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*
Servizio di Immunologia Clinica and
Servizio di Citometria Centro Biotechnologie Avanzate, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy;
Dipartimento di Oncologia, Biologia e Genetica, Università degli Studi di Genova, Genova, Italy;
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Divisione ORL, Istituto Giannina Gaslini, Genova, Italy; and
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Department of Medicine, North Shore University Hospital and New York University School of Medicine, Manhasset, NY, 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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transcripts of activated SE
B cells. Therefore, SE B cells are heterogeneous, being comprised of B
cells with mutated Ig VH4 genes, that are Ag-experienced B
cells, and a subset of B cells with unmutated VH4 genes
that are either virgin cells or cells driven by Ags that did not induce
or select for V gene mutations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MZ B cells of experimental animals are heterogeneous and are comprised of virgin and memory B cells and of cells that are capable of both T cell-dependent and T cell-independent responses (8, 9, 12, 13, 14, 15). As a result of obvious experimental constraints, studies of these issues are difficult in humans, although some information has been provided by analyses of VH gene expression (16, 17). During a T cell-dependent response, B cells expand in germinal centers (GC) where they accumulate point mutations in their VH and VL genes and are selected for increased affinity for the stimulating Ag (16, 17, 18, 19, 20). The presence of somatic mutations in VH or VL genes is, therefore, taken as evidence that a cell has been previously stimulated and has become a memory B cell. The absence of VH or VL somatic mutation can imply either that a cell is a virgin B cell or a B cell that was stimulated by an Ag that did not elicit T cell help. The presence of clonal amplification would help to distinguish these two possibilities. B cells stimulated and expanded by TI-2 Ags likely fall into this category. Studies on single MZ B cells have demonstrated that some of these cells express mutated and others unmutated V genes, thus indicating that they are heterogeneous (16, 17, 21).
Cells with the morphology and immune phenotype of splenic MZ B cells have been described in the extrafollicular areas of other lymphoid tissues in humans, including the dome region of Peyer’s patches, the subcapsular sinus of lymph nodes, the thymic medulla, and the subepithelial (SE) region of tonsils (22, 23, 24, 25). These are often referred to as MZ or MZ-like B cells. The similarities of these cells and those from the splenic MZ are reinforced by the observation that tonsillar SE B cells respond efficiently to TI-2 Ags in vitro (26).
In previous studies, we defined conditions to isolate highly purified suspensions of tonsillar SE B cells that were used for further functional studies in vitro (25, 26). In the present study, we have analyzed the expressed VH genes of subsets of tonsillar SE B cells as a means to elucidate their activation histories and maturation stages. The results obtained support the notion that SE B cells are heterogeneous since they are comprised of both unmutated and mutated B cells that can be distinguished based upon surface phenotype. An unexpected finding of these studies was the observation that a subset of SE B cells that express mutated VH4 genes bear low levels of surface IgD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tonsils were obtained from 5- to 12-year-old children undergoing
routine tonsillectomies. B cells were purified and further fractionated
by discontinuous Percoll (Pharmacia Biotech, Uppsala, Sweden) density
gradients as detailed previously (25). Briefly, purified B
cells were separated into fractions of different density by
centrifugation through 30, 40, 50, 60, and 100% Percoll fractions (see
Fig. 1
). The high-density B cells collected on the surface of 100%
Percoll were fractionated into CD5+ cells
(follicular mantle B cells (FM)) and CD5- cells
(SE B cells) by immune rosetting (25, 27). GC B cells were
purified from low-density B cells (30 and 40% Percoll fractions) by
depletion of cells expressing CD39 and IgD using immune rosetting. To
obtain the cells operationally called activated B cells, the medium
size B cells from the 50% Percoll fractions were depleted of cells
expressing CD5, CD38, and IgD by magnetic cell separation. Briefly, the
B cells were incubated with the corresponding mAbs for 30 min at 4°C,
followed by goat anti-mouse Ig microbeads (Miltenyi Biotec,
Bergisch Gladbach, Germany) for 10 min at 4°C. CD5-IgD-CD38-negative
B cells were recovered by negative selection by collecting cells not
retained on the magnetic column using the MiniMACS system (Miltenyi
Biotec).
