150 ng random hexamers, 0.4 mM 2’-deoxynucleoside 5’-triphosphate, 100 U SuperScript III, and 3.5 ml water per
well and incubating the plates at 42˚C for 10 min, 25˚C for 10 min,
50˚C for 60 min, and 94˚C for 5 min. Nested PCR was performed on 3 ml
cDNA in 25-ml reactions with the HotStar Taq Plus Kit (QIAGEN), using
59 leader sequence–specific and 39 IgG-specific primers. In the secondround PCR, 1.5 ml PCR product was used as template. Nested PCR
products were evaluated on 2% 96-well agarose gels, and positive wells
with a specific band of ∼450 bp were PCR purified and sequenced. Generated sequences are available with accession numbers KF947536KF948098 (Env-specific) and KF948099-KF948514 (Total) at GenBank
(http://www.ncbi.nlm.nih.gov/genbank/).
Ab cloning and expression
Cloning sites were introduced in the V and J regions following verification
of V(D)J sequence identity and primer align. The cloning PCR was performed in a total volume of 25 ml on 2 ml nested PCR product, using the
Phusion Hot Start II High-Fidelity PCR Kit according to the manufacturer’s instructions (Thermo Scientific). Briefly, the PCR consisted of
5 ml 53 Phusion GC Buffer, 1 ml 10 mM 2’-deoxynucleoside 5’-triphosphate, 1 ml each of 10 mM 39 and 59 cloning primers, 2 ml nested PCR
product, and water to 25 ml. The PCR had an initial denaturation at 98˚C
for 30 s, followed by 35 cycles of 98˚C for 10 s, 55˚C (10 cycles) and 60˚C
(25 cycles) for 30 s, and 72˚C for 30 s. There was a final elongation at
72˚C for 7 min before evaluation on 1% agarose gel. Positive bands [∼400
bp for the H chain V(D)J and ∼350 bp for the k and l L chain VJ] were
then gel extracted (Thermo Scientific).
Restriction enzyme digestion of PCR products and cloning into
eukaryotic expression vectors containing human Igg1 H, Igl2, or Igk1 L
chain Ab expression cassettes were performed as previously described
(15), with some modifications. Restriction digestions were performed using
FastDigest enzymes (Thermo Scientific) according to the manufacturer’s
instructions. Following digestion, products were purified (Thermo Scientific) and ligated into linearized, shrimp alkaline phosphatase–treated
expression vectors using T4-DNA ligase (Thermo Scientific). XL10-Gold
ultracompetent bacteria were transformed according to the manufacturer’s
instructions (Agilent Technologies). Colonies were screened for positive
insert by PCR, and plasmids containing products of the correct size were
expanded and sequenced (GATC Biotech).
Ab expression and specificity mapping
H and L chain vectors with functional inserts were transfected at equal
ratio into 293F cells, cultured to a density of 1.2 million cells per milliliter,
using FreeStyle MAX Reagent (Life Technologies) according to the manufacturer’s instructions. The cell culture supernatants were tested for the presence of secreted IgG and Ag binding via ELISA 4–5 d following transfection
and then harvested and purified by Protein G Sepharose columns (GE
Healthcare) 7 d following transfection. The purified Abs were further
tested for sensitivity and specificity by ELISA. MaxiSorp plates (Nunc)
were coated with 1–2 mg/ml Goat–anti-IgGFcg (Jackson ImmunoResearch), gp140-F (the Ag used for immunizations), and influenza
hemagglutinin 1, included as a control protein, in PBS overnight at 4˚C.
The samples were added in a 5-fold dilution series, starting from 5 mg/ml
purified mAb or 1:10 diluted culture supernatant, and incubated for 1.5 h at
room temperature. HRP-conjugated goat–anti-rhesus IgG (Nordic
MUbio) was then added at 1:5000 dilution in wash buffer and incubated for
1 h at room temperature. The bound Ab was detected by adding tetramethylbenzidine+ (Life Technologies) for 5 min before stopping the reaction
with an equal volume of 1 M H2SO4. The OD was measured at 450 nm.
