The Journal of Immunology
VH4 Gene Segments Dominate the Intrathecal Humoral Immune Response in Multiple Sclerosis1 Gregory P. Owens,2* Kimberly M. Winges,* Alanna M. Ritchie,* Sydni Edwards,* Mark P. Burgoon,* Laura Lehnhoff,* Kirsten Nielsen,* John Corboy,* Donald H. Gilden,*† and Jeffrey L. Bennett*‡ A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138ⴙ plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138ⴙ cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138ⴙ cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138ⴙ cells with functionally rearranged VH4 gene segments as an overriding feature of MS CeSF repertoires. VH4 dominance was attributed to the preferential selection of specific VH4 genes, particularly gene segment VH4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in VH4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis. The Journal of Immunology, 2007, 179: 6343– 6351.
M
ultiple sclerosis (MS)3 is the most common inflammatory demyelinating disease of humans. Although genetic susceptibility and environmental factors play a role in the development of MS, the cause remains unknown. The intrathecal synthesis of Ig and the presence of oligoclonal bands (OCBs) are among the most reliable immunochemical markers of disease (reviewed in Ref. 1). Intrathecal IgG synthesis and OCBs are routinely found in chronic infections of the CNS, where Ab is directed against the cause of disease (2). In MS, evidence for the antigenic specificity of intrathecal IgG has been inconclusive. Nevertheless, mounting evidence supports a role for humoral immunity in MS pathogenesis. Immunohistologic studies indicate immune effector functions for plaque IgG (3–7) and MS patients with a high B cell-to-monocyte ratio in cerebrospinal fluid (CeSF) show a more rapid disease progression (8). Oligoclonal IgM bands are also present in CeSF of some MS patients and correlate with a more aggressive form of disease (9, 10). Phenotypic analyses of B cell subsets and sequence analyses of Ig V regions expressed by B and plasma cells in MS CeSF and
*Department of Neurology, †Department of Microbiology, and ‡Department of Ophthalmology, University of Colorado Health Sciences Center, Denver, CO 80262 Received for publication June 18, 2007. Accepted for publication August 13, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Public Health Service Grants NS32623 (to D.H.G., G.P.O.), EY014573 (to J.L.B.), NS041549 (to M.P.B.), and the National Multiple Sclerosis Society Research Grant RG3908 (to J.L.B.). K.M.W. is a medical student supported by a Howard Hughes Medical Institute Fellowship Award. 2 Address correspondence and reprint requests to Dr. Gregory P. Owens, Department of Neurology, University of Colorado Health Sciences Center, Mail Stop B-182, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address:
[email protected] 3 Abbreviations used in this paper: MS, multiple sclerosis; CeSF, cerebrospinal fluid; OCB, oligoclonal band; CIS, clinically isolated demyelinating syndrome; SSPE, subacute sclerosing panencephalitis; RT, reverse transcription; MNC, mononuclear cell.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org
brain indicate a T cell-dependent Ag-specific response (reviewed in Ref. 11). Compared with matching peripheral blood, there is a significant enrichment of memory B cells (12–15), particularly class-switched IgM⫺IgD⫺ memory cells (14) and the features of oligoclonal H chain V-region sequences (VH) recovered from MS plaques (16 –18) and CeSF (19 –23) are also consistent with a postgerminal center B cell response. VH sequences are somatically mutated, some quite extensively, and intraclonal sequence diversification within B and plasma cell clones has been observed repeatedly. The primary Ab-secreting cell type in MS CeSF carries surface markers consistent with short-lived plasma blasts (13). Clonal expansion has also been observed in the CeSF of individuals after a clinically isolated demyelinating syndrome (CIS), suggesting that B cell responses are established early in the disease course (22, 24, 25). To identify molecular aspects of CNS humoral immunity that might distinguish the disease process in MS, we compared blood and CeSF repertoires from a wide spectrum of clinically definite MS patients and from two control individuals with other chronic inflammatory CNS diseases. The preferential selection of plasma cells using specific sets of VH4 gene segments was a prevailing feature of the MS CeSF repertoire.
Materials and Methods MS and control patients CeSF (⬃20 ml) was collected from 15 MS patients (11 with relapsingremitting MS, 3 with primary progressive disease, and 1 with secondary progressive MS), 1 patient with chronic meningitis (IC05-2) of unknown etiology, and 1 patient with subacute sclerosing panencephalitis (SSPE; Table I). The duration of disease at the time of lumbar puncture varied from 6 mo to 25 years. Informed consent was obtained for all study participants. All MS patients had multiple white matter lesions demonstrated by brain magnetic resonance imaging, and their CeSF contained both OCBs and elevated percentages of IgG (Table I). With the exception of patient MS05-1, who was being treated with -IFN at the time of sampling, none of the donors had received steroids or immunomodulatory drugs for at least 1 mo before sampling. Both inflammatory control CeSFs contained increased levels of IgG and OCBs. Blood was also collected from five MS patients and from three healthy adult controls for peripheral B cell analysis.
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HUMORAL IMMUNITY IN MS CeSF
Table I. Clinical and CeSF features of MS patients undergoing single-cell analysis Patient
Age
Sex
Diagnosis
CeSF Cells
% IgG
OCBs
Disease Duration at LPa
MS02-2 MS02-6 MS02-11 MS02-14 MS02-19 MS02-24 MS03-7 MS05-3 MS04-2 MS05-6 MS05-1 MS06-2 MS06-1 MS06-3 MS06-6 IC05-2 IC06-1
38 42 22 51 46 39 42 30 26 62 25 32 36 52 51 31 12
M F F F F F F F F M F F F F F M M
Relapsing-remitting MS Relapsing-remitting MS Relapsing-remitting MS Relapsing-remitting MS Primary-progressive MS Secondary-progressive MS Relapsing-remitting MS Relapsing-remitting MS Primary-progressive MS Primary-progressive MS Relapsing-remitting MS Relapsing-remitting MS Relapsing-remitting MS Relapsing-remitting MS Relapsing-remitting MS Chronic meningitis of unknown etiology Subacute sclerosing panencephalitis
1 9 18 10 10 17 10 18 0 1 23 2 11 3 9 30 7
8 13 21 15 15 16.9 32 23.7 25 18 18.5 5.5 21 3.2 33.4 20.2 59.2
2–3 5 3 5 6 1 3 21 5 14 23 12 20 13 19 21 17
2 years 1 year 1–2 years 22 years 3 years 20 years 4 years 1 year 6 months 3 years 2.5 years 3 years 8 months 25 years 5 years 4 years 1 year
a
LP, Lumbar puncture.
Cell labeling and FACS ⫹
⫹
CD19 B cells and CD138 plasma cells were sorted from CeSF using a MoFlo flow cytometer (Cytomation) as described (21, 22). To avoid deposition of multiple cells into a single well, forward scatter integration was used to eliminate cell clusters during the sorting protocol. Immediately after collection, CeSF was centrifuged at ⬃500 ⫻ g for 10 min in a tabletop centrifuge. Under sterile conditions, the supernatant was removed and the cell pellet was suspended in ⬃200 l of residual CeSF. The cell suspension was incubated at room temperature for 30 min with anti-human CD19allophycocyanin, anti-human CD138-PE, and anti-human CD3-FITC (Caltag Laboratories) diluted with PBS and placed on ice. After gating for cells in the size range of lymphocytes and plasma cells by light scattering, single CD138⫹ plasma or CD19⫹ B cells were deposited into wells of a 200-l, 96-well PCR plate (ISC Bioexpress) containing 20 l of 1⫻ reverse transcriptase (RT) buffer (Invitrogen Life Technologies). Samples were sorted until one to three plates of CD138⫹ and CD19⫹ cells were obtained. Because CD138⫹ cells also express varying levels of CD19, a phenotype indicative of plasma blasts (13), CD19⫹CD138⫺ cell populations were also sorted from some MS CeSFs (MS04-2, MS05-1). Peripheral blood was isolated in either EDTA or CPT Cell Separation Vacutainer tubes (BD Biosciences). Peripheral blood collected in EDTAcontaining tubes was washed repeatedly with red cell lysis buffer (0.017 M Tris-HCl (pH 6.5), 0.014 M NH4Cl) and the remaining mononuclear cells (MNCs) were recovered by centrifugation at ⬃500 ⫻ g. MNCs were prepared from CPT tubes per the manufacturer’s instructions and washed once in red cell lysis buffer. CD19⫹ peripheral blood MNCs were sorted as described for CeSF B cells. Memory B cell repertoires from peripheral blood were obtained by IgG- and IgM-specific PCR amplification of sorted CD19⫹CD27⫹ cells using anti-human CD19-allophycocyanin and anti-human CD27-PE Abs. In patient MS02-24, an additional antihuman IgG-FITC mAb staining was used to enrich for IgG⫹ memory cells.
