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Departments of
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Neurology and
Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262; and
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we used cell sorting and single-cell RT-PCR to study clonal B cell expansion in CSF of four MS patients and two control subjects with non-MS inflammatory disease. Analysis of V region sequences expressed in randomly sorted, single B cells reduces potential bias when amplifying Ab repertoires from cDNA or cDNA libraries (14, 15), which may overrepresent sequences expressed by activated B cells or plasma cells. A second and more distinct advantage of single-cell PCR is that the clonal B cell H and L chain pairings found in vivo can be faithfully duplicated in vitro and used to produce novel Abs potentially useful in identifying disease relevant Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All MS and inflammatory control CSFs (2–30 ml) were collected in the outpatient clinic of Department of Neurology using procedures approved by the University of Colorado School of Medicine Institutional Review Board. All MS patients had relapsing-remitting disease, magnetic resonance imaging scans that revealed multiple white matter lesions, and CSFs containing two or more OGBs of IgG. The duration of MS varied from 1 to 22 years (Table I). None of the MS patients had received steroids or an immunomodulatory drug for at least 1 mo before CSF sampling. The two control CSFs were from patients with acute viral meningitis.
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CSF obtained by lumbar puncture was immediately centrifuged for 10 min at 1500 rpm. Pelleted cells were resuspended in 200 µl of residual CSF, placed on ice, and sorted within 1–3 h. A three-color premixed combination (15 µl) of fluorescence-tagged murine Abs (Caltag Laboratories, Burlingame, CA) specific for the human white blood cell surface markers CD45 (Tri-Color), CD19 (r-PE), and CD3 (FITC) was added to the CSF cell suspension and incubated for 20 min at room temperature. Labeled cells were diluted with 500 µl of sterile PBS, placed on ice, and sorted using a MoFlo cytometer (Cytomations, Fort Collins, CO). Cells in the approximate size range and granularity of lymphocytes were first selected by light scattering, followed by a second selection for all CD45+ cells. B cells (CD19+, CD3-) were further separated from T cells (CD19-, CD3+) and other CD45+ cells (CD19-, CD3-), and individually expelled into single wells of a 200-µl, 96-well PCR plate (catalog no. T-3060; ISC Bioexpress, Kaysville, UT) containing 20 µl of 1x reverse-transcription (RT) reaction buffer. From one to four plates of sorted cells were collected for each CSF sample. When possible, the lysis and RT steps were conducted directly. Additional plates were stored at -70°C until processed for cDNA synthesis and PCR. To determine the probability of depositing more than one cell per well, fluorescent beads were also sorted into 96-well plates. Because none of the wells were found to contain more than one bead, the deposition of more than one cell into a single well was unlikely.
Synthesis of cDNA and amplification of VH and VL sequences
The protocol used for cDNA synthesis and PCR amplification was a modification of that described by Wang and Stollar (15). cDNA synthesis and PCR were performed using the I-Cycler (BioRad, Hercules, CA) thermocycler equipped with a 96-well block. Cells in each well were lysed in 4.5 µl of a detergent mixture containing 2 µl of 5x RT buffer (Invitrogen, Carlsbad, CA), 0.5 µl of random hexamers (3 µg/µl), 0.5 µl of an IgG antisense C region primer CH5 (10 pmol/µl) conserved among all four IgG isotypes, and 1 µl of a 22.5% solution of Nonidet P-40. Plates were incubated for 3 min at 65°C, cooled to 25°C, incubated for 3 min in the thermocycler, and immediately placed on ice. A mixture of 3 µl of 0.1 M DTT, 0.5 µl RNAsin (50 U/µl), 1 µl 10 mM dNTPs, and 0.5 µl of Superscript II RT (200 U/µl) was added to each well (final volume of 30 µl). Plates were incubated in the thermocyler for 1 h at 37°C, heated at 70°C for 10 min to inactivate RT, and cooled to 4°C. Plates containing cDNA were stored at 4°C.