Between each step the ELISA plates were washed six times with PBS
supplemented with 0.05% Tween 20.
Library preparation for 454-pyrosequencing
The 454-library preparation was largely performed as described before
(16), with some modifications. Total RNA was extracted from 12 million
PBMCs from NHP F128 following the fifth immunization. mRNA was
isolated from the purified total RNA, using the Oligotex Kit (QIAGEN).
Then cDNA synthesis was performed in three reactions at 40 ml, using
SuperScript III and Oligo(dT)18 primers according to the manufacturer’s
instructions (Life technologies). The cDNA was then pooled and PCR
purified, followed by elution with 30 ml 10 mM Tris-HCl (pH 8.5), making
each microliter correspond to ∼0.4 million input PBMCs.
Separate PCR reactions were set up for the IGHV1, 3, and 4 families,
using mixes of primers covering the rhesus germline genes (Supplemental
Table I). Library amplification was performed on 5 ml cDNA, corresponding to 2 million input PBMCs, in 50-ml reactions using the Phusion
High-Fidelity PCR Kit with the GC Buffer according to instructions. The
PCR program was initiated with a 98˚C incubation for 30 s, followed by 10
cycles of 98˚C for 10 s, 51˚C (for VH3) or 55˚C (for VH1 and 4) for 30 s,
and 72˚C for 30 s and 15 cycles of 98˚C for 10 s, 65˚C for 30 s, and 72˚C
for 30 s. Following cycling there was a 10-min elongation at 72˚C and,
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we similarly analyzed the Ag-specific B cells isolated from nonhuman primates (NHPs) immunized with soluble HIV-1 Env
trimers in adjuvant. For total IgG-switched B cells, we used two
independent methods: ultradeep 454-pyrosequencing of V(D)J
transcripts generated from mRNA isolated from PBMCs and
single-cell sorting of IgG-switched memory B cells followed by
nested PCR of V(D)J sequences. We observed highly congruent
results with the two methods, allowing us to identify a large number
of genetically distinct VH gene segments that were frequently or
less frequently used. Furthermore, when we examined the gene
segment use of Env-specific IgG+ memory B cells obtained from
highly specific flow cytometric sorting (11), we observed a similar
broad pattern of VH usage. These data demonstrate that the
polyclonal B cell response to the HIV-1 trimers used in this study
is genetically highly diverse, providing a basis for studies aimed to
activate selected VH gene segments in a more specific manner.
These results represent a first comprehensive analysis of Ig VH
gene usage in IgG-switched rhesus macaque memory B cells, as
well as in the Ag-specific B cell response to HIV-1 Env protein
immunization.
Ig GENE USAGE IN IMMUNIZED RHESUS MACAQUES
The Journal of Immunology
3
finally, cooling to 4˚C. PCR products were evaluated on a 1% agarose gel,
the bands were then excised, and the DNA was extracted. To remove potential gel residues, the PCR products were further purified by phenol/
chloroform extraction followed by ethanol precipitation. The DNA was
quantified using Qubit (Life Technologies) before sequencing in a GS FLX
instrument (Roche 454 Life Sciences), using the manufacturer’s suggested
methods and reagents. Image collection and subsequent signal processing,
quality filtering, and generation of nucleotide sequence quality scores were
performed as described previously (10). The sequences are available under
accession number SRP033611 at the National Center for Biotechnology
Information SRA database (http://www.ncbi.nlm.nih.gov/sra).
Sequence analysis
Statistical analysis
The p values for comparing total and Env-specific V(D)J-family distributions were calculated with the x2 test. Comparisons of specific V-gene
segments were made with two-way ANOVA followed by the Bonferroni
multiple comparison posttest. Correlation was determined with the nonparametric Spearman correlation test, with a two-tailed p value calculated
for significance.