were of sufficient homology to amplify VH6 and VH7 gene segments. Both IgM and IgG amplifications were done on CD19⫹ and CD27⫹ cells, whereas only IgG amplifications were performed on CD138⫹ cells.
cDNA synthesis and amplification of VH sequences First-strand cDNA synthesis and subsequent nested PCR amplification of IgG and IgM VH-region sequences were performed using the I-Cycler (Bio-Rad) in a 96-well format as described (21, 22). All PCRs were performed in a 50-l reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.01% gelatin, 200 M dNTPs, ⬃10 pM of each primer (100 ng per PCR), and 2 U of Taq polymerase (Applied Biosystems). Cycle conditions for the primary PCR which included VH leader primers in conjunction with a conserved IgG or IgM constant region primer (CH1) were as follows: an initial denaturation cycle at 95°C for 5 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a final extension cycle at 72°C for 7 min. Nested PCR followed the same protocol except that the annealing step was conducted at 56.5°C for optimum performance of the VH framework 1 primers and a second conserved IgG or IgM constant region primer (CH2). Because single germline segments comprise the VH6 and VH7 families and are rarely used in the human VH repertoire, primers specific for these families were not incorporated into the PCR primer mix. Nonetheless, V region primers specific to other VH families
FIGURE 1. PCR efficiency of VH leader primers. The indicated copy numbers of pooled plasmid DNA containing somatically mutated VH sequences cloned from MS and SSPE brain cDNA libraries were amplified by real-time PCR using the respective VH family leader primers, IgG C region primer (CH1) and PCR cycling conditions used in the primary PCR of our standard single-cell RT-PCR. Family specific plasmid DNA pools included 8 of 11 VH1 gene segments, 3 of 3 VH2 gene segments, 14 of 22 VH3 gene segments, 9 of 11 VH4 gene segments, and 1 of 2 VH5 gene segments. Both the dose-response curves and regression analysis are included and demonstrate a strong inverse relationship between DNA copy number and cycle time. Pairwise comparisons showed that the entire range of VH family amplification efficiencies varied by ⬍15%.
The Journal of Immunology
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Table II. Features of mononuclear cells from MS and non-MS inflammatory CeSF VH Repertoire CeSF
B Cell Type
% CD19 or CD138
Total number of VH regions analyzed
% sequences in VH clonesa
No. VH clones
Average % Homologyb
MS02-2 MS02-6 MS02-11 MS02-14 MS02-19
CD19 CD19 CD19 CD19 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138 CD19 CD138
1.5 2.6 3.9 1.7 3.5 0.7 3.7 3.5 2.5 0.8 2.4 1.1 7.4 1.8 2.5 0.9 1.8 1.3 4.3 1 2.7 1.7 7 1.5 4.6 1.3 2.5 0.3 3.9 1.25
25c 35e 39e 45e 26c 23 42c 76 Nd 65 33e 76 Nd 109 23e 55 Nd 36 Nd 50 Nd 47 Nd 30 Nd 194 Nd 48 Nd 71
81 80 11 50 35 78 12 33 Nd 38 0 58 Nd 68 0 73 Nd 86 Nd 74 Nd 79 Nd 74 Nd 62 Nd 35 Nd 70
3 2 1 7 4 8 2 10 Nd 11 0 10 Nd 26 0 11 Nd 3 Nd 5 Nd 8 Nd 4 Nd 35 Nd 8 Nd 7
Ndd Nd Nd Nd Nd 94.2 Nd 94.8 Nd 94.6 Nd 92.4 Nd 92.2 Nd 93.5 Nd 94.2 Nd 93.8 Nd 94.5 Nd 92.5 Nd Nd Nd 95.1 Nd 93.3
MS02-24 MS03-7f MS04-2f,g MS05-3 MS05-1f,g MS05-6 MS06-1f MS06-2 MS06-3 MS06-6 IC05-2 IC06-1
a Clonal populations are operationally defined as cells from the same CeSF expressing an identical V(D)J recombination based on the VH CDR3 amino acid sequence and refers only to IgG-expressing cells. IgM⫹ clones were rarely detected in CeSF CD19⫹ B cell repertoires and were not examined in CD138⫹ cells. b Values represent the average degree of homology to germline for unique CD138⫹ VDJ rearrangements. c Analysis includes IgG⫹ cells only. d Nd, Not done. e Analysis includes IgG⫹ and IgM⫹ cells. f Additional repertoire analyses of peripheral blood CD19⫹ B lymphocytes and/or CD27⫹ memory B cells were also performed and are described in subsequent tables. g Unlike CD19⫹ cell populations from previous donors, only CD19⫹, CD138⫺ B lymphocytes were sorted and analyzed. These repertoires are prominently IgM-expressing cells.
Because B and plasma cells express a single functional VH rearrangement and occasionally a nonfunctional rearrangement, there was no direct competition between different VH family H chain leader and framework 1 primers in the mixtures used for nested amplification of VH region sequences. Instead, VH family primers only needed to be sufficiently efficient to amplify the correct V regions from input cDNA obtained from a single-cell RT reaction. Thus, it was not unexpected that amplification of V region sequences from plasma cells, which contain increased Ig mRNA levels, was more efficient than amplification from peripheral blood and CeSF CD19⫹ B cells. The average efficiency of VH amplification was 56% for single CeSF CD138⫹ cells (range 19 – 80%) and 36% for peripheral blood CD19⫹ cells (range 18 – 66%). To further ensure that there were no gross differences in amplification efficiency with different VH family primers which might introduce bias into B cell repertoires, individual family VH family leader primers were tested in dose-response curves using real-time PCR (data not shown). VH gene segments were cloned from MS and SSPE brain cDNA libraries (16, 17) and the purified plasmid DNA clones were pooled based upon their family germline identity (VH1–VH5). The individual VH clones contained varying degrees of somatic mutation and comprised 8 of 11 VH1 family gene segments, 3 of 3 VH2 gene segments, 14 of 22 VH3 gene segments, 9 of 11 VH4 gene segments, and 1 of 2 VH5 gene segments. Serial dilutions of each VH gene segment pool containing 102–106 copies of total plasmid DNA were subjected to real-time PCR using family specific leader sequence primers, CH1 primer, and cycling conditions used in the first PCR of our standard single-cell RT-PCR (21). Real-time PCR was performed on an ABI 7500 Fast Real-Time PCR System using Power SYBR Green PCR Master Mix (Applied Biosystems). To calculate the efficiency of each VH family PCR, linear regression was performed on the real-time PCR dose-response curve (Intercooled Stata 9.2).
Regression lines demonstrated a strong inverse relationship between the log concentration of target sequence and cycle time (Fig. 1). VH3 family leader sequences demonstrated the highest efficiency while VH4 and VH5 family leader sequences were lowest. Pairwise comparisons demonstrated significant differences in PCR efficiency for VH2–VH5, VH3–VH4, and VH3–VH5 reactions. The magnitude of the difference, however, was minimal with the entire range of VH family amplification efficiencies, varying by ⬍15%.