Table II lists the primers used for cDNA synthesis, specific amplification of VH and L chain V (VL) regions, and sequencing. The VH and V
leader and framework 1 PCR primers were family based and identical with or modifications of well-established primer sets designed for single-cell amplification of V region sequences from peripheral blood B cells (14, 16). Conserved family-specific V
leader and framework 1 primers were obtained from alignments of family germline segments found in V BASE, an online database (V BASE, http:/www.mrc-cpe.cam.ac.uk/) containing all known human VH, V, V germline segments. To amplify VH sequences, conserved family-based leader sequence primers for VH families 1–5 were used in a single primary PCR with the conserved C region primer CH
1 for IgG amplification. A separate primary PCR with the C region primer CHµ1 was used to amplify IgM sequences. Single-cell PCR amplifications were performed in a 50-µl vol containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.01% gelatin, 100 µM dNTPs, 100 pmol of each primer, 2 U Taq polymerase, and 5 µl of cDNA reaction mix. Cycling conditions included a single 5-min denaturation step at 94°C, followed by 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, ending with a 7-min incubation at 72°C. A nested PCR using 2 µl of the primary PCR product and a pool of family-based framework 1 primers in conjunction with a second, more 5'-conserved IgG C region primer (CH2) or IgM C region primer (CHµ2) was used to amplify VH region products. To amplify full-length or L chain sequences, sets of family-based leader and framework 1 primers were used in conjunction with C region primers complementary to the C-terminal portion of the L chain coding sequence.
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In analysis of patient MS02-2 CSF, specific VH sequences from some B cell populations were reamplified using clone-specific primers complementary to known nucleotide sequences within the complementarity-determining region 3 (CDR3). For those studies, antisense CDR3 primers specific for each MS02-2 clone 2 VH sequence (see Table III), identified by the CDR3 amino acid sequences PRNRYQDGLFDS (5'-GTCCTGATACCTGTTCCGGGG) and DGGSDYASQFYDY (5'-GAGACCGGTAGTCGCTCCCACC), were used in conjunction with the appropriate VH framework 1 primer in nested PCR, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Single CD19+ B cells were isolated from MS and inflammatory control CSF using Abs to a combination of human white blood cell markers (CD45, CD3, CD19), as shown in Fig. 1 for MS02-11 CSF. CD19+ cells accounted for 3.9% of the selected CD45+ cells in this patient and varied from 1.5 to 3.9% among the four MS CSF samples. In the lymphocyte population of control subjects with aseptic meningitis, CD19+ B cells represented 0.4% for donor IC02-1 and 1.2% for donor IC02-3.
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CD19+ B cell repertoires of CSF from two patients with acute viral meningitis were analyzed as a non-MS inflammatory control. In both patients, the H chain repertoires consisted of about equal numbers of IgG- and IgM-expressing B cells. The amplified H chain V region product from each positive PCR of the CSF as well as L chain V region products amplified from VH-positive wells were purified and sequenced. Approximately 80% of the VH-positive wells of the IC02-1 CSF and 54% of VH-positive wells from IC02-3 CSF also revealed a L chain sequence. Only two VH and two VL nonproductive rearrangements were detected among the 174 V region sequences analyzed from the two inflammatory controls.
Each VH and VL sequence was aligned to a database containing all known functional human H and L chain germline segments to determine the family and most homologous germline segment, J segment (MS patients only), and the extent of somatic mutation for each sequence. Because of its unique nature, the CDR3 amino acid sequence was used as a clonal marker to categorize V region sequences in the repertoire. No clonally related B cell populations were detected among the 27 IgG and 22 IgM VH sequences analyzed from IC02-1 CSF, and only 2 VH sequences of the 50 analyzed in the IC02-3 repertoire were clonally related, indicating a predominantly polyclonal response in CSF of both patients. Table V gives the characteristics of V region sequences obtained from the IC02-1 CSF analysis.
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The relative number of IgG- and IgM-expressing B cells varied among the four MS patients. Whereas the B cells from donors MS02-2 and MS02-14 were predominantly IgG expressing, a more balanced or IgM dominant response was observed in MS02-11 and MS02-6 CSF, respectively (Table IV). Consistent with previous characterization of OGBs in MS CSF, L chains represented the predominant L chain of each MS donor. Of the combined 344 V region sequences analyzed from the 4 MS donors, only 12 were nonproductive rearrangements.
Clonally expanded B cell populations were detected in the IgG repertoire of each of the MS samples, although to varying degrees (Table IV). For example, the diversity of the IgG B cell repertoires of both MS02-2 and MS02-14 CSF was limited, with clonal populations comprising 20 of 25 and 15 of 30 of the VH region sequences amplified from these respective donors. In contrast, the repertoire of MS02-11 was predominately polyclonal, with only 2 of 18 IgG VH region sequences comprising a single clonal population. With the exception of a single clonal population detected in MS02-6 CSF, clonally expanded IgM B cell populations were not found in any other MS CSFs. Overall, and particularly for donors MS02-11 and MS02-14, the amplification of VL regions from single B cells was relatively more efficient than the corresponding H chain amplification. Thus, some amplified L chain sequences could not be paired with the partnering H chain sequence (Table IV). With the exception of donor MS02-11 in which two B cells expressing identical L chain sequence were detected, the sequencing of these L chains revealed no additional clonal populations in the other MS CSFs (data not shown).