Results
Ab sequence isolation and purification
To investigate the representation of the recently described H chain
Ig V-gene segments in the rhesus macaque B cell repertoire (11,
14), IgG-switched B cells were interrogated using two independent
approaches: 454-pyrosequencing of expressed V(D)J sequences from
bulk PBMCs of NHP F128 (12) (Fig. 1A) and flow cytometric
sorting of single memory B cells from five different NHPs—F124,
F126, F128, F129, and F130 (12)—followed by RT-PCR of
expressed H chain V(D)J sequences (Fig. 1B). The 454-pyrosequencing was performed for the major IGHV families (IGHV1, 3,
and 4), constituting 53 of 62 described germline V-genes (11). Each
IGHV1, 3, and 4 library was prepared from cDNA corresponding to
2 million PBMCs and sequenced separately to reduce PCR amplification bias. For single-cell analysis, IgG specificity was achieved
by sorting memory B cells based on positive surface expression of
CD20, CD27, and IgG and using 39 primers specific for rhesus
IgG1-4. Single-cell RT-PCR for IGHV families 1–7 was performed using recently described rhesus-specific VH primers (14).
To analyze the generated Ab sequences, we implemented a
bioinformatics pipeline for NHP H chain sequences (Fig. 1C).
Briefly, sequence reads were first filtered for length, whereby only
sequences between 300 and 600 bp were included. The sequences
were then mapped to the current rhesus germline VH database and
FIGURE 1. IGHV sequence generation and analysis. (A) Schematic of the
library preparation for deep sequencing using 454-pyrosequencing. mRNA
was isolated from PBMCs and reverse transcribed. Individual PCR reactions were performed for the IGHV1, 3, and 4 families using high-fidelity
enzyme and family-specific primers with 454-adaptor tags. The PCR products were purified and sequenced separately using a GS FLX 454 instrument.
(B) Schematic of the single-cell sort followed by Ig V(D)J RT-PCR. IgGswitched memory B cells, defined as CD20+CD27+IgG+, were sorted at
single-cell density into 96-well plates containing lysis buffer. Following
reverse transcription with random hexamers, nested PCR was performed
with a mix of IGHV family–specific primers. PCR reactions producing
specific bands of ∼450 bp were sequenced and used in subsequent analysis. (C) To obtain full-length functional Ig V-gene segments for subsequent analysis, raw sequences were first filtered for length (300–600 bp).
They were then translated to amino acid sequence to exclude sequences
containing stop codons and to include the conserved WGXG and CXR/K
domains. In addition, included sequences had an amino acid/nucleotide
ratio $ 0.7. Functional sequences were aligned to the rhesus germline
IGHV database, using ClustalW2 to calculate SHM and provide the sequence with a V-gene assignment. To determine that a diverse pool of B cells
was sampled, only cells expressing unique B cell receptors, as quantified by
CD-HIT clustering, were included.
retained only if they included the conserved amino acid CXR/K
and WGXG motifs present at the beginning and end of the CDRH3
region. Sequences with premature stop codons were discarded, as
were sequences with amino acid/nucleotide ratios of #0.7 for
identity to the assigned germline sequence, owing to the likelihood that insertions or deletions resulted in a change of reading
frame. In addition, to reduce potential PCR bias of specific
amplicons, only nonredundant sequences were included in the
analysis. After application of these filters, the 454-pyrosequencing
yielded a large number of sequences for each V-gene family:
IGHV1 (20,166), IGHV3 (51,320), and IGHV4 (85,645). Singlecell sorting and PCR amplification of total memory B cells generated 480 raw sequences that, following bioinformatic processing, resulted in 416 unique sequences.