Screening and purification of PCR products PCR were screened by agarose gel electrophoresis for appropriate V-region sizes and positive PCR products were purified from primers using either Montage PCR purification columns (Millipore) or Qiavac 96-well PCR purification plates (Qiagen). PCR products were sequenced by the University of Colorado Cancer Center DNA Sequencing and Analysis Core (University of Colorado Health Sciences Center, Denver, CO) with conserved C-region primers as described (21). Individual sequences were analyzed with DNASIS Max software and aligned with human Ab V-region germline segments in the V Base 1 (www.mrc-cpe.cam.ac.uk) or V Base 2 (http://vbase2.org/) databases. This online service was used to identify the closest V-region germline segments and to determine the extent of sequence homology for each of the VH Ab sequences analyzed. For consistency, homologies to germline segment alleles of donor DP were used when applicable. Germline designations for individual V region sequences were assigned by locus.
Statistics Statistical analyses were performed using Microsoft Excel and Intercooled Stata 9.2. Based on independent studies showing that the observed VH
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FIGURE 2. VH family distributions in blood and CeSF repertoires. A, IgM VH family distributions in normal (n ⫽ 3) and MS (n ⫽ 5) peripheral blood CD19⫹ B cells as compared with the expected germline distributions. No significant differences in VH family distributions were observed between normal and MS blood repertoires. B, Comparison of VH family distributions in MS CeSF CD138⫹ (n ⫽ 11) and MS peripheral blood CD19⫹ (n ⫽ 5) repertoires. The frequency of CeSF CD138⫹ cells expressing VH4 was significantly higher than that of blood CD19⫹ cells whether total (T) or unique (U) sequences were considered. ⴱ, p ⬍ 0.0005; ⴱⴱ, p ⬍ 0.00001 (t test). No difference (p ⫽ 0.29, sign test) in the percentage of VH4⫹ CeSF plasma cells was observed in MS CeSF when total CD138⫹ cells were compared with unique V(D)J rearrangements.
family distribution in adult peripheral blood CD19⫹ B lymphocytes approximates germline family prevalence (26 –28), the probability of an observed VH family distribution in MS or inflammatory control repertoires was calculated by 2 goodness of fit using the functional family germline prevalence published by Cook and Tomlinson (29). With the exception of a single sequence observed in the MS06-6 CeSF CD138 repertoire, VH6 family germlines were not amplified from any B cell or plasma cell population and thus not included in statistical calculations. The probability of an observed VH4 population in a specific repertoire was calculated by binomial distribution. After a Bonferroni correction for multiple comparisons of each VH family, differences were considered significant at p ⬍ 0.008. Comparison of VH family and individual germline use in peripheral blood and CeSF was determined by t test with unequal variances using the method of Satterthwaite. A Bonferroni correction for multiple comparisons was applied to the reported p values.
Results Features of the B cell response in MS and inflammatory control CeSF CeSF was collected from 15 MS patients, from 1 patient with chronic meningitis (IC05-2) of unknown etiology and from 1 patient (IC06-1) with SSPE (Table I). The MS patient pool included individuals with widely differing disease durations (6 mo–25 years) and clinical courses. Regardless of their clinical profiles and levels of CeSF pleocytosis, CD19⫹ B lymphocytes and/or CD138⫹ plasma cells were readily detected and sorted from each CeSF sample (Table II). CD19⫹ cells accounted for 1.5–9% of MS CeSF MNCs, and CD138⫹ cells varied from 0.4 –3.5%. The percentage of B lymphocytes and plasma cells observed in the CeSF of the inflammatory control patients also fell within this range.
HUMORAL IMMUNITY IN MS CeSF Table II lists the number of functional VH region sequences amplified and sequenced from each CeSF. Comparative CD138 and CD19 repertoire analyses were not available or not performed for some CeSF sorts. A total of 11 different CD138⫹ analyses and 8 different CD19⫹ analyses were performed. Most amplified Vregion sequences were productive rearrangements and were in the correct translational reading frame into the C region domain. The recovery of more than one functional V(D)J rearrangement from a single well was not observed in any CeSF analyses indicating that deposition of multiple cells into a single well was a rare event. Occasionally, a nonproductive VH sequence containing either an in-frame stop codon or an out-of-frame V(D)J rearrangement was also amplified from isolated wells including those yielding productive VH sequences. As reported (22) and extended in Table II, clonally expanded plasma cell populations were prominent in each CD138 repertoire. The number of clones identified in each repertoire was in part dependent on the number of functional sequences analyzed. The repertoires of MS05-3 and MS06-6, which analyzed 100 –200 single plasma cells, revealed the largest number of plasma cell clones. Features of sequences comprising the MS02-19 and MS02-24 repertoires have been described in detail (22). Alignment of V-region sequences to their closest germline segment revealed that almost all CD138⫹ VH sequences contained some degree of somatic mutation. The degree of somatic hypermutation for CD138 repertoires averaged for unique V(D)J rearrangements is presented in Table II. There was little evidence of extensive receptor editing within CeSF CD138⫹ populations in which both H and L chain V-region sequences were analyzed. One clone in the SSPE CeSF (IC06-1) repertoire was associated with two different L chain sequences (30) and the largest VH clone in MS05-6 CeSF was associated with multiple L chains. We previously reported a CD19⫹ clone from MS02-2 CeSF which expressed multiple L chain sequences (21). Amplification and sequencing of both IgG- and IgM-expressing CD19⫹ cells performed for a subset of patients revealed the presence of clonal populations in CD19⫹ CeSF cells, but at a lower frequency than in the matching CD138 repertoire. Expanded B cell clones were observed almost exclusively within class-switched IgG⫹ B cells, while a more polyclonal response was observed for IgM-expressing B cells. Among all MS CeSF CD19⫹ repertoires analyzed, only a single small IgM clone (MS02-6) was identified. Because CD138⫹ cells in CeSF express varying levels of CD19 (13), they were present to some degree in the CD19 repertoires of MS patients and probably contributed to the CD19⫹ clonal populations. Analysis of CD19⫹CD138⫺ B lymphocytes sorted from the CeSF of MS04-2 and MS05-1 showed that most functional VH rearrangements were IgM, accounting for 24 of 33 B cell sequences from MS04-2 CeSF and 21 of 23 sequences recovered from MS05-1 CeSF. No IgM or IgG clones were observed in either repertoire. The mature CD19⫹ MS peripheral blood repertoire does not differ from healthy adult controls The human Ab repertoire uses 51 functional germline genes which are divided into families based on sequence homology (26). Of the seven VH families, the largest is VH3 with 22 gene segments, followed by VH1 and VH4 with 11 gene segments each. Consistent with previous independent observations on VH usage in peripheral blood CD19⫹ B lymphocytes (27–29), CD19⫹IgM⫹ B cells sorted from the peripheral blood of both normal healthy adults and MS patients displayed VH family distributions that approximated germline prevalence or were slightly biased toward excess VH3 family sequences (Fig. 2A). No significant differences in VH distribution were observed between healthy subjects and MS patients.
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Table III. VH family representation of normal and MS peripheral blood CD19⫹ B lymphocytesa Germline
NC05-1
NC05-2
NC05-3
MS03-12
MS03-7
MS04-2
MS05-1
MS06-1
VH Family
n ⫽ 51
n ⫽ 50
n ⫽ 35
n ⫽ 63
n ⫽ 33
n ⫽ 62
n ⫽ 41
n ⫽ 50
n ⫽ 35
1 2 3 4 5 7 2c p⫽
21.6% 5.9% 43.1% 21.6% 3.9% 2.0%
20.6% 3.2% 52.4% 17.5% 6.3% 0% 4.78 0.44
6.1% 6.1% 60.6% 21.2% 3% 3% 6.25 0.28
28.3% 5% 41.7% 23.3% 5% 0% 2.65 0.75
29.3% 7.3% 43.9% 12.2% 7.3% 0% 4.96 0.42
25% 0% 55.6% 16.7% 2.8% 0% 8.6 0.13
20% 2.9% 54.3% 17.1% 5.7% 0% 2.9 0.72
20% 2% 58%b 10% 4% 6% 10.99 0.05
17.1% 0% 57.1% 25.7% 0% 0% 6.33 0.28
a Single CD19⫹ peripheral blood B lymphocytes were sorted from the blood of normal healthy controls (NC) and MS patients, and the respective IgM VH regions amplified, sequenced, and analyzed as described in Materials and Methods. The majority of cells in each repertoire expressed nonmutated germline sequences and probably represented naive B lymphocytes. Each column indicates the blood donor, the number of functional VH sequences analyzed (n), and their distribution among different VH families. No VH6 germline segments were detected. b Binomial distribution: p ⫽ 0.025. c A 2 goodness of fit (5 degrees of freedom) was used to compare the observed VH family distribution to the expected VH family distributions using the published germline prevalence of the 51 functional VH gene segments (26).