Table VI compiles the entire VH and matching VL region repertoires amplified from B cells recovered from the CSF of donor MS02-14. The VH sequences could be organized into seven discrete clonally expanded B cell populations. For example, MS02-14 clone 1 (identified by the CDR3 sequence VPRDGYNYFDF) was amplified on four occasions and comprised the largest clonal population. Each B cell within this population used the same VH and JH germline segments, and shared an identical set of 18 somatic mutations (data not shown). Three of these cells expressed an identical L chain sequence that shared a common set of somatic mutations; no L chain product was amplified from the fourth cell. Similar observations were made for B cells comprising other clonal populations in MS02-14 CSF. However, limited sequence heterogeneity was detected in V region sequences of some clonally related B cell progeny, indicating a degree of clonal variation among these expanded populations. Although the VH sequence of B cells in MS02-14 clones 6 and 7 was detected only once, the L chain rearrangement expressed by each of these cells was detected multiple times, indicating a clonally expanded population. Table III lists the features of VH and VL sequences belonging to clonal B cell populations rescued from the CSF of the three additional MS donors studied. Three distinct clonal populations were detected in MS02-2 and MS02-6 CSF, and one or possibly two distinct clonal populations were detected in MS02-11 CSF. The repertoire in MS02-2 CSF was the most restricted of the four MS CSFs studied. Clones 1 and 2 accounted for 70% of the H chain V regions analyzed.
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and VHµ genes amplified from the MS donors was 94 and 97%, respectively, irrespective of disease duration. Evidence of receptor editing in MS02-2 B cell repertoire
Several features of the MS02-2 IgG repertoire distinguished it from the other clonal populations identified in Tables III and VI. In B cells of MS02-2 clone 1, characterized by the H chain CDR3 sequence VKASFDY, no single dominant L chain sequence was identified. Although it is possible that an existing dominant clone 1 L chain sequence failed to amplify, four different productive L chain rearrangements and one nonproductive rearrangement were detected in those B cells from which an L chain sequence was amplified (Table III). It is unlikely that these sequences represented PCR contamination or artifact. These L chains were not found in B cells from any other CSF repertoires analyzed in this study, and MS02-2 clone 1 was the only clonal population in which we detected multiple productive L chain rearrangements. Interestingly, whereas the VH region of this population had accumulated 21–22 different somatic mutations, rearranged L chain sequences were highly homologous to, and in one cell, identical with their germline sequence. The difference in mutational frequencies between clone 1 H and L chain sequences (mean ratio VH/VL = 0.94 ± 0.01; n = 4) was significantly greater (p > 0.0002, nonpaired t test) than the differences found in the remaining MS02-2 B cell population (mean ratio VH/VL = 0.98 ± 0.02, n = 12), suggesting that some B cells of the clone 1 population had recently undergone receptor editing to produce the multiple L chain rearrangements detected.
A second clonal B cell population of MS02-2 CSF showed evidence of H chain receptor editing. All of the B cells expressing the VH rearrangement with the CDR3 sequence PRNRYQDGLFDS coexpressed the same L chain (QQYGILPWT) or a clonal variant of this sequence. However, a subset of B cells that paired this L chain with a different VH rearrangement (DGGSDYASQFYFDY) was also detected. Interestingly, an in-frame nonsense codon was present in the first amino acid position of the CDR2 region of each of this second group of H chain sequences (data not shown), indicating that the VH sequence was nonfunctional. To determine whether B cells in the MS02-2 clone 2 population contained mRNAs encoding both the PRNRYQDGLFDS and DGGSDYASQFYFDY IgG H chains, we repeated the nested H chain PCR amplifications using clone-specific primers hybridizing to the distinct CDR3 nucleotide sequences of both these H chains. Fig. 2 shows that products corresponding to the PRNRYQDGLFDS H chain were readily amplified from seven of seven B cells expressing the QQYGILPWT L chain, but not from B cells comprising MS02-2 clone 1 (VKASYFDY). Products corresponding to the nonfunctional DGGSDYASQFYFDY H chain were only detected in the same clone 2 B cells from which this VH sequence was originally identified. Thus, two B cells in the clone 2 population were confirmed to contain distinct mRNA encoding both the functional PRNRYQDGLFDS VH rearrangement and the corrupted DGGSDYASQFYFDY sequence. The lower frequency of a second nonfunctional VH rearrangement suggests that H chain receptor editing occurred in a precursor of the clone 2 B cell population. This second H chain rearrangement most likely occurred during B cell development, but could also have occurred peripherally in response to generation of the nonsense mutation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The synthesis of IgM in the CSF of inflammatory and infectious diseases is thought to indicate active antigenic stimulation within the CNS (18, 19). IgM OGBs have been detected in the CSF of 50% of MS patients and appear to correlate with onset of relapses and a worsening disease course (20, 21). IgM-positive B cells constituted a significant portion of the B cell repertoire in three of the four MS patients studied in this work. Such IgM-positive cells were more abundant than IgG-positive B cells in MS02-6 and MS02-11, and comprised 33% of the B cell repertoire of MS02-14. Because we detected only one clonal IgM population in the four MS patients, it is still unknown whether the presence of IgM-positive B cells is due to antigenic stimulation or breakdown of the blood-brain barrier.
