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n2:pmcid
PMC0
bibo:doi
10.1084%2Fjem.20101499
n6:contains
_:vb9789189 _:vb9789166 _:vb9789172
Subject Item
_:vb9789166
rdf:type
n6:Section
dc:title
materials and methods
n6:contains
_:vb9789168 _:vb9789169 _:vb9789170 _:vb9789171 _:vb9789167
Subject Item
_:vb9789167
rdf:type
n2:Context
rdf:value
Ig secretion was determined by ELISPOT assay as previously described (Tumang et al., >>2005<<), using MultiScreen-IP plates (Millipore) coated with goat anti–human IgM (SouthernBiotech), and blocked with 5% bovine serum albumin (Sigma-Aldrich).
n2:mentions
n3:15749846
Subject Item
_:vb9789168
rdf:type
n2:Context
rdf:value
Ig secretion was determined by ELISA assay, as previously described (Hastings et al., >>2006<<). In brief, sort-purified B cells were cultured for 5 d at 1 × 106 per ml in RPMI medium (supplemented as above).
n2:mentions
n3:16609926
Subject Item
_:vb9789169
rdf:type
n2:Context
rdf:value
Mononuclear cells were analyzed after phosphatase inhibition, as previously described (Holodick et al., 2009b). In brief, cells were treated with sodium pervanadate for varying periods of time, after which they were fixed with paraformaldehyde, permeabilized with methanol, and stained for surface antigens and intracellular phosphorylated proteins
n2:mentions
n3:19592097
Subject Item
_:vb9789170
rdf:type
n2:Context
rdf:value
Individual cells were sort-purified onto a 48-well Ampligrid (Beckman Coulter) and Ig sequences were PCR-amplified in a semi-nested approach as previously described (Holodick et al., 2009a), using primers designed for human Ig gene transcripts (Wang and Stollar, 2000).
n2:mentions
n3:19714574
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_:vb9789171
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onto a 48-well Ampligrid (Beckman Coulter) and Ig sequences were PCR-amplified in a semi-nested approach as previously described (Holodick et al., 2009a), using primers designed for human Ig gene transcripts (Wang and Stollar, >>2000<<). Products were sequenced (GENEWIZ), and sequences were analyzed using the International ImMunoGeneTics Information System.
n2:mentions
n3:11033034
Subject Item
_:vb9789172
rdf:type
n6:Section
dc:title
results
n6:contains
_:vb9789188 _:vb9789184 _:vb9789185 _:vb9789186 _:vb9789187 _:vb9789173 _:vb9789174 _:vb9789175 _:vb9789180 _:vb9789181 _:vb9789182 _:vb9789183 _:vb9789176 _:vb9789177 _:vb9789178 _:vb9789179
Subject Item
_:vb9789173
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In the murine system, B1 cell progenitors are more abundant in fetal hematopoietic tissues than in adult hematopoietic tissues, and B1 cells emerge before the bulk of B2 cell production occurs (Montecino-Rodriguez et al., >>2006<<). Following this paradigm, we first examined umbilical cord blood specimens for spontaneous IgM secretion by ELISPOT assay, which we used for initial screening, and then looked for efficient T cell stimulation and tonic intracellular
n2:mentions
n3:16429139
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_:vb9789174
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These CD20+CD27+ cord blood B cells uniquely express CD43, a well-described marker for murine B1 cells (Wells et al., >>1994<<), whereas CD20+CD27− cord blood B cells are CD43− (Fig.
n2:mentions
n3:7989752
Subject Item
_:vb9789175
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In the human system, CD27 expression is generally considered to mark memory B cells (Agematsu et al., >>2000<<), which suggests incongruity in the identification of CD27+ B cells in cord blood that is obtained at birth before exogenous antigen exposure.
n2:mentions
n3:10782048
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_:vb9789176
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In contrast to the large number of somatic mutations present in adult CD27+ B cell–derived antibodies (Klein et al., >>1998<<), we found that all cord blood B cells express antibodies with very low levels of somatic mutation, both CD20+CD27+CD43+ and CD20+CD27−CD43− B cells (Table I, Fig.