When CD19 blood repertoires were examined individually by 2 analysis (Table III), only in one repertoire (NC05-1) was deviation ( p ⫽ 0.05) from germline distribution observed, which was attributable to an increased percentage of VH3 sequences ( p ⫽ 0.025). Most (⬃73%) IgM VH sequences in blood CD19⫹ repertoires of control and MS patients were nonmutated germline sequences (0 –1 mutations) and presumably represented mature naive B cells. The CD138⫹ VH repertoire in MS CeSF is biased for VH4 family germlines Compared with VH4 prevalence in peripheral blood CD19⫹IgM⫹ B cells (mean ⫾ SD: 21 ⫾ 8.6%), representation of the VH4 family was significantly elevated ( p ⬍ 0.00001) in MS CeSF CD138 repertoires (mean ⫾ SD: 70.7 ⫾ 11.4% for total CeSF CD138⫹ cells). This increase in VH4 use was significant ( p ⬍ 0.00001) whether comparing the total or the unique number of CD138⫹ VH rearrangements in CeSF (Fig. 2B). The increased number of VH4⫹ CD138 cells in MS CeSF was accompanied by a significant re-
duction ( p ⬍ 0.0005) in cells expressing VH3 germline segments. Reduced VH1 germline use was also evident, but was not significant ( p ⫽ 0.06). Analysis of each MS CeSF CD138 repertoire individually (Table IV) revealed a significant VH4 bias in 11 of 11 patients when distributions were based on total CD138⫹ cell counts and in 10 of 11 CeSF repertoires when distributions were compiled using unique V(D)J rearrangements. The single discrepancy in VH4 prevalence between total and unique CD138⫹ plasma cell repertoires was in MS06-3 CeSF which displayed VH4 dominance when viewing all CD138⫹ sequences and VH3 dominance when viewing unique VH sequences. This difference was due to the presence of a single large VH4 clone in the MS06-3 repertoire that accounted for ⬎50% of CD138⫹ cells analyzed. A VH4 bias was also found in the chronic meningitis CeSF repertoire (IC05-2), but not in the SSPE CeSF repertoire (IC06-1) which was biased for VH1 family gene segments. VH4 bias was not always prominent in MS CeSF CD19⫹ repertoires, which displayed various VH family distributions (Table
Table IV. VH family representation in CeSF CD138⫹ cells from MS and other inflammatory CNS diseasesa VH Family
MS02-19
MS02-24
MS03-7
MS04-2
MS05-1
MS05-3
MS05-6
MS06-1
MS06-2
MS06-3
MS06-6
IC05-2
IC06-1
Total
n ⫽ 23
n ⫽ 76
n ⫽ 66
n ⫽ 66
n ⫽ 55
n ⫽ 109
n ⫽ 36
n ⫽ 50
n ⫽ 47
n ⫽ 30
n ⫽194
n ⫽ 48
n ⫽ 71
1 2 3 4 5 7 2e p⬍ Unique 1 2 3 4 5 7 2e p⬍
0% 13% 4.3% 82.6%b 0% 0% 56.7 0.0001 n ⫽ 13 0% 15.4% 7.7% 76.9%b 0% 0% 27.9 0.0001
2.6% 5.3% 27.6% 64.5%b 0% 0% 86.6 0.0001 n ⫽ 62 1.6% 6.6% 26.2% 65.6%b 0% 0% 74 0.0001
1.5% 7.6% 15.2% 75.8%b 0% 0% 118.8 0.0001 n ⫽ 39 2.6% 12.8% 15.4% 69.2%b 0% 0% 60.2 0.0001
12.1% 1.5% 25.8% 50%b 7.6% 3% 37 0.0001 n ⫽ 53 7.5% 1.9% 24.5% 56.6%b 5.7% 3.8% 42 0.0001
0% 20.0%c 16.4% 63.6%b 0% 0% 88.1 0.0001 n ⫽ 27 0% 14.8% 22.2% 63%b 0% 0% 35.4 0.0001
6.4% 7.3% 17.4% 68.8%b 0% 0% 148.5 0.0001 n ⫽ 64 4.7% 10.9% 14.1% 70.3%b 0% 0% 98.4 0.0001
0% 5.6% 2.8% 91.7%b 0% 0% 105.9 0.0001 n⫽7 0% 14.3% 14.3% 71.4%d 0% 0% 12.1 0.03
2% 6% 14% 78%b 0% 0% 95.8 0.0001 n ⫽ 18 5.6% 11.1% 16.7% 66.7%b 0% 0% 24 0.0002
2.1% 17.0%d 10.6% 70.2%b 0% 0.0% 84.2 0.0001 n ⫽ 18 5.6% 11.1% 27.8% 55.6%d 0% 0% 14.7 0.01
3.3% 0.0% 36.7% 60%b 0% 0% 29.1 0.0001 n ⫽ 11 9.1% 0% 63.6% 27.3% 0% 0% 3.3 0.65
1.5% 6.7% 17% 70.6%b 3.6% 0% 286.7 0.0001 n ⫽ 110 1.8% 8.2% 17.3% 70.9%b 0.9% 0% 167.4 0.0001
0% 8.3% 35.4% 56.2%b 0% 0% 41.2 0.0001 n ⫽35 0% 8.6% 37.1% 54.3%b 0% 0% 27.8 0.0001
59.2%b 0% 21.1% 15.5% 4.2% 0% 61.6 0.0001 n ⫽ 28 42.9%f 0% 35.7% 10.7% 10.7% 0% 13.4 0.02
a Single CD138⫹ cells were sorted from CeSF of 11 MS patients and from patients with SSPE (IC06-1) and chronic meningitis of unknown etiology (IC05-2). The respective IgG VH regions were amplified, sequenced, and analyzed as described in Materials and Methods. Each column indicates the CeSF donor, the number of functional VH sequences analyzed (n), and their percent distribution among different families calculated using the total and unique number of VH sequences analyzed. With the exception of MS06-6 where a single VH6 germline was detected, no VH6 segments were detected in any other repertoire. Numbers in bold represent the predominant VH family in that repertoire. b VH4 family binomial distribution: p ⬍ 0.0001. c VH4 family binomial distribution: p ⬍ 0.0003. d VH4 family binomial distribution: p ⬍ 0.006. e A 2 goodness of fit (5 degrees of freedom) was used to compare the observed to the expected VH family distributions calculated from the germline prevalence published by Cook and Tomlinson (29). f VH4 family binomial distribution: p ⫽ 0.009.