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-chain Ab (Dako, Glostrup,
Denmark) and sorting on a FACS (Epics-Elite; Coulter, Hialeah, FL). In
selected experiments, the IgDlow SE B cells were
stained with CD27 mAb and a PE-conjugated secondary reagent and sorted
into CD27low and CD27high
cell subsets. Morphology of the cells was analyzed on cytospin preparations stained with Giemsa and analyzed by light microscopy (25).
Flow cytometry
Cells were stained with the following Ab: anti-human
µ-chain, anti-human -chain, anti-CD23, anti-CD10,
anti-CD38 from Becton Dickinson (San Jose, CA); anti-CD27 from
Jansenn Biochimica (Beerse, Belgium); anti-CD24 from Ortho
Diagnostics (Raritan, NJ); anti-CD80 and anti-CD95 from
Immunotech (Marseille, France); and FITC-conjugated goat anti-human
-chain from Southern Biotechnology Associates (Birmingham, AL).
Anti-CD38, IB4 (28) and anti-CD39 (29)
mAb were kindly provided by Dr. F. Malavasi (University of Torino,
Torino, Italy) and Dr. G. Aversa (DNAX, Palo Alto, CA), respectively.
Indirect immunofluorescence was performed using FITC- or PE-conjugated
goat anti-mouse Ig isotypes as secondary reagents
(30). Samples were analyzed by flow cytometry using a
FACSort (Becton Dickinson).
PCR
RNA was isolated from 0.5 to 5.0 x
106 cells using RNA-Clean method (TIB Molbiol,
Genoa, Italy) according to the manufacturer’s instructions. For
first-strand cDNA synthesis, the total RNA yield was reverse
transcribed using oligo(dT) as primer and murine myeloblastosis virus
reverse transcriptase (Life Technologies, Paisley, Scotland) in a
40-µl final volume (31). First-strand cDNA (1–5 µl)
was amplified using a sense VH4 family-specific
primer (5'-atgaaacacctgtggttcttcctcc) with the appropriate antisense
CH constant region (µ:
5'-ttagacgagggggaaaagggtt; : 5'-gtaggacagc(ct)gggaaggtgtgcac). PCR
was conducted for 35 cycles using the following conditions:
denaturation for 1 min at 94°C, annealing for 1 min at 57°C,
and extension for 2 min at 72°C. All amplifications were performed in
a Mastercycler 330 (Eppendorf Scientific, Hamburg, Germany). The
calculated internal Taq polymerase error rate (corresponding
to the frequency of nucleotide exchange introduced by Taq)
in our system is 0.15%.
Genomic DNA was purified from resting IgM-only SE B cells by cell lysis and digestion with proteinase K, "salting out" extraction and precipitation with ethanol, and used as a template for the amplification of the VH4DJH genes as described previously (32).
DNA cloning and sequencing
The PCR products were purified with Advantage PCR Pure kit (Clontech Laboratories, Palo Alto, CA), ligated into TOPO TA vector, and then transformed into TOP10F' competent bacteria (Invitrogen, Carlsbad, CA). Multiple colonies were picked randomly and sequenced in both directions automatically using the Ready Reaction DyeDeoxy-Terminator or BigDye cycle sequencing kit and a sequenator (both from Perkin-Elmer Applied Biosystem, Milan, Italy). Nucleotide sequences were analyzed using the MacVector (Oxford Molecular Group, Oxford, U.K.) software and compared with those in the V Base Gold database (I. Tomlinson, Medical Research Council Centre for Protein Engineering, Cambridge, U.K.). D genes were assigned to the appropriate family based upon the criteria of Klein et al. (33). Reading frames of D segment were classified as yielding either a hydrophilic, a hydrophobic, or a stop codon segment (34).
All of the sequences were deposited in the European Molecular Biology Laboratory (EMBL) under the accession numbers AJ244928–AJ245073 and AJ279513–AJ279553.