n2:mentions
n3:9802980
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_:vb9789177
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contained N region additions at the V–D and D–J junctions, much like CD27−CD43− B cell Ig sequences (Table I) reflecting the presence of terminal deoxynucleotidyl transferase throughout ontogeny in the human system (Asma et al., >>1986<<). Among cord blood B cells, CD27 expression does not correlate with increased Ig mutational status and does not correspond to memory B cell status, and thus is presumably indicative of another B cell population.
n2:mentions
n3:3742878
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_:vb9789178
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The production of particular antimicrobial and autoantibody specificities is a well-described characteristic of murine B1 cells (Hayakawa et al., >>1984<<) and it has been reported that autoreactive Ig is preferentially produced by human CD5+ B cells (Casali and Notkins, 1989), although it has also been reported that human CD5− B cells produce autoantibodies (Mackenzie et al., 1991; Kasaian
n2:mentions
n3:6609363
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_:vb9789179
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and autoantibody specificities is a well-described characteristic of murine B1 cells (Hayakawa et al., 1984) and it has been reported that autoreactive Ig is preferentially produced by human CD5+ B cells (Casali and Notkins, >>1989<<), although it has also been reported that human CD5− B cells produce autoantibodies (Mackenzie et al., 1991; Kasaian et al., 1992).
n2:mentions
n3:2469441
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_:vb9789180
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rdf:value
al., 1984) and it has been reported that autoreactive Ig is preferentially produced by human CD5+ B cells (Casali and Notkins, 1989), although it has also been reported that human CD5− B cells produce autoantibodies (Mackenzie et al., >>1991<<; Kasaian et al., 1992).
n2:mentions
n3:1707183
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_:vb9789181
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been reported that autoreactive Ig is preferentially produced by human CD5+ B cells (Casali and Notkins, 1989), although it has also been reported that human CD5− B cells produce autoantibodies (Mackenzie et al., 1991; Kasaian et al., >>1992<<). Because the repertoire of human B1 cells might well differ from murine B1 cells in terms of pathogenic microbes and self-antigens in view of the tens of millions of years that have passed since murine and human evolution diverged, we
n2:mentions
n3:1374094
Subject Item
_:vb9789182
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To address this issue, we exposed human adult peripheral blood mononuclear cells to phosphorylcholine (PC; Lalor and Morahan, >>1990<<) and to aspartate-tryptophan-glutamate-tyrosine-serine (DWEYS) tetramer DNA mimetope (Shirai et al., 1991; Jacobi et al., 2009) and determined the level of binding to CD20+CD27+CD43+ (B1), CD20+CD27+CD43− (memory), and CD20+CD27−CD43−
n2:mentions
n3:1690657
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_:vb9789183
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To address this issue, we exposed human adult peripheral blood mononuclear cells to phosphorylcholine (PC; Lalor and Morahan, 1990) and to aspartate-tryptophan-glutamate-tyrosine-serine (DWEYS) tetramer DNA mimetope (Shirai et al., >>1991<<; Jacobi et al., 2009) and determined the level of binding to CD20+CD27+CD43+ (B1), CD20+CD27+CD43− (memory), and CD20+CD27−CD43− (naive) B cells, as described in Materials and methods.
n2:mentions
n3:1706969
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_:vb9789184
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we exposed human adult peripheral blood mononuclear cells to phosphorylcholine (PC; Lalor and Morahan, 1990) and to aspartate-tryptophan-glutamate-tyrosine-serine (DWEYS) tetramer DNA mimetope (Shirai et al., 1991; Jacobi et al., >>2009<<) and determined the level of binding to CD20+CD27+CD43+ (B1), CD20+CD27+CD43− (memory), and CD20+CD27−CD43− (naive) B cells, as described in Materials and methods.
n2:mentions
n3:19488401
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_:vb9789185
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Although in the murine system CD43 marks B1 cells and only appears on mature B2 cells during terminal differentiation (Gulley et al., >>1988<<), we considered the possibility that CD43 might be inducible in the human system, much like CD5 (Freedman et al., 1989; Cong et al., 1991).