Ig gene contribution to the IgG-switched B cell repertoire
The combination of deep sequencing and single-cell RT-PCR
allows for qualitative and quantitative assessment of the Ab V(D)J
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The 454-pyrosequencing and single-cell RT-PCR–generated sequences
were first filtered for read lengths of 300–600 bp. Germline V-genes for
each sequence were then assigned using a BLAST procedure with parameters set to mimic IgBLAST (17). V region sequences were extracted
based on the ClustalW2 global alignment of each sequence to single-cell RTPCR–generated Ab sequences. Incomplete sequences and problematic
sequences (sequences with internal stop codons, frameshifts, or loss of
any of the conserved CDRH3-flanking motifs CXR/K and WGXG) were
removed from downstream analysis. The clustering program CD-HIT
(18) was next used with 100% sequence coverage and identity to
remove redundant reads. Finally, the divergence (percent nucleotide mutation from the assigned germline V-gene) was calculated based on
ClustalW2 global alignment to the V region of the most homologous
germline V-gene segment in a germline database constructed from previously annotated rhesus macaque Ig genes (11, 14). V, D, and J assignments
were additionally performed by IMGT/HighV-Quest (19) (version 1.1.3,
reference directory release 201338-1) using the rhesus macaque option and
further analyzed in the recently published Ig Analysis Tool (IgAT, version
1.14) (20) program. However, individual V-gene assignments could not be
performed using the IMGT tools, as their database is still limited compared
to published rhesus germlines (11).
Clonality within the sorted populations (total and Env-specific memory
B cells) was determined by IgAT with the criteria that clonally related
sequences 1) use the same VH and JH genes, 2) have the same CDRH3
length, and 3) have a CDRH3 nucleotide sequence homology of $90%.
4
spectively. The percentage SHM of each contributing sequence
was calculated relative to the assigned germline sequence, and the
average SHM levels were determined (Fig. 3A–C). These analyses
show that unmutated variants, or variants very close to the
assigned germline sequences, were found for the majority of gene
segments. However, some exceptions did exist, such as VH1.61,
for which the closest variants were ∼5% mutated from the putative
germline, suggesting that these sequences may have derived from
a V-gene that deviates from the published VH1.61 germline sequence. Overall, 42 of the possible 53 annotated V-gene segments
were found to contribute to the circulating IgG-switched B cell
repertoire, although usage of some gene segments was clearly
preferred relative to others, closely resembling the pattern of
V-gene usage observed in humans (21).
Highly diverse V-gene usage in the HIV-1 Env-specific
IgG-switched memory B cell repertoire
We next investigated the range of V(D)J gene segments used in
vaccine-induced responses, using samples from rhesus macaques
inoculated with well-described recombinant HIV-1 Env protein–
based trimers (12, 13). Both total and Env-specific memory B cells
were sorted to allow comparisons within individual animals. To
isolate the Env-specific memory repertoire, B cells were sorted
at single-cell density based on expression of cell-surface CD20,
CD27, and IgG, as well as binding to a fluorochrome-conjugated
Env trimer probe, as previously described (11). The Env-specific
population was found to be ∼4% of the total memory population
(Fig. 4A). Next, H chain V(D)J sequences from sorted Envspecific memory B cells were rescued with nested RT-PCR, and
FIGURE 2. High concordance in Ig VDJ gene usage by single-cell RT-PCR and deep sequencing. The frequency of H chain VDJ gene contribution
(Y-axis) was determined for the individual V-gene segments (A) or D- and J-families (B and C) (X-axis) within the three major V-gene families, IGHV1, 3,
and 4, for 454-pyrosequencing–generated sequences (open bars) and single-cell flow-sorted total memory B cells (filled bars). The number of sequences
included in the V-gene analysis (A) were for 454-pyrosequencing: VH1, n = 20,226; VH3, n = 51,320; VH4, n = 85,645; and for single-cell RT-PCR: VH1,
n = 41; VH3, n = 152; VH4, n = 188. Individual D-family and J-gene (B, C) contribution to all analyzed sequences was determined following pooling of the
sequences (454-pyrosequencing, n = 148,105; single-cell RT-PCR, n = 379). (D) The correlation of the V-gene, D-family, and J-gene contribution to the
analyzed sequences (A–C), as determined by 454-pyrosequencing (Y-axis) and single-cell RT-PCR (X-axis), was calculated by determining the nonparametric Spearman correlation coefficient (r) and the p value.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
gene contributions. Deep sequencing provides the capacity to
ascertain, with both depth and high accuracy, genes that are
expressed or not, whereas single-cell RT-PCR provides a quantitative measurement of how often specific gene segments are used.