HCDR3 analysis
CDR3 length was determined by counting the number of amino acids between codon 94 at the end of FR3 and codon 102 at the beginning of FR4. The estimated isoelectric point value defined the acidic and basic aa composition of the CDR3 and was determined using the MacVector software (version 6.01).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
summarizes the purification
methods, phenotype, and typical yield of the various subset populations
studied. In evaluating the latter data, it should be stressed that the
more abundant cell subsets were represented by the large
IgD+CD39+ cells (range,
25–30%), the medium size CD5+ cells (range,
15–20%), and the medium size
IgD+CD38+CD5-
cells (range, 15–20%). The major phenotypic features of each B cell
subset have been described in detail previously (see Ref.
25) and are summarized in Fig. 1. Notably, resting SE B
cells and resting FM B cells failed to express surface activation
markers such as CD80 and CD95. Resting SE B cells were
CD10-CD38+/-CD23-CD39-,
and CD24+/- while resting FM B cells expressed
CD23 and CD39 but not CD38 or CD10. GC B cells expressed CD38, CD10,
and CD77. The B cells recovered from the 50% Percoll fraction and
depleted of IgD+CD5+ and
CD38+ cells expressed surface activation markers
(CD80 and CD95, Fig. 1). Phenotypically, these B cells were identical
to the memory B cells of the tonsillar SE area described previously
(25, 35) and will be referred to as activated SE B
cells.
FM B cells expressed both surface IgM and IgD, whereas GC and activated
SE B cells displayed mostly surface IgG (Fig. 2). Resting SE B cells were comprised
mostly of IgM+ B cells and of a smaller fraction
of cells that also stained weakly for IgD (Fig. 2). In a typical
experiment, the IgDlow cells represented 20–30%
of the total resting SE B cells. Resting IgM-only and resting
IgDlow SE B cells were purified by sorting and
were restained for a number of surface markers (Fig. 3). Resting IgM-only SE B cells expressed
lower levels of surface IgM than the resting
IgDlow SE B cells. Consistent with their origin,
both SE B cell subsets had low levels of CD23 and CD38 and a bimodal
distribution of CD44 (see also Ref. 25). CD24 was slightly
more abundant on the resting IgDlow SE B cell
fraction. Morphologically, both resting IgM-only and
IgDlow SE B cells were comprised of relatively
large lymphocytes, with indented nuclei and a well-represented
cytoplasm (Fig. 3 and Ref. 25). Collectively, these
features were those of SE (monocytoid) B cells previously reported by
several groups including our own (25, 36).
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The cDNA sequences of 32 molecular clones containing
VH4DJH gene segments from
the IgM-only SE B cells of three different tonsils were determined and
compared with the corresponding germline genes (GL) (Table I). A number of identical sequences were
found among different molecular clones (2-A2 and 2-A10, 2-A3 and 2-A4,
2-A7 and 2-A9; 3-A2 and 3-A7, 3-A3 and 3-A4 and 3-A12), suggesting an
expansion of certain B cell clones in vivo. The
VH4-34 gene was predominantly used by the B cells
from all of the tonsils (12/26 of the clones analyzed, 46%) and
particularly from tonsil 2 (7/8 of the clones, 87.5%). Overuse of this
particular gene by tonsillar B cells has been noted (37).
The sequences that deviated by >2% from the GL were considered as
"mutated," while those with <2% mutations were defined
"unmutated." Accordingly, 25 of 26 of the individual sequences of
the IgM-only SE B cells analyzed (96.2%) were unmutated. The one
mutated clone (3-A9) differed from the most similar germline gene by
3% (Table I).
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Resting IgDlow SE B cells use mutated and unmutated VH4 genes
Twenty-nine VH4 cDNA clones were generated
from the resting IgDlow SE B cells (Table II). VH4-34 was the
most frequently encountered VH gene (12/29
clones; 41.4%) followed by VH4-31 (6/29 clones;
20.7%). A significant level of somatic mutation was observed in this B
cell subset. Thirteen of 29 sequences (44.8%) displayed >2%
difference from the most similar germline counterpart. In a total of 28
functional rearranged genes (1 of the 29 sequences had a stop codon,
clone 2-B22, see Table II), there were 173 point mutations resulting in
an average mutation frequency of 2.09% (3.6% average degree of
mutation among the mutated sequences).