n2:mentions
n3:2897394
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_:vb9789186
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system CD43 marks B1 cells and only appears on mature B2 cells during terminal differentiation (Gulley et al., 1988), we considered the possibility that CD43 might be inducible in the human system, much like CD5 (Freedman et al., >>1989<<; Cong et al., 1991). In such an instance, CD20+CD27+CD43+ B cell populations could contain an unknown number of activated B cells in addition to B1 cells.
n2:mentions
n3:2462935
Subject Item
_:vb9789187
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B1 cells and only appears on mature B2 cells during terminal differentiation (Gulley et al., 1988), we considered the possibility that CD43 might be inducible in the human system, much like CD5 (Freedman et al., 1989; Cong et al., >>1991<<). In such an instance, CD20+CD27+CD43+ B cell populations could contain an unknown number of activated B cells in addition to B1 cells.
n2:mentions
n3:1716972
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_:vb9789188
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In identifying human B1 cells we focused on CD20+ B cells to avoid the few circulating terminally differentiating (CD20−) plasma cells that have been reported in peripheral blood samples and that can secrete Ig (Radbruch et al., >>2006<<). Umbilical cord blood lacks CD19+CD20− B cells (Fig. 9 A), and so it is clear that no version of human B1 cells exists among putative CD20− B cells in cord blood because CD20− B cells do not exist in cord blood.
n2:mentions
n3:16977339
Subject Item
_:vb9789189
rdf:type
n6:Section
dc:title
discussion
n6:contains
_:vb9789200 _:vb9789201 _:vb9789202 _:vb9789203 _:vb9789204 _:vb9789205 _:vb9789206 _:vb9789207 _:vb9789208 _:vb9789209 _:vb9789210 _:vb9789211 _:vb9789212 _:vb9789213 _:vb9789190 _:vb9789191 _:vb9789192 _:vb9789193 _:vb9789194 _:vb9789195 _:vb9789196 _:vb9789197 _:vb9789198 _:vb9789199
Subject Item
_:vb9789190
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to differentiated follicular B cells that might secrete IgM, such as plasma cells or plasmablasts, for the following reasons: human B1 cells expressed CD20 (which is lost during differentiation at the plasmablast stage; Jego et al., >>2001<<); human B1 cells did not express CD138 (which is acquired by differentiated plasma cells; Klein and Dalla-Favera, 2007); and neither CD20 loss nor CD138 acquisition occurred during culture of human B1 cells with IL-6 for 5 d (unpublished
n2:mentions
n3:11238125
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_:vb9789191
rdf:type
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reasons: human B1 cells expressed CD20 (which is lost during differentiation at the plasmablast stage; Jego et al., 2001); human B1 cells did not express CD138 (which is acquired by differentiated plasma cells; Klein and Dalla-Favera, >>2007<<); and neither CD20 loss nor CD138 acquisition occurred during culture of human B1 cells with IL-6 for 5 d (unpublished data).
n2:mentions
n3:17521580
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_:vb9789192
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For this reason, in addition to the many other differences between CD20− plasma cells and CD20+ B cells (Horst et al., >>2002<<; Hogerkorp and Borrebaeck, 2006; Radbruch et al., 2006), there is no evidence to suggest that any significant number of human B1 cells reside within the CD20− B cell population.
n2:mentions
n3:12452825
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_:vb9789193
rdf:type
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rdf:value
For this reason, in addition to the many other differences between CD20− plasma cells and CD20+ B cells (Horst et al., 2002; Hogerkorp and Borrebaeck, >>2006<<; Radbruch et al., 2006), there is no evidence to suggest that any significant number of human B1 cells reside within the CD20− B cell population.
n2:mentions
n3:16982868
Subject Item
_:vb9789194
rdf:type
n2:Context
rdf:value
For this reason, in addition to the many other differences between CD20− plasma cells and CD20+ B cells (Horst et al., 2002; Hogerkorp and Borrebaeck, 2006; Radbruch et al., >>2006<<), there is no evidence to suggest that any significant number of human B1 cells reside within the CD20− B cell population.