The latter means of analysis provides nonbiased PCR amplification
from a single cell in which a unique V(D)J recombination event has
occurred. The V-, D-, and J-gene contribution of known annotated
germline genes in IGHV families 1, 3, and 4 was analyzed for
sequences generated by 454-pyrosequencing and single-cell RTPCR (Fig. 2A–C). Owing to the challenge of correctly assigning
D-gene segments, the D-gene contribution was determined at the
family level. Overall, the expression of Ig genes varied greatly;
some were abundantly used, whereas others contributed to a lesser
extent to the expressed IgG-switched B cell repertoire. In addition,
for the V-gene segments, some were not detected at all in our data
set, despite having high homology to the PCR primers used for
library preparation or single-cell RT-PCR. Generally, a very high
concordance was found between the 454-pyrosequencing and the
single-cell RT-PCR, resulting in a highly correlated pattern of
gene segment contribution (Spearman r = 0.92, p , 0.0001;
Fig. 2D). This finding suggests that the PCR protocol used for
454-pyrosequencing had no major bias and that both data sets
therefore can be used for quantitative assessments and qualitative
observations.
To further assess the germline VH gene segments contributing
to the functional IgG-switched B cell repertoire, the 454-pyrosequencing–derived sequences corresponding to unique V(D)J recombination events were plotted from highest to lowest frequency
of contribution within each of the IGHV1, 3, and 4 families, re-
Ig GENE USAGE IN IMMUNIZED RHESUS MACAQUES
The Journal of Immunology
5
Discussion
FIGURE 3. Differential usage of germline Ig V-gene segments. Frequencies of the individual V-gene segment contribution to the IGHV1 (A),
IGHV3 (B), and IGHV4 (C) families, as determined by 454-pyrosequencing, are plotted in descending order. The number of sequences (n)
and mean SHM, based on nucleotide alignment to closest germline sequence, of each V-gene segment are indicated in the table. The scatter plot
(right) shows SHM for each V-gene segment with mean values indicated
by the gray line.
a total of 638 VH sequences were generated. After processing in
the bioinformatics pipeline (Fig. 1) for annotation of individual
V-gene segments and IMGT/HighV-Quest, followed by analysis in
IgAT for V(D)J-gene family assignment, 563 and 570 unique Ab
sequences were obtained, respectively. Of these, 33 H chain V(D)J
sequences from two NHP donors were cloned, along with the
corresponding L chain VJ sequences from the same cell to allow
expression of the complete mAbs to verify the specificity of the
Ag-specific sort. The resulting mAbs were evaluated for binding
to HIV-1 Env by ELISA and were all confirmed positive, indicating very high specificity of the flow cytometry–based sort
(Supplemental Fig. 1).