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Tables III and
IV summarize the analyses on 45
VH4 sequences from µ (n = 25)
and (n = 21) cDNA, respectively. As for the other
SE B cell fractions, VH4-34 was the most
frequently used VH4 gene (10/25 clones, 40%),
followed by VH4-61 and
VH4-39 (both 24%). There were repeated sequences
in the µ transcripts (clones 1-C10 and 1-C11). The majority of
VH genes were unmutated (14/25 clones, 56%). The
overall average mutation frequency was 1.7% (4.8% average degree of
mutation among mutated sequences).
In the transcripts, 17 of 21 clones (81%) analyzed were mutated.
The calculated mutation frequency was 5.7%, with a range of 2–14%.
One sequence (clone 2-E127) showed a 3-bp insertion in CDR1, a
phenomenon that is considered characteristic of cells that have gone
through a germinal center (38, 39).
VH4 gene usage by FM and GC B cells
FM B cells are generally assumed to be virgin B cells, whereas GC B cells are located in the site where mutation of VH genes takes place (19, 40, 41). In these experiments, VH4 genes utilized by FM and GC B cells were analyzed and compared with the VH4 genes used by resting SE B cells (data not shown; sequences deposited in EMBL under accession numbers AJ245008–245052). Among the 23 sequences generated from FM B cells, 2 were identical. Of the 21 individual cDNA clones obtained from the FM B cells, 5 displayed between 1 and 2 bp substitutions and only 1 had three- point differences. The 12 total changes observed gave rise to an average mutation frequency of 0.19%. Usage of individual VH4 genes was random.
Twenty-four of the genes obtained from GC B cells were functional, whereas one had a stop codon in the CDR2 generated by somatic mutation. One sequence was found twice. VH4-34 accounted for 66.7% of the sequences analyzed in GC B cells (16/24 clones). The 23 individual sequences had accumulated a total of 193 substitutions with a mutation frequency of 2.7% (range, 1–6.5%). Two clonally related sequences that shared the same CDR3 and the same VH gene exhibited a number of different VH point mutations, indicating that they represented intraclonal variants of a common ancestral B cell (data not shown).
HCDR3 composition
CDR3 length was not significantly different within the various B
cell subsets (range, 11.8–13 aa; Table V). D segments were identified in 63%
(92/146) of the sequences generated from the different B cell subsets
and their distribution reflected the estimated D gene composition in
genomic DNA (42). D2, D3, and D4 gene segments were the
most frequently used (17.4, 30.4, and 18.5%, respectively).
Interestingly, only 1 sequence of the 92 analyzed (1.08%) contained a
rearranged D7 segment (DQ52) that is typical of fetal B cells
(43). This sequence was observed in the FM B cells. D
segments were used mostly in their hydrophilic reading frame (56/92
sequences; 60.8%), with some differences in the various B cell subsets
analyzed (see Table V). Although unmutated sequences utilize
preferentially the hydrophilic reading frame (34), in the
resting IgM-only SE B cells there was a balance between the hydrophilic
reading frame (RF) and the hydrophobic RF. Such balance was also
observed in the GC B cells (Table V).
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The long stretches of tyrosines encoded at the 3' end by JH6 were mostly conserved in the resting IgM-only SE B cells, in GC B cells, and especially in the activated SE IgM+ B cells that had a minimum of three Y residues per JH6 sequence. In contrast, the tyrosines in the JH6 sequences were significantly altered in the resting IgDlow SE B cells, where only one of six sequences retained these residues. The FM and the activated IgG+ B cells were excluded from this analysis due to the low presence of JH6 gene segments (3/21 clones and 2/21 clones, respectively).