An interesting characteristic of the MS02-2 B cell repertoire was evidence suggestive of L chain receptor editing. Receptor editing occurs in primary lymphoid tissue during B cell development as an attempt to rescue self-reactive B cells from destruction by altering their receptor specificity (reviewed in Ref.22). Through renewed or continual expression of the recombinase genes, a secondary rearrangement at the Ig loci can result in new Ab rearrangements to form a functional, but nonautoreactive Ig receptor (23, 24, 25, 26). Secondary rearrangements can also contribute to receptor diversification in germinal centers during an immune response. In clone 1 of the MS02-2 repertoire, we did not detect a predominant L chain sequence, but instead found four distinct and productive L chain rearrangements expressed in different B cells with the same H chain rearrangement (clone 1, Table III). Because an L chain product was detected in only 5 of 11 clone 1 B cells, it is possible that our primers failed to amplify a dominant L chain sequence from this population. However, at least some clone 1 B cells appear to have undergone additional L chain rearrangements. Compared with the extensively mutated VH sequence, the rearranged VL sequences did not deviate significantly from germline, suggesting that rearrangement of these L chains did not occur during B cell development, but rather as a more recent event. Ig receptor editing might represent an attempt either to alter autoreactivity of this B cell population or to improve affinity for Ag. Because there are no organized germinal centers in brain, it is interesting that no dominant clonal L chain rearrangement was found in clone 1 and may offer insights as to where B cell responses in the CNS of MS patients mature. In model systems containing an intact blood-brain barrier, specific B cell responses to Ag introduced intracerebrally develop in peripheral germinal centers (27) and then migrate as activated B cells back into the CNS, where they are retained and differentiate at the site of Ag deposition (28). Our observations suggest either that Ag recognition is largely independent of the L chain sequence in clone 1, or that these secondary L chain rearrangements may actually be occurring within the CNS. The significance of L chain receptor editing in modifying the CNS immune response in MS requires further study. Meanwhile, this phenomenon does not appear to be widespread because MS02-2 clone 1 was the only MS B cell population in which evidence of L chain editing was found.
The significance of a functional and nonfunctional VH rearrangement in MS02-2 clone 2 is not clear. This second rearrangement most likely occurred during B cell development to generate a functional B cell receptor at the pre-B cell stage or to alter a self-reactive B cell. The stop codon in the nonfunctional VH rearrangement may be present in one of the DP-14 germline alleles or simply may have occurred during the germinal center reaction in the absence of selective pressure.
The relationship between the clonal B cell populations in MS CSF and OGB production remains to be determined. The number of OGBs in MS02-6 and MS02-11 CSF exceeded that of clonal populations detected in our CSF analysis. Some of these OGBs may be synthesized by B or plasma cells located in plaques that extravasate into the CSF. The degree of relatedness between B cells populating plaques and those in CSF is still unknown. Plasma cells are also present in CSF at levels that are 25% of those observed for B cells (29) and probably account for the bulk of Ab production. Their similarity to B cell populations in MS CSF also remains to be determined.
Overall, B cell clonal expansion is a prominent feature of the MS humoral response and has been found in both MS plaques (8, 10) and CSF (12, 13). The clonal variation observed within some populations and the somatic mutations found in rearranged V region segments indicate that a targeted response occurs in MS. Identification of rearranged Ig sequences is the first step in accurately recreating recombinant Abs from these overrepresented H and L chains for use in efforts to discover the relevant Ag(s) in MS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregory P. Owens, Department of Neurology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Mail Stop Box B182, Denver, CO 80262. E-mail address: greg.owens{at}uchsc.edu
3 Abbreviations used in this paper: CSF, cerebrospinal fluid; CDR, complementarity-determining region; MS, multiple sclerosis; OGB, oligoclonal band; RT, reverse transcription.
Received for publication March 13, 2003. Accepted for publication June 26, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene repertoire of IgM+ cells. J. Clin. Invest. 99:1614.[Medline]
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