V-gene segments of all IGHV families except VH6 were detected in the Env-sorted populations, although at variable frequencies, similar to the distribution observed in the total IgG
repertoire (Fig. 4B). However, a significant overrepresentation of
the VH5 family (p , 0.01, x2 test) was noted in the Env-specific
group compared with the total IgG-switched B cells. Upon closer
Rhesus macaques are frequently used as models of human immunology and are important for the evaluation of primate responses
to infection and vaccination (22–24). Until recently, systems to
investigate elicited B cell responses in NHPs at the cellular and
genetic level were poorly developed. With the publishing of the
rhesus macaque genome (25), the annotation of rhesus Ig germline
gene segments, and the description of methods to phenotype and
sort rhesus macaque B cell populations (11), the ability to dissect
vaccine- and infection-induced B cell responses in NHPs is now
available. This capability, coupled with methods to clone and
express Ag-specific human mAbs (6, 15), recently adapted to the
rhesus macaque system (14), and recent developments in deep
sequencing approaches to study human Ig repertoires (16, 21, 26–
39), motivated us to investigate VH gene usage in IgG-switched
and Ag-specific repertoires in rhesus macaques. Although isolated
studies of VH gene segment use in rhesus macaque B cells were
reported (40–43), to our knowledge, this is the first comprehensive
report describing the IgG-switched B cell repertoire in NHPs at
a level of high resolution to more specifically elucidate the genetic
diversity of rhesus macaque memory B cells.
Another unique aspect of our study is that we use both 454pyrosequencing and single-cell sorting for genetic characterization
of IgG-switched memory B cells. Despite performing the PCR
under very different conditions by these two distinct means of
analysis, we obtained remarkably similar results with the two
methods, demonstrating VH gene segments that were frequently
used, rarely used, or not detectably used. Most notable was the lack
of representation of several gene segments of the VH3 family, the
largest VH family in both humans and macaques, in agreement with
results previously obtained from 454-pyrosequencing of the human
Ab repertoire (21). It is possible that a greater sequencing depth, or
the analysis of more animals, would reveal the use of additional
gene segments, as we observed some variation in the five different
macaques analyzed in this study (Supplemental Figs. 2, 3). Although 454-pyrosequencing provides a large number of sequences,
allowing a substantial coverage of the VH repertoire, single-cell
PCR allows the isolation of matched heavy and light V(D)J
sequences for subsequent cloning and expression of functional
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examination of the individual V-gene segments (Fig. 4C), VH5.46
was found to be used with significantly greater frequency in the
Env-specific memory B cell pool than in the total IgG memory
B cell pool (p , 0.001, two-way ANOVA followed by the Bonferroni posttest). D- and J-genes from all families were found to
contribute to the B cell pool, with similar frequencies observed in
the Env-specific and total IgG-switched memory B cells, with the
exception of JH5 and 6, which were slightly overrepresented
among the Env-specific memory B cells (p , 0.05, x2 test). The
reasons for the overrepresented gene segments will be investigated in our future studies aimed at characterizing the elicited
specificities in greater detail. When investigating specific V-gene
segment contribution to the IGHV families, the sorted total
memory B cells were found to engage 42 of 62 described
germline genes (Fig. 4C, black bars; and Supplemental Fig. 2).
Remarkably, the Ag-specific memory B cell repertoire elicited
by HIV-1 Env immunization engaged a similar breadth of
V-genes, highlighting the extensive genetic diversity in the B cell
response to a complex but single protein-based vaccine Ag (Fig. 4C,
red bars; and Supplemental Fig. 3). Furthermore, by calculating the
number of participating clonal lineages in the Env-specific V(D)J
sequences, we found that 502 of 606 functional sequences represented unique clonotypes (Table I), indicating a high diversity of Ab
specificities elicited toward the trimeric Env Ag.
6
Ig GENE USAGE IN IMMUNIZED RHESUS MACAQUES
mAbs. This is particularly important for the analyses of Agspecific repertoires in which it is critical to confirm the specificity of the flow cytometry–based sort by evaluating the binding
capacity and specificity of the resulting mAbs. In the current
study, we observed 100% specificity of a panel of 33 cloned
mAbs, strongly supporting the conclusions in regard to the Envspecific VH repertoire presented in this article.