CD27 expression
In these experiments, we determined whether the CD27, usually
found on somatically mutated B cells (45, 46), was
differentially expressed by the various B cell subsets. Analyses of
CD27 expression revealed that virtually all FM B cells were CD27
negative, whereas CD27 was found on both GC B cells and activated SE B
cells. Resting IgM-only SE B cells displayed a small shift in the
fluorescence profile when stained with anti-CD27 mAb, while the
flow cytometry profile of resting IgDlow SE B
cells indicated the presence of two subsets characterized by different
expression of this markers (Fig. 4).
Resting IgDlow SE B cells were stained for CD27
and sorted as depicted in Fig. 4. Two subsets of
CD27low- and
CD27high-IgDlow SE B cells,
respectively, could be isolated. They were subsequently analyzed for
the level of somatic mutation in their rearranged
VH4 genes. Eleven molecular clones were obtained
from the µ transcripts of the
CD27low-IgDlow SE B cells.
All of them were in germline configuration (average, 99.75% of
homology with the germline genes). In contrast, 11 of the 14 molecular
clones (78.5%) obtained from the
CD27high-IgDlow SE B cells
showed somatic mutations (5–20 bp differences from the most homologous
germline counterparts), and only 3 sequences (21.5%) were virtually
unmutated (range, 99–100% homology) (data not shown; sequences in
EMBL under accession numbers AJ279513-AJ279537).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All but 1 (96.2%) of 26 molecular clones obtained from resting
IgM-only SE B cells expressed unmutated VH4
genes. This could not be accounted for by contamination of FM B cells
based on surface marker analyses (
Figs. 1–3) and morphological,
cytochemical, and functional studies (25, 26). The
presence of unmutated B cells among tonsillar SE B cells apparently
went unnoticed in previous molecular studies aimed at characterizing
the memory cell compartment of SE B cells (40). In the
present study, the detection of a substantial number of unmutated
VH4 sequences was possibly facilitated by the
separation of SE B cells into resting and activated cells and the
subsequent purification of the IgM-only B cells. Previous studies on
the B cells from the dome region of Peyer’s patches (another source of
MZ-like B cells) indicated that up to 25% of these cells exhibited
unmutated VH4 genes, a result that is more in
line with our data and the most recent findings on the splenic MZ B
cells and on the B cells from subcapsular sinuses of lymph nodes
(16, 17, 21).
The finding of unmutated SE B cells suggests the following scenario regarding the antigenic stimulation and subsequent fate of cells. When virgin unmutated SE B cells first reach the tonsils, they seed beneath the epithelial linings where they are constantly exposed to incoming pathogens. Here, antigenic stimulation of two types can occur. The first involves foreign proteins which can elicit the help of T cells and other accessory cells to execute a T cell-dependent immune reaction that can result in somatic mutation. The second is initiated by bacterial polysaccharides (TI-2 Ags) that promote the proliferation and plasma cell differentiation of SE B cells in the absence of T cell help. The two types of response are not mutually exclusive, although it is not known whether the same resting SE B cells can be involved in the two different pathways.
The observations that numerous molecular clones (11/32) from the
resting IgM-only SE B cells had identical unmutated sequences provides
strong evidence for local amplification of this subset. Moreover, a
similar finding was noticed when analyzing the DNA (rather than cDNA
from RNA) prepared from resting IgM-only SE B cells. Since this
approach circumvents the artifact that can be introduced by plasma
cells, that have markedly enhanced IgV gene mRNA, these data
unequivocally confirmed the in situ proliferation and accumulation of B
cells with identical unmutated
VHDJH sequences. In
addition, the finding of a number of molecular clones with unmutated
VH4 sequences in the µ transcripts from
activated SE B cells (see Tables III and IV) also suggests local
activation and expansion of these cells. Finally, the presence of
plasma cells in the SE area supports the notion of local plasma cell
differentiation (25). Based on these findings, it is very
unlikely that the clonally expanded unmutated B cell clones are virgin
cells. It is more likely that they represent B cells that have been
activated, expanded, and matured by Ags that did not or could not
induce and select for V gene mutations. TI-2 Ags are the most probable
candidates in the case of SE (MZ-like) B cells. The finding that
resting IgM-only SE B cells express some (albeit low) levels of CD27
(see Fig. 4) is in line with the notion of previous antigenic
stimulation.