n2:mentions
n3:16977339
Subject Item
_:vb9789195
rdf:type
n2:Context
rdf:value
Although CD43 is associated with B cell differentiation, and not activation, in the murine system (Gulley et al., >>1988<<), in the human system this surface antigen is up-regulated in previously CD43− B cells by some, but not all, activating stimuli, and so must be considered an “activation antigen.
n2:mentions
n3:2897394
Subject Item
_:vb9789196
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” In this sense, CD43 behaves much like CD5, which is also induced by activating stimuli (Freedman et al., >>1989<<; Cong et al., 1991; but which, however, is still used effectively to delineate murine B1 cells).
n2:mentions
n3:2462935
Subject Item
_:vb9789197
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” In this sense, CD43 behaves much like CD5, which is also induced by activating stimuli (Freedman et al., 1989; Cong et al., >>1991<<; but which, however, is still used effectively to delineate murine B1 cells).
n2:mentions
n3:1716972
Subject Item
_:vb9789198
rdf:type
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type, albeit not activated B cells, could be intermixed with this population, particularly in view of ELISPOT results on murine B1 cells, which detect almost an order of magnitude greater proportion of secreting cells (Tumang et al., >>2005<<; Holodick et al., 2010).
n2:mentions
n3:15749846
Subject Item
_:vb9789199
rdf:type
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B cells, could be intermixed with this population, particularly in view of ELISPOT results on murine B1 cells, which detect almost an order of magnitude greater proportion of secreting cells (Tumang et al., 2005; Holodick et al., >>2010<<). It seems more likely, however, that the ELISPOT assay is not detecting all secreting B1 cells, inasmuch as detection depends on several variables, including the affinities of capture and detecting antibodies, the rate of Ig secretion,
n2:mentions
n3:21061433
Subject Item
_:vb9789200
rdf:type
n2:Context
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First, whereas peripheral (cord) blood at birth would seem to be necessarily devoid of memory B cells, as has been previously reported (Maurer et al., >>1990<<; Agematsu et al., 1997), our results indicate that a population of CD27+ B1 cells is, in fact, present, as has been suggested (Shi et al., 2005), furthering the notion that CD27 is not an immutable indicator of memory B cell status, as
n2:mentions
n3:1702722
Subject Item
_:vb9789201
rdf:type
n2:Context
rdf:value
First, whereas peripheral (cord) blood at birth would seem to be necessarily devoid of memory B cells, as has been previously reported (Maurer et al., 1990; Agematsu et al., >>1997<<), our results indicate that a population of CD27+ B1 cells is, in fact, present, as has been suggested (Shi et al., 2005), furthering the notion that CD27 is not an immutable indicator of memory B cell status, as also suggested by its
n2:mentions
n3:9295047
Subject Item
_:vb9789202
rdf:type
n2:Context
rdf:value
be necessarily devoid of memory B cells, as has been previously reported (Maurer et al., 1990; Agematsu et al., 1997), our results indicate that a population of CD27+ B1 cells is, in fact, present, as has been suggested (Shi et al., >>2005<<), furthering the notion that CD27 is not an immutable indicator of memory B cell status, as also suggested by its presence on developing B cells (Nilsson et al., 2005).
n2:mentions
n3:16116217
Subject Item
_:vb9789203
rdf:type
n2:Context
rdf:value
B1 cells is, in fact, present, as has been suggested (Shi et al., 2005), furthering the notion that CD27 is not an immutable indicator of memory B cell status, as also suggested by its presence on developing B cells (Nilsson et al., >>2005<<). Second, whereas adult peripheral blood memory B cells have been reported to efficiently stimulate T cells (Good et al., 2009), our results indicate that this function is actually contributed by B1 cells contained within the CD27+
n2:mentions
n3:16338493
Subject Item
_:vb9789204
rdf:type
n2:Context
rdf:value
Second, whereas adult peripheral blood memory B cells have been reported to efficiently stimulate T cells (Good et al., >>2009<<), our results indicate that this function is actually contributed by B1 cells contained within the CD27+ population and not by memory B cells, per se.