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HUMORAL IMMUNITY IN MS CeSF
Table V. VH family representation in MS CeSF CD19⫹ cellsa MS02-2
MS02-6
MS02-11
MS02-14
MS02-19
MS02-24
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
IgM
IgG
VH Family
n ⫽ 25
n⫽4
n ⫽ 13
n ⫽ 21
n ⫽ 18
n ⫽ 21
n ⫽ 30
n ⫽ 15
n ⫽ 26
n⫽4
n ⫽ 41
1 2 3 4 5 7 2c p⬍
48% 8% 4% 32% 8% 0% 19.9 0.001
ND ND ND ND ND ND
0% 0% 69.2% 30.8% 0% 0% 4.8 0.23
14.3% 0% 47.6% 33.3% 4.8% 0% 3.6 0.61
16.7% 5.6% 66.7% 11.1% 0% 0% 4.5 0.48
9.5% 0% 52.4% 28.6% 9.5% 0% 5.63 0.34
3.3% 0% 30% 60%b 6.7% 0% 29.2 0.0001
0% 0% 86.7% 6.7% 6.7% 0% 12.84 0.02
3.8% 26.9% 38.5% 23.1% 7.7% 0% 24.8 0.0001
ND ND ND ND ND ND
2.4% 9.8% 17.1% 70.7%b 0% 0% 62.6 0.0001
IgM
ND ND ND ND ND ND
MS04-2
MS05-1
IgG
IgM
IgG
IgM
n⫽9
n ⫽ 24
n⫽2
n ⫽ 21
11.1% 11.1% 66.7% 12.5% 8.3% 0% 3.0 0.7
12.5% 0% 66.7% 12.5% 8.3% 0% 7.99 0.16
ND ND ND ND ND ND
14.3% 9.5% 57.1% 9.5% 9.5% 0% 5.45 0.36
a Single CD19⫹ B lymphocytes were sorted from MS CeSF and the respective IgM and IgG V regions were amplified and analyzed as described in Materials and Methods. Each column indicates the CeSF donor, the total number of functional V sequences analyzed (n), and their distribution among different VH families. The most prevalent VH family is in bold for each analysis. The repertoire analyses of MS02-2, MS02-6, MS02-11, and MS02-14 were reported previously (21) and included CD19⫹CD138⫹ cells, whereas the CD19⫹ B cell repertoires reported for MS04-2 and MS05-1 CeSF did not include CD19⫹CD138⫹ cells. b Binomial distributions calculated for VH4 family: p ⬍ 0.0001. c A 2 goodness of fit was used to compare the observed to the expected VH family distributions based on germline prevalence reported by Cook and Tomlinson (29).
V). No IgM B cell repertoires showed VH4 bias. Instead, VH distributions approximated germline prevalence or displayed an excess of VH3 gene segments. The IgG⫹CD19⫹ B cell repertoire from MS02-24 CeSF showed significant VH4 bias like that seen in CD138⫹ cells. A VH4 bias was also found in MS02-14 CeSF, but was not observed in any other IgG⫹CD19⫹ repertoire, whether tabulating total (Table V) or unique (data not shown) sequences. For example, comparison of the CD19 and CD138 repertoires from MS02-19 CeSF revealed significant differences in their VH family distributions (2 ⫽ 16.6, p ⫽ 0.002). Although the CD138⫹ repertoire was strongly biased toward VH4 (Table IV), the CD19⫹ repertoire for MS02-19 displayed a VH2 bias. Comparative CD138 repertoires were not available for MS02-2, MS02-6, MS02-11, and MS02-14 CeSF. Because CD138⫹ cells in MS CeSF also express varying levels of CD19, the fraction of cells coexpressing CD138 in the CD19 repertoire might contribute to the VH4 bias found in some CD19 repertoires. MS02-24 CeSF, which displayed a strong VH4 bias in both CD138⫹ and CD19⫹ repertoires, contained a robust CD138⫹ cell population that was almost equivalent in num-
bers to CD19⫹ cells (Table II), whereas the CD138:CD19 ratio in MS02-19 CeSF was 5-fold lower. When CD19⫹ cells from MS04-2 and MS05-1 CeSF were sorted to exclude CD138⫹ cells (Table V), a smaller fraction (10 –27%) of the CD19 pool was IgG⫹ B cells. No VH4 bias was observed among the few IgG⫹ B cells recovered from MS04-2 CeSF. VH representation in peripheral blood memory cells Most CeSF B cells, including CD138⫹ cells, in CNS inflammatory diseases display a class-switched memory cell phenotype (12–14). We therefore examined peripheral blood CD27⫹ memory B cells for any perturbations in VH family distribution. Both IgM- and IgG-expressing CD19⫹, CD27⫹ blood cells from two MS patients (MS02-24 and MS05-1) and an adult control (NC05-3) were analyzed; the matching peripheral blood CD19⫹ IgM repertoires and CeSF CD138 repertoires of each donor are also presented for comparison (Table VI). As observed with blood CD19⫹ cells, the repertoires of both IgM and IgG memory cells were essentially polyclonal. Only a single clone of two identical sequences was
Table VI. Comparison of VH family representation in peripheral blood CD27⫹ memory cellsa NC05-3
MS02-24b
MS05-1
PBLs
PBLs
CeSF
PBLs
CeSF
CD19
CD27
CD27
CD19
CD27
CD27
CD138
CD19
CD27
CD27
CD138
IgM
IgM
IgG
IgM
IgM
IgG
IgG
IgM
IgM
IgG
IgG
VH Family
n ⫽ 63
n ⫽ 46
n ⫽ 32
n ⫽ 50
n ⫽ 45
n ⫽ 46
n ⫽ 55
n ⫽ 33
n ⫽ 64
n ⫽ 31
n ⫽ 75
1 2 3 4 5 7 2e p⫽
20.6% 3.2% 52.4% 17.5% 6.3% 0% 4.78 0.44
10.9% 2.2% 65.2% 15.2% 6.5% 0.0% 11.26 0.05
12.5% 0% 68.8% 12.5% 6.3% 0% 3.86 0.57
12% 2% 42% 34% 6% 4% 8.6 0.13
8.7% 2.2% 71.7% 13% 4.3% 0% 15.79 0.007
20% 2.2% 33.3% 35.6%c 8.9% 0% 9.82 0.08
0% 14.8% 22.2% 63.6%d 0% 0% 88.1 0.0001
6.1% 6.1% 60.6% 21.2% 3% 3% 6.25 0.28
14.1% 10.9% 57.8% 14.1% 3.1% 0% 10.58 0.06
19.4% 0% 45.2% 32.3% 3.2% 0% 4.16 0.53
2.7% 9.3% 41.3% 46.7%d 0% 0% 40.1 0.0001
a Single CD19⫹CD27⫹ memory cell B lymphocytes from one normal control and two MS patients were sorted from PBLs into IgM and IgG populations, and the respective V regions were amplified and analyzed as described in Materials and Methods. Each column indicates the blood and/or CeSF donor, the total number of functional VH sequences analyzed (n), and their distribution among different VH families in blood CD19⫹ cells, blood CD19⫹CD27⫹ cells and for comparison, matching CeSF CD138⫹ cells. The most prevalent VH family is in bold for each analysis. b Serial CeSF and blood analyses were performed on MS02-24. The CeSF CD138 repertoire and blood CD27 repertoires listed were analyzed 3 years after the MS02-24 repertoire reported in Table IV. Note that the CeSF CD138⫹ cell repertoire remains significantly biased for VH4 germline segments. The blood CD19⫹ cells were obtained 1 year before the second CeSF analysis. c VH4 family binomial distribution: p ⬍ 0.02. d VH4 family binomial distribution: p ⬍ 0.0001. e A 2 goodness of fit was used to compare the observed and expected VH family distributions based on reported germline prevalence (26).