Stimulation of SE B cells by T cell-dependent Ags may result in their
migration to GCs where the B cells could accumulate point mutations and
undergo isotype switching. A minority of mutated SE B cells could also
derive from TI-2 Ag stimulation as suggested by certain studies
(47, 48). Mutated SE B cells may move again from the GCs
to the SE area or migrate to the peripheral blood or to the splenic MZ
or to other equivalent sites. Indeed, a minority of IgM-only cells
sharing some of the phenotypic features of SE B cells have been
detected in the circulation (41, 49, 50). Interestingly,
VH and VL gene analyses
have determined that these cells are almost invariably mutated,
suggesting that primarily memory cells tend to recirculate. The
mutation frequency of VH genes found in the
peripheral blood IgM-only B cells is comparable to that determined here
for the µ transcripts from tonsillar activated SE B cells. The degree
of antigenic selection to which circulating IgM-only B cells were
subjected was calculated by determining the overall ratio between
replacement (R) and silent (S) mutations in the framework region (FR)
region. Since the FRs are important for the integrity of the Ab
combining site, R mutations should be counterselected during antigenic
stimulation in GCs, resulting in a low R:S ratio in memory cells
(51, 52, 53, 54). Based on these criteria, the observed overall FR
R:S ratio in circulating IgM-only B cells was 1.5 (55). We
found an almost identical value by applying the same type of approach
to the µ transcripts from activated SE B cells (Table VI). The notion that SE B cells generate
memory cells with point mutations in their VH
genes through a passage within the GC is not in conflict with the
hypothesis that also FM B cells give origin to memory B cells with the
same features. Unfortunately, it is impossible at present to determine
the reciprocal contribution to the mutated memory cell pool by these
two subsets, since clonally related sequences were not found across the
different subsets. This is similar to the observations of other groups
(20, 40, 41).
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and
Ref. 25). Furthermore, these cells had much higher
VH4 mutation frequencies than the FM and the
resting IgM-only SE B cells (Table VI). Analyses of CD27 expression by
resting IgDlow SE B cells revealed that they
could be subdivided into two distinct subsets
(CD27low- and CD27high-
IgDlow SE B cells, see Fig. 4) that differed in
mutation frequencies in their VH4 genes. Indeed,
these mutations were found mainly in the
CD27high- IgDlow
SE B cells. This raises a number of unresolved questions on the mutual
relationship of these two subsets of IgDlow SE B
cells. Two groups independently reported the existence of
IgM+ IgD+ memory B cells in
the human bone marrow and peripheral blood, respectively (55, 57). These cells had an overall percentage of V gene mutations
comparable to that of the resting
CD27high-IgDlow SE B cells
described here and, at least in the case of peripheral blood B cells, a
comparable phenotype (57). As in the case of our mutated
tonsillar IgDlow SE B cells, peripheral blood
IgM+ IgD+ memory B cells
were found to express CD27 (45). The origin of resting IgDlow SE B cells is not known. One possibility is that they derive from a rare type of tonsil GC subset that contains IgM+ IgD+ CD38+ B cells (58, 59). The virgin cells that seed and expand in these GC have not, however, been delineated. Moreover, the mode of recirculation of these cells, and hence the relationship between resting IgDlow tonsillar SE B cells and those from other sites, is not known. Whatever their origin and relationship with other B cell subsets may be, it is clear that IgM+ IgD+ B cells collectively represent a substantial part of the memory B cell pool (45).