n2:mentions
n3:19124732
Subject Item
_:vb9789205
rdf:type
n2:Context
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Notably, it has been reported that IgM memory B cells are responsible for controlling infections produced by Streptococcus pneumoniae and other encapsulated organisms (Kruetzmann et al., >>2003<<); however, the population of IgM memory B cells studied in previous work presumably included B1 cells because prior investigations used CD27 to identify memory B cells.
n2:mentions
n3:12682112
Subject Item
_:vb9789206
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rdf:value
The report that anti-Streptococcus pneumoniae IgM memory B cells are generated in the spleen (Kruetzmann et al., >>2003<<) is reminiscent of previous reports on mouse B1 cells indicating they also require the spleen for development (Wardemann et al., 2002), and again raises the possibility that at least some B cells previously described as IgM memory B cells
n2:mentions
n3:12682112
Subject Item
_:vb9789207
rdf:type
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anti-Streptococcus pneumoniae IgM memory B cells are generated in the spleen (Kruetzmann et al., 2003) is reminiscent of previous reports on mouse B1 cells indicating they also require the spleen for development (Wardemann et al., >>2002<<), and again raises the possibility that at least some B cells previously described as IgM memory B cells may actually be human B1 cells.
n2:mentions
n3:11901202
Subject Item
_:vb9789208
rdf:type
n2:Context
rdf:value
Regardless of the relationship between human B1 cells and IgM memory B cells, the number of CD27+ B cells declines with advancing age (Shi et al., >>2005<<), and our results indicate that the fraction of CD43+ B1 cells within the CD20+CD27+ population declines even more precipitously, particularly in the very old age group.
n2:mentions
n3:16116217
Subject Item
_:vb9789209
rdf:type
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For example, both are CD20+CD27+CD43+CD70−; most normal B1 cells express CD5, as do malignant CLL cells; and, both express relatively nonmutated Ig (Damle et al., >>1999<<; Hamblin et al., 1999; Jung et al., 2003).
n2:mentions
n3:10477712
Subject Item
_:vb9789210
rdf:type
n2:Context
rdf:value
For example, both are CD20+CD27+CD43+CD70−; most normal B1 cells express CD5, as do malignant CLL cells; and, both express relatively nonmutated Ig (Damle et al., 1999; Hamblin et al., >>1999<<; Jung et al., 2003).
n2:mentions
n3:10477713
Subject Item
_:vb9789211
rdf:type
n2:Context
rdf:value
For example, both are CD20+CD27+CD43+CD70−; most normal B1 cells express CD5, as do malignant CLL cells; and, both express relatively nonmutated Ig (Damle et al., 1999; Hamblin et al., 1999; Jung et al., >>2003<<). In addition, we have found that normal human B1 cells ZAP-70 and ILT3 (unpublished data) like CLL cells (Best et al., 2006; Colovai et al., 2007). And, in respect to pathophysiology, the chronically activated phenotype of normal B1
n2:mentions
n3:12580968
Subject Item
_:vb9789212
rdf:type
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In addition, we have found that normal human B1 cells ZAP-70 and ILT3 (unpublished data) like CLL cells (Best et al., >>2006<<; Colovai et al., 2007).
n2:mentions
n3:16906573
Subject Item
_:vb9789213
rdf:type
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In addition, we have found that normal human B1 cells ZAP-70 and ILT3 (unpublished data) like CLL cells (Best et al., 2006; Colovai et al., >>2007<<). And, in respect to pathophysiology, the chronically activated phenotype of normal B1 cells may predispose to malignant transformation. Thus, in regard to CLL, in some respects we may be back to the future that was postulated 30 yr ago;
n2:mentions
n3:17266150
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29
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21
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17
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16
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15
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15
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15
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14
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14
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13
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13
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13
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13
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13
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13
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12
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12
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12
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11
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11
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11
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11
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10
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10
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10
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10
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10
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10
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9
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9
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9
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9
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9
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9
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9
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9
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9
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9
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9
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8
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8
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