The Journal of Immunology
6349
Table VII. VH4 germline representation in CD138⫹ cells from MS and inflammatory CNS disease CeSFa
MS02-19 (n ⫽ 13) MS02-24 (n ⫽ 62) MS03-7 (n ⫽ 39) MS04-2 (n ⫽ 53) MS05-1 (n ⫽ 27) MS05-6 (n ⫽ 7) MS05-3 (n ⫽ 65) MS06-1 (n ⫽ 18) MS06-2 (n ⫽ 18) MS06-3 (n ⫽11) MS06-6 (n ⫽ 110) MS CeSFb Mean ⫾ SD MS PBLsb mean ⫾ SD IC05-2 (n ⫽ 35) IC06-1 (n ⫽ 28)
% VH4-04
% VH4-28
% VH4-31/30.1
% VH4-30.2
7.7
0
0
0
3.3
0
0
0
0
3.8
% VH4-34
% VH4-39
% VH4-59
0
15.4
38.5
15.4
0
0
11.5
0
3.3
37.7
4.9
0
4.9
17.9
0
0
0
20.5
23.1
5.1
2.6
0
13.2
0
5.7
5.7
15.1
5.7
3.8
3.8
0
0
7
0
0
0
33
22
0
0
0
0
28.6
0
0
14.3
14.3
14.3
0
0
3.1
0
24.6
0
0
6.2
21.5
13.8
0
0
0
0
22.2
0
0
0
33.3
22
0
0
5.6
0
11.1
0
0
0
5.6
27.8
5.6
5.6
18.2
0
0
0
0
0
0
0
9.1
0
3.6
14.5
25.5
0
0
0.9
19.1
8.2
0
0
4.1 ⫾ 5.3
1.3 ⫾ 4.4
13.7 ⫾ 10.9
21.7 ⴞ 12.8c
13.3 ⫾ 8.5
2.1 ⫾ 3.2
1 ⫾ 1.8
2.3 ⫾ 1.4
0⫾0
5.1 ⫾ 3.7
2.4 ⫾ 3.3
5.3 ⫾ 3.5
1.4 ⫾ 1.3
0.3 ⫾ 0.7
13.5
0
2.8
8.6
0
0
0
0
20 3.6
% VH4-30.4
1 ⫾ 3.5
0.5 ⫾ 1.7
4.2 ⫾ 5.8
0.5 ⫾ 1.1
0.5 ⫾ 1.1
4 ⫾ 3.7
0
0
0
0
11.4
0
0
0
0
7.1
% VH4-61
% VH4-b
a
The use of individual VH4 gene segments is given as a percentage of the unique V(D)J rearrangements analyzed from each donor CeSF. Values indicate the mean percentage ⫾ SD of each VH4 gene for all MS donors analyzed, and compares their prevalence in CeSF CD138⫹ cell repertoires (n ⫽ 11) to their prevalence in the MS peripheral blood CD19⫹ cell repertoires (n ⫽ 5) listed in Table III. c Value of p ⫽ 0.005; t test comparing means of VH4-39 use in MS CeSF CD138⫹ cells and MS PBL CD19⫹ B lymphocytes. b
identified in the IgG memory cell repertoire of MS05-1. In control and MS patients, VH3 family gene segments were the predominant H chains used by IgM memory cells. In MS peripheral IgG memory cell repertoires, there was a modest elevation in VH4 family sequences (⬃36 and 32%) compared with germline distributions. The percentage approached statistical significance for MS05-1 ( p ⫽ 0.02), but not for MS02-24 ( p ⫽ 0.113) probably because of the smaller sample size. VH4 prevalence in IgG memory cells of the control patient (NC05-3) was 12.5%. In a recent and more extensive single-cell analysis of IgG⫹ memory cells from three healthy adult controls, the percentage of VH4 family sequences also approximated germline prevalence at 23.3% (31). The CD138 VH distribution given in Table VI for MS02-24 CeSF, analyzed 3 years after the initial determination (Table IV), remained significantly biased for VH4 family germline segments. Preferential use of Individual VH4 germline segments in the MS B cell response A comparison of individual VH gene segments used by peripheral blood CD19⫹ B cells from healthy adult controls and MS patients revealed no significant differences between the two populations (data not shown). On average, the most abundantly used VH3 germlines in MS and controls were 3–23 (⬃10%), followed by 3-30, 3-07, and 3-48 (⬃6% each). Within the VH4 family, 4-34, 4-59, and 4-39 genes were encountered at about equal frequency (2–5%). In MS CeSF, the high proportion of VH4 CD138⫹ plasma cells resulted from the preferential activation of B cells using germline segments 4-39, 4-31, and 4-59 (Table VII). Compared with its prevalence in peripheral blood CD19⫹ B cells (averaged from the
MS blood repertoires listed in Table III), the 4-39 germline was significantly enriched ( p ⫽ 0.005) in CeSF plasma cells and accounted, on average, for 22% of all unique H chain V(D)J rearrangements analyzed. The 4-39 germline was not detected in CD138⫹ cells recovered from the chronic meningitis CeSF (IC05-2) or from SSPE CeSF (IC06-1), strongly suggesting that the use of this germline in MS was disease related rather than a consequence of chronic CNS inflammation. Although the 4-31 and 4-59 germlines also showed clear increases in MS CeSF compared with blood, the increases were not statistically significant.
Discussion B lymphocytes are typically rare in the CeSF of healthy individuals, but are found in significant numbers in both chronic and acute inflammatory CNS diseases (8, 12, 13, 15, 21–22). Herein, we used cell sorting and single-cell RT-PCR to analyze H chain rearrangements of a large number of single CD138⫹ and/or CD19⫹ CeSF B cells from 15 MS patients and 2 patients with other CNS inflammatory diseases. Several experimental lines of evidence indicate that the primer sets and methodology used for single-cell VH amplification did not introduce significant experimental error into the observed B and plasma cell repertoires. Amplification of cloned H chain V regions using the primary PCR leader sequence primers showed minimal differences (⬃15%) in amplification efficiency of different VH families (Fig. 1). More importantly, our single-cell analyses of peripheral blood B cells from both healthy adult controls and MS patients yielded VH family distributions consistent with observations obtained by other independent studies using single-cell PCR amplification from B cell genomic DNA (26, 27), single-cell PCR amplification from B cell transcripts (28,
6350 31), VH amplification from blood MNC RNA (32), and screening of blood lymphocyte cDNA libraries (33). In most blood repertoires, the use of VH family gene segments approximated their germline distributions with VH3 family genes being used most frequently followed by VH4 and VH1 families. Thus, any strong bias that might be introduced into our analyses because of failure to amplify specific VH families due to differential primer efficiencies should have been apparent in the naive B cell populations where the levels of B cell transcripts are limited in comparison to CD138⫹ cells. The primary Ab-secreting B cell subtype in inflammatory CeSF, CD138 plasma blasts, was characterized by prominent IgG clonal expansion; overall, 64% of CD138 cells in MS CeSF were in clonal populations. Because each repertoire is a representation of the CD138⫹ cell population in CeSF and is more likely to reveal the largest clones, the number of VH clones reported in Table II is probably underestimated. The large repertoires generated from MS05-3 and MS06-6 CeSF, which included 100 –200 single cells per patient, revealed 25 and 35 distinct IgG clonotypes, respectively. Clonal expansion was not as prominent in the CD19 repertoire, particularly in IgM cells where clones were rarely detected. A striking feature of the CD138 repertoire in MS CeSF was the accumulation of cells expressing VH4 family gene segments. On average, ⬃70% of CD138⫹ cells used a functional VH4 H chain sequence and most (⬃50%) were V(D)J rearrangements of three specific gene segments, 4-39, 4-31, and 4-59. This VH4 bias was not found in mature peripheral blood IgM⫹ B cells or IgM⫹ CeSF B cells, indicating that selective pressures, rather than inherent biases in the available B cell pool, were shaping the class-switched CD138⫹ repertoire in MS patients. In SSPE CeSF, VH1 was the predominant germline family used by CD138⫹ cells, consistent with the notion that MS VH4 bias is more than a consequence of chronic CNS inflammation. A VH4 bias was found in the chronic meningitis (IC05-2) CeSF, but used a set of VH4 H chains distinct from the typical MS donor (Table VII). VH4 bias in MS CeSF CD19⫹ cells was not as pronounced as in CD138⫹ cells. Only MS02-14 and MS02-24 CeSF showed significant VH4 bias of the 6 CD19⫹ B cell repertoires examined. Differences in VH family germline use in MS CeSF CD19⫹ cells from that observed in CD138⫹ cells probably reflects B cell dynamics and the composition of B cell subtypes, particularly the fraction of CD19⫹ CD138⫹ plasma blasts (13) comprising the more complex CD19⫹ cell population. The variable use of H chain family germline segments among different MS patients was also found in a separate single-cell study of CeSF CD19⫹ cells in which genomic DNA amplification was used and where only one of five CeSF B cell repertoires showed clear VH4 bias (15). Phenotypic characterization of B cells in MS CeSF and in CeSF of other chronic and acute human CNS inflammatory diseases identified 80 –90% of CeSF B cells as CD19⫹CD27⫹ memory cells (12, 13, 15). Of these cells, class-switched IgM⫺IgD⫺ memory cells were the most abundant, accounting for ⬃50% of CeSF B cells compared with 10% in peripheral blood (14). Thus, it is not surprising that MS CeSF CD138⫹ cells display markers indicative of recent memory cell activation and expansion. These cells express high levels of CD27 and CD38, high to intermediate levels of CD19, and retain MHC class II expression, a phenotype characteristic of plasma blasts (13). CD138⫹ CeSF cells are extensively mutated and display mutational patterns consistent with germinal center selection (15). Thus a scenario to explain the B cell dynamics in MS CeSF might include entry into the CNS of activated memory B cells, first generated after exposure to Ag in germinal centers of draining cervical lymph nodes (34 –36), followed by differentiation into CD138⫹ Ab-secreting cells after a second exposure to Ag. Whether activated memory cells from the periphery differentiate directly into