In the past few years a number of studies were performed using
454-pyrosequencing to investigate the Ab repertoires in HIV-1–
infected individuals, with the aim to trace the evolution of broadly
neutralizing Abs in these individuals (16, 27–31). These analyses
provide valuable information about the antibodyome induced and
shaped by chronic HIV-1 infection and highlight the extreme levels
of Ab affinity maturation that can develop under these conditions
(44), likely a common feature of, and a consequence of, chronic
infection (45). Deep sequencing approaches were also recently used
to investigate human Ab responses to vaccination (32, 46), but, so
Table I. Clonal lineage analysis of Env-specific memory B cells
Donor
Functionala
Uniqueb
Clonotypesc
F124
F125
F128
Total
217
185
204
606
200
175
195
570
164
156
180
502
Env refers to CD20+CD27+IgG+gp140-F+ B cells.
a
Number of functional sequences as determined by IMGT/V-Quest and IgAT.
b
Number of unique sequences as determined by IMGT/V-Quest and IgAT.
c
Defined as 1) use the same VH and JH genes, 2) identical CDRH3 length, 3) 90%
CDRH3 nucleotide homology.
far, such studies have not been reported from immunization trials
in NHPs. Our results obtained by deep sequencing of rhesus
macaque B cell responses therefore provide timely information,
which will greatly facilitate future studies of B cell/Ab repertoires in the NHP model.
In parallel, we generated .1000 sequences from single-cell
sorted total and Ag-specific memory B cells from animals inoculated with soluble HIV-1 Env trimers (12). In contrast to studies
of infection-induced B cell responses, in which the Env Ag is
highly variable owing to the continuously changing virus population and therefore not easily definable (6, 47), we used a probe
for FACS sorting that was identical to the protein used for immunization, allowing isolation of all potential Env-elicited subspecificities. Clonal lineage analysis of the isolated sequences
estimated that 502 unique Ab clonotypes were present in 606 Envspecific sequences, suggesting that vaccination results in a highly
polyclonal response. In addition, the overall VDJ gene distribution
of the Env-specific sequences was highly similar to that observed
in the IgG-switched total memory B cell repertoire, with the exception of an increase of VH5-using sequences, especially of
VH5.46, in Env-immunized macaques, potentially signifying that
Env-specific Abs using this V-gene segment might have a selective
advantage following immunization, a finding that needs further
investigation.
A similar estimation of the clonality of vaccine-induced B cell
responses was reported following single-cell sorting and Ab isolation from plasmablasts of humans repeatedly immunized with the
protein-based vaccine tetanus toxoid (7, 8). In that study, immunization also induced a highly polyclonal B cell response consisting of ∼100 different Ab clonotypes using V gene segments
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 4. The HIV-1 Env-specific memory B cell Ig V-gene segment usage is highly polyclonal. (A) Env-specific memory B cells were sorted at singlecell density based on the positive surface expression of CD20, IgG, CD27, and Env (red gate). Gate frequencies (percent) correspond to number of cells of
total input. Streptavidin-allophycocyanin–conjugated trimeric gp140-F–biotin was used as the Env-specific probe. (B) IGHV-, D-, and J-family distribution
of RT-PCR–amplified sorted cells analyzed with IMGT/HighV-Quest and IgAT. Families are color coded, with family 1, red; 2, gray; 3, blue; 4, green; 5,
yellow; 6, pink; and 7, black. The size of the colored area corresponds to the frequency (percent) out of the total number of sequences, indicated in the
center of the graphs. Differences in the gene family distribution between the Total and Env-specific sequences were evaluated with the x2 test, with *p ,
0.05, **p , 0.01. (C) V-gene contribution to the total number of sequences (percent of total) shown as mean + SEM for sorted Total (black, n = 5 donors)
and Env-specific (red, n = 3 donors) memory B cells is shown for the individual V-gene segments. For statistical analysis, two-way ANOVA was used,
followed by the Bonferroni posttest for multiple comparisons, with ***p , 0.001.
The Journal of Immunology
Disclosures
The authors have no financial conflicts of interest.
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