The present study delineates a somewhat surprising heterogeneity of tonsillar SE B cells that are comprised of subsets differentiated by surface markers, states of activation, isotype expression, and functional properties. The relationships of these subsets among themselves and with the other B cells from the peripheral blood and lymphoid organs are not completely clear, although tonsillar SE B cells may represent a suitable source of MZ-equivalent B cells for further functional studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mariella Dono, Servizio di Immunologia Clinica, Istituto Nazionale per la Ricerca sul Cancro, IST, Largo go Rosanna Benzi, no. 10, 16132 Genova GE, Italy.
3 Abbreviations used in this paper: MZ, marginal zone; SE, subepithelial; FM, follicular mantle; GC, germinal center; RF, reading frame; FR, framework region; R, replacement; S, silent.
Received for publication June 3, 1999. Accepted for publication March 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1
6)dextran. Proc. Natl. Acad. Sci. USA 91:2502.
genes with no or little somatic mutation throughout life. Eur. J. Immunol. 23:3272.[Medline]
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C.-G. Liu, G. A. Calin, B. Meloon, N. Gamliel, C. Sevignani, M. Ferracin, C. D. Dumitru, M. Shimizu, S. Zupo, M. Dono, et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues PNAS, June 29, 2004; 101(26): 9740 - 9744. [Abstract] [Full Text] [PDF]
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B. T. Messmer, E. Albesiano, D. Messmer, and N. Chiorazzi The pattern and distribution of immunoglobulin VH gene mutations in chronic lymphocytic leukemia B cells are consistent with the canonical somatic hypermutation process Blood, May 1, 2004; 103(9): 3490 - 3495. [Abstract] [Full Text] [PDF]
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A. M. Collins, M. Ikutani, D. Puiu, G. A. Buck, A. Nadkarni, and B. Gaeta Partitioning of Rearranged Ig Genes by Mutation Analysis Demonstrates D-D Fusion and V Gene Replacement in the Expressed Human Repertoire J. Immunol., January 1, 2004; 172(1): 340 - 348. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, A. E. Jackson, S. Tolani, R. J. Fattah, and J. K. Inman Role of Complement-Binding CD21/CD19/CD81 in Enhancing Human B Cell Protection from Fas-Mediated Apoptosis J. Immunol., November 15, 2003; 171(10): 5244 - 5254. [Abstract] [Full Text] [PDF]
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B. Falini, E. Tiacci, A. Pucciarini, B. Bigerna, J. Kurth, G. Hatzivassiliou, S. Droetto, B. V. Galletti, M. Gambacorta, A. Orazi, et al. Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT) Blood, November 15, 2003; 102(10): 3684 - 3692. [Abstract] [Full Text] [PDF]
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U. Klein, Y. Tu, G. A. Stolovitzky, J. L. Keller, J. Haddad Jr., V. Miljkovic, G. Cattoretti, A. Califano, and R. Dalla-Favera Transcriptional analysis of the B cell germinal center reaction PNAS, March 4, 2003; 100(5): 2639 - 2644. [Abstract] [Full Text] [PDF]
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G. A. Calin, C. D. Dumitru, M. Shimizu, R. Bichi, S. Zupo, E. Noch, H. Aldler, S. Rattan, M. Keating, K. Rai, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia PNAS, November 26, 2002; 99(24): 15524 - 15529. [Abstract] [Full Text] [PDF]
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 U. Klein, Y. Tu, G. A. Stolovitzky, M. Mattioli, G. Cattoretti, H. Husson, A. Freedman, G. Inghirami, L. Cro, L. Baldini, et al. Gene Expression Profiling of B Cell Chronic Lymphocytic Leukemia Reveals a Homogeneous Phenotype Related to Memory B Cells J. Exp. Med., December 3, 2001; 194(11): 1625 - 1638. [Abstract] [Full Text] [PDF]
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M. B. Bjorgan, J. E. Thoen, J. B. Natvig, and K. M. Thompson Bm1-Bm5 Classification of Peripheral Blood B Cells Reveals Circulating Germinal Center Founder Cells in Healthy Individuals and Disturbance in the B Cell Subpopulations in Patients with Primary Sjogren's Syndrome J. Immunol., October 1, 2001; 167(7): 3610 - 3618. [Abstract] [Full Text] [PDF]
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