HUMORAL IMMUNITY IN MS CeSF Ab-secreting cells or instead undergo T cell-dependent germinal center-like reactions within the CNS before undergoing clonal expansion and differentiation remains unknown (12). Germinal center-like reactions may continuously renew and refine the CD138⫹ Ab-secreting cell population from a long-lived memory cell pool in MS CNS (37). Alternatively or coincident with CNS immune processes, memory B cells may also be constantly produced or activated in the periphery and serve to refresh or modify the existing CNS repertoire. In that context, it was interesting that the peripheral blood IgG⫹CD27⫹ memory cell pool of two MS patients, but not normal controls, displayed some evidence of VH4 bias, suggesting that factors shaping the CD138 repertoire in CeSF may also influence the peripheral IgG memory cell repertoire. The identification of a single memory cell in the MS05-1 blood repertoire using the exact same V(D)J rearrangement as a MS05-1 CeSF IgG clone further illustrates the potential nexus between peripheral blood memory cells and CeSF plasma blasts. More extensive serial repertoire analyses are needed to define further any ongoing relationships between peripheral and CeSF memory B cell pools. IgG repertoires in CeSF demonstrate features of a T cell-dependent Ag-driven humoral response (16 –22). Although expansion of memory B cell pools can occur in the absence of specific Ag through mechanisms such as bystander T cell or TLR activation (38), the bias for VH4 dominant plasma cells in MS CeSF is consistent with antigenic stimulation through preferential binding to VH4 BCRs and not polyclonal activation. The restricted accumulation of plasma cells expressing VH4 germline segments in MS CeSF is generally accompanied by a reduction in VH3 and, to a lesser extent, VH1 gene segments. However, expanded CD138⫹ clones expressing most VH families were observed to some degree in MS patients, arguing against nonconventional modes of activation operating through binding to VH4 framework regions on the BCR. Restricted VH family repertoires have been observed in response to both infectious agents and in putative autoimmune diseases. B cells reactive against rotavirus proteins 6 and 7 have been shown to preferentially use specific VH1 and VH4 germline segments (39). Herein, we found a bias for VH1 germline segments in a repertoire from SSPE CeSF that has been shown to contain anti-measles virus CD138⫹ clones (30). In systemic lupus erythematosus, splenic germinal center B lymphocytes from patients demonstrate a bias in VH5 gene family usage and an underrepresentation of VH1 family segments (40), whereas in synovial tissue of patients with rheumatoid arthritis, memory B cells with a high proportion of VH4 family segments are observed (41). The global VH4 predominance we found in MS CeSF suggests chronic B cell stimulation by a common mechanism or antigenic target. Possibly, there are dominant epitopes in MS that are preferentially recognized by the hypervariable loop structure of VH4 gene segments. Plasma blast populations in MS CeSF persist throughout disease (13), and the VH4 bias we observed was independent of disease duration and clinical course. Thus, once established, the driving force for B cell clonal expansion might also persist, indicating that antigenic drift may not play a prominent role in molding the humoral immune response over time. One future aim is to determine whether VH4 bias is present in all MS patients, indicates a more aggressive disease course or correlates with ongoing disease activity. Quantitation of immune cell types in MS CeSF shows the B cell to monocyte ratio to be one of the most variable CeSF parameters and that high B cell numbers correlate with a more aggressive disease course (8). Although we did not quantitate monocyte numbers in this study, it is likely that we were primarily studying patients with a high B cell to monocyte ratio because we do encounter patients in which recovery of B cells (particularly CD138⫹ plasma blasts) is poor. Such patients, of
The Journal of Immunology course, would not be included in our survey. There was some variation in CeSF plasma blast numbers (0.9 –3.5% of sorted cells) within our patient pool, but they did not correlate with the extent of VH4 bias. Our previous study of VH repertoires obtained from three different MS brain cDNA libraries identified a clear VH4 bias (and a predominance of VH4-39 germline use) in only one (16, 17). However, the inflammatory state of MS lesions used for cDNA library construction was not well-characterized, and the repertoires generated in that study reflected total IgG mRNA levels in brain plaques, with no distinction between B cell subtypes. We recently analyzed CeSF B cell repertoires in 10 individuals after a CIS; patients with VH4 and/or VH2 repertoire bias progressed rapidly to definite MS over a subsequent 6-mo period, whereas none of the CIS subjects without CeSF repertoire bias progressed to MS in 3 years (J. Bennett, unpublished observation). Thus, VH4 bias may prove to be a valuable biomarker for MS disease progression. Identifying the role of B cells and their antigenic targets remains an important goal in MS and may yield insights into the etiology and pathogenesis of disease.
Acknowledgments We thank Marina Hoffman for editorial assistance and Cathy Allen for assistance in manuscript preparation.
Disclosures The authors have no financial conflict of interest.
References 1. Correale, J., and M. de los Milagros Bassani Molinas. 2002. Oligoclonal bands and antibody responses in multiple sclerosis. J. Neurol. 249: 375–389. 2. Gilden, D. H., M. E. Devlin, M. P. Burgoon, and G. P. Owens. 1996. The search for virus in multiple sclerosis brain. Mult. Scler. 2: 179 –183. 3. Archelos, J. J., M. K. Storch, and H. P. Hartung. 2000. The role of B cells and autoantibodies in multiple sclerosis. Ann. Neurol. 47: 694 –706. 4. Prineas, J. W., and J. S. Grahm. 1981. Capping of surface immunoglobulin G on macrophages engaged in myelin breakdown. Ann. Neurol. 10: 149 –158. 5. Gay, F. W., T. J. Drye, G. W. A. Dick, and M. M. Esiri. 1997. The application of multifactorial clustering analysis in the staging of plaques in early multiple sclerosis: identification and characterization of the primary demyelinating lesion. Brain 120: 1461–1483. 6. Storch, M. K., S. Piddlesden, and M. Haltia. 1998. Multiple sclerosis: in situ evidence for antibody and complement mediated demyelination. Ann. Neurol. 43: 465– 471. 7. Luchinetti, C., W. Bruck, J. Parisis, B. Schiethauer, M. Rodriguez, and H. Lassmann. 2000. Heterogeneity of multiple sclerosis lesions: Implications for the pathogenesis of demyelination. Ann. Neurol. 47: 707–717. 8. Cepok, S., M. Jacobsen, S. Schock, B. Omer, S. Jaekal, I. Boddeke, W. H. Oertel, N. Sommer, and B. Hemmer. 2001. Patterns of cerebrospinal fluid correlate with disease progression in multiple sclerosis. Brain 124: 2169 –2176. 9. Villar, L. M., J. Masjuan, P. Gonzalez-Porque, J. Plaza, B. S. Sadaba, E. Roldan, A. Bootello, and J. C. Alvarez-Cermeno. 2002. Intrathecal IgM synthesis in neurologic diseases: relationship with disability in MS. Neurology 58: 824 – 826. 10. Villar, L. M., J. Masjuan, P. Gonzalez-Porque, J. Plaza, B. S. Sadaba, E. Roldan, A. Bootello, and J. C. Alvarez-Cermeno. 2003. Intrathecal IgM synthesis is a prognostic factor in multiple sclerosis. Ann. Neurol. 53: 222–226. 11. Owens, G. P., J. L. Bennett, D. H. Gilden, and M. P. Burgoon. 2006. The B cell response in multiple sclerosis. Neurol. Res. 28: 236 –244. 12. Corcione, A., S. Cassaza, E. Ferretti, D. Giunti, E. Zappia, A. Pistorio, C. Gambini, G. L. Mancardi, A. Uccelli, and V. Pistoia. 2004. Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis. Proc. Natl. Acad. Sci. USA 101: 11064 –11069. 13. Cepok, S., B. Rosche, V. Grummel, F. Vogel, D. Zhou, J. Sayn, N. Sommer, H. Hartung, and B. Hemmer. 2005. Short-lived plasma blasts are the main B cell effector subset during the course of multiple sclerosis. Brain 128: 1667–1676. 14. Cepok, S., G. von Geldern, V. Grummel, S. Hochgesand, H. Celik, H. Hartung, and B. Hemmer. 2006. Accumulation of class switched IgD⫺IgM⫺ memory B cells in the cerebrospinal fluid during neuroinflammation. J. Neuroimmunol. 180: 33–39. 15. Harp, C., J. Lee, D. Lambracht-Washington, E. Cameron, G. Olsen, E. Frohman, M. Racke, and N. Monson. 2007. Cerebrospinal fluid B cells from multiple sclerosis patients are subject to normal germinal center selection. J. Neuroimmunol. 183: 189 –199. 16. Owens, G. P., H. Kraus, M. P. Burgoon, T. Smith-Jensen, M. E. Devlin, and D. H. Gilden. 1998. Restricted use of VH4 germline segments in an acute multiple sclerosis brain. Ann. Neurol. 43: 236 –243. 17. Smith-Jensen, T., M. P. Burgoon, J. Anthony, H. Kraus, D. H. Gilden, and G. P. Owens. 2000. Comparison of the IgG heavy chain sequences in MS and SSPE brains reveals an antigen-driven response. Neurology 54: 1227–1232.
6351 18. Baranzini, S., M. C. Jeong, C. Butunoi, R. S. Murray, C. C. Bernard, and J. R. Oksenberg. 1999. B cell repertoire diversity and clonal expansion in multiple sclerosis brain lesions. J. Immunol. 163: 5133–5144. 19. Qin, Y., P. Duquette, Y. Zhang, P. Talbot, R. Poole, and J. Antel. 1998. Clonal expansion and somatic hypermutation of VH genes of B cells from cerebrospinal fluid in multiple sclerosis. J. Clin. Invest. 102: 1045–1050. 20. Colombo, M., M. Dono, P. Gazzola, S. Roncella, A. Valetto, N. Chiorazzi, G. Mancardi, and M. Ferrarini. 2000. Accumulation of clonally related B lymphocytes in the cerebrospinal fluid of multiple sclerosis patients. J. Immunol. 164: 2782–2789. 21. Owens, G. P., A. M. Ritchie, M. P. Burgoon, R. A. Williamson, J. R. Corboy, and D. H. Gilden. 2003. Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid. J. Immunol. 171: 2725–2733. 22. Ritchie, A. M., D. H. Gilden, R. A. Williamson, M. P. Burgoon, X. Yu, K. Helm, J. Corboy, and G. P. Owens. 2003. Comparative analysis of the CD19⫹ and CD138⫹ cell antibody repertoires in the cerebrospinal fluid of patients with multiple sclerosis. J. Immunol. 173: 649 – 656. 23. Monson, N. L., H. P. Brezinchek, R. Brezinchek, A. Mobley, G. K. Vaughan, E. M. Frohman, M. K. Racke, and P. E. Lipsky. 2005. Receptor revision and atypical mutational characteristics in clonally expanded B cells from the cerebrospinal fluid of recently diagnosed multiple sclerosis patients. J. Neuroimmunol. 158: 170 –181. 24. Haubold, K., G. P. Owens, P. Kaur, A. M. Ritchie, D. H. Gilden, and J. L. Bennett. 2004. B-lymphocyte and plasma cell clonal expansion in monosymptomatic optic neuritis cerebrospinal fluid. Ann. Neurol. 56: 97–107. 25. Qin, Y., P. Duquette, Y. Zhang, M. Olek, R.-R. Da, J. Richardson, J. P. Antel, P. Talbot, N. R. Cashman, W. W. Tourtellotte, et al. 2003. Intrathecal B-cell clonal expansion, an early sign of humoral immunity, in the cerebrospinal fluid of patients with clinically isolated syndrome suggestive of multiple sclerosis. Lab. Invest. 83: 1081–1088. 26. Brezinschek, H. P., R. I. Brezinschek, and P. E. Lipsky. 1995. Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J. Immunol. 155: 190 –202. 27. Brezinschek, H. P., S. J. Foster, R. I. Brezinschek, T. Dorner, R. Domiati-Saad, and P. E. Lipsky. 1998. Analysis of the human VH gene repertoire. J. Clin. Invest. 99: 2488 –2501. 28. Wardemann, H., S. Yurasov, A. Schaefer, J. W. Young, E. Meffre, and M. C. Nussenzweig. 2003. Predominant autoantibody production by early B cell precursors. Science 301: 1374 –1377. 29. Cook, G. P., and I. M. Tomlinson. 1995. The human immunoglobulin VH repertoire. Immunol. Today 16: 237–242. 30. Owens, G. P., A. M. Ritchie, D. H. Gilden, M. P. Burgoon, D. Becker, and J. L. Bennett. 2007. Measles virus-specific plasma cells are prominent in subacute sclerosing panencephalitis cerebrospinal fluid. Neurology 68: 815– 819. 31. Tiller, T., M. Tsuiji, S. Yurasov, K. Velinzon, M. C. Nussensweig, and H. Wardemann. 2007. Autoreactivity in human IgG⫹ memory B cells. Immunity 26: 205–213. 32. Kohsaka, H., D. A. Carson, L. Z. Rassenti, W. E. R. Ollier, P. P. Chen, and T. J. Kipps. 1996. The human immunoglobulin VH gene repertoire is genetically controlled and unaltered by chronic autoimmune stimulation. J. Clin. Invest. 98: 2794 –2800. 33. Huang, C., and D. Stollar. 1993. A majority of Ig H chain cDNA of normal human adult lymphocytes resembles cDNA for fetal Ig and natural autoantibodies. J. Immunol. 151: 5290 –5300. 34. Knopf, P. M., C. J. Harling-Berg, H. F. Cserr, D. Basu, E. J. Sirulnick, S. C. Nolan, J. T. Park, G. Keir, E. J. Thompson, and W. F. Hickey. 1998. Antigen-dependent intrathecal antibody synthesis in the normal rat brain: tissue entry and local retention of antigen-specific B cells. J. Immunol. 161: 692–701. 35. de Vos, A. F., M. van Meurs, H. P. Brok, L. A. Boven, R. Q. Hintzen, P. van der Valk, R. Ravid, S. Resning, L. Boon, B. A. ‘t Hart, and J. D. Laman. 2002. Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs. J. Immunol. 169: 5415–5423. 36. Karman, J., C. Ling, M. Sandor, and Z. Fabry. 2004. Initiation of immune responses in brain is promoted by local dendritic cells. J. Immunol. 173: 2353–2361. 37. Uccelli, A., F. Aloisi, and V. Pistoia. 2005. Unveiling the enigma of the CNS as a B-cell fostering environment. Trends Immunol. 26: 254 –259. 38. Bernasconi, N., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298: 2199 –2202. 39. Weitkamp, J. H., N. L. Kallewaard, K. Kusuhara, E. Bures, J. V. Williams, B. La Fleur, H. B. Greenberg, and J. E. Crowe Jr. 2003. Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires. J. Immunol. 171: 4680 – 4688. 40. Fraser, N. L., G. Rowley, M. Field, and D. I. Stott. 2003. The VH gene repertoire of splenic B cells and somatic hypermutation in systemic lupus erythematosus. Arthritis Res. Ther. 5: R114 –R121. 41. Voswinkel, J., L. Trumper, G. Carbon, T. Hopf, M. Pfreundschuh, and A. Gause. 1996. Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA). Clin. Exp. Immunol. 106: 5–12.
