The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kato, J.
Right arrow Articles by Watanabe, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kato, J.
Right arrow Articles by Watanabe, T.
The Journal of Immunology, 1998, 160: 4788-4795.
Copyright © 1998 by The American Association of Immunologists

Affinity Maturation in Lyn Kinase-Deficient Mice with Defective Germinal Center Formation

Jun Kato, Noboru Motoyama, Ichiro Taniuchi, Hiromichi Takeshita, Masaki Toyoda, Keiji Masuda and Takeshi Watanabe1

Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Higashi-Ku, Fukuoka, Japan 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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lyn kinase-deficient (lyn-/-) mice show several abnormalities such as reduced numbers of circulating B cells, hyper-IgM, and low proliferative responses induced by CD40 ligand. Lyn-/- mice also develop splenomegaly, produce autoreactive Abs with age, and finally develop glomerulonephritis. Another abnormality observed in lyn-/- mice is that their disability to form germinal centers (GCs). It has been considered that GCs play an important role in affinity maturation and differentiation to B cell memory upon immunization with thymus-dependent Ag. Since Lyn kinase has been thought to be downstream of the signals from the B cell Ag receptor as well as CD40, we studied whether or not lyn-/- mice could exhibit normal Ag-specific class switching and affinity maturation following somatic hypermutation. The mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken {gamma}-globulin (NP-CG). Production of NP-specific IgG1 Abs was slightly reduced but clearly detectable. The affinity of Abs produced was comparable to that in wild-type mice. Furthermore, somatic hypermutation occurred in the heavy-chain variable region at the same level as that in wild-type mice. Therefore, we conclude that isotype switching and affinity maturation occur normally in lyn-/- mice without the formation of GCs. The results lead to a speculation that Lyn may not play a role in induction of isotype switching or affinity maturation, despite being downstream of the signals from the B cell Ag receptor complex and CD40, and that GC architecture may not be absolutely essential for affinity maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The B cell Ag receptor (BCR)2 consists of surface IgM (sIgM) noncovalently associated with heterodimers of Ig{alpha} (CD79a) and Igß (CD79b) subunits, which couple sIgM to protein tyrosine kinases (PTKs) (1). The Srk family kinases, one class of PTKs, are activated by cross-linking of the BCR (2, 3, 4), resulting in phosphorylation of several proteins including themselves, Ig{alpha}, and Igß (5), phospholipase C{gamma} (PLC{gamma}) (6), phosphatidylinositol-3 (PI-3) kinase (7), the proto-oncoprotein Vav (8), Ras GTPase-activating protein (9), Shc (10), HS1 (11), and Syk kinase (12).

One of the Src family kinase members, Lyn, is expressed preferentially in hemopoietic cells (13), especially in B cells, in monocytes/macrophages, and in neural tissue cells (14). Recent studies have shown that Lyn is physically associated with a number of hemopoietic cell surface receptors such as the BCR (2, 3), CD40 (15), LPS receptor (CD14) (16), high affinity Fc{epsilon}RI complex (17), IL-2R (IL-2R) (18), and the granulocyte-CSF receptor (19). Lyn kinase is activated shortly after the cross-linking of BCR (2, 3). The function of Lyn in BCR-mediated signaling was first demonstrated in a Lyn-deficient chicken B cell line, DT40 (20), in which cross-linking of the BCR on Lyn-deficient DT40 cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of inositol-triphosphate turnover. Further, in murine and human B-lineage lymphoma cells, Lyn was shown to be required for anti-sIgM-mediated cell cycle arrest (21). Recent studies have also suggested a function of Lyn in the signaling pathway through CD40 (15, 22), a member of the TNFR superfamily, which plays an important role in B cell survival, proliferation, memory, isotype switching, and apoptosis (23, 24). Lyn was shown to be tyrosine phosphorylated directly by CD40 cross-linking, resulting in activation of PLC{gamma}2 and PI-3 kinase in human Daudi cells (15).

Lyn-deficient (lyn-/-) mice, recently generated by gene targeting, showed splenomegaly with age and accumulated splenic lymphoblastoid cells expressing Mac1 Ag on the surface and IgM in the cytoplasm (25, 26). Despite reduced numbers of circulating B cells, lyn-/- mice had high concentrations of serum IgM, and many of them exhibited severe glomerulonephritis caused by immune complex-containing autoreactive Abs. Furthermore, B cells of lyn-/- mice showed several abnormalities in vitro (25, 26, 27). Splenic B cells from young lyn-/- mice were hyperresponsive to sIgM cross-linking (27), whereas the cells from aged lyn-/- mice were hyporesponsive (25, 26). The proliferative response to CD40 ligand (CD40L) was also greatly reduced in the B cells of lyn-/- mice, although addition of IL-4 restored the response (26, 27). In addition, splenic B cells of lyn-/- mice were impaired in the induction of Fas expression after CD40L stimulation, resulting in a reduced susceptibility to Fas-mediated apoptosis (27).

One of the phenotypes observed in lyn-/- mice was a defect in the ability to form germinal centers (GCs) (25). GCs are dynamic microenvironments of B cell differentiation that arise transiently in the B cell follicles of secondary lymphoid organs during thymus-dependent (TD) Ag responses (28). It is believed that GCs play an important role in affinity maturation following somatic hypermutation and generation of memory B cells (29, 30, 31, 32). The GCs have also been reported to play a role in isotype switching in human tonsils (33). However, there is a study demonstrated that GCs are not necessary for isotype switching in the mouse (34), and this was confirmed by the work of Toellner et al. showing the appearance of sterile switching transcripts in Ag-activated B cells of the outer periarteriolar lymphatic sheath (35). The help of activated T cells is required for these responses (32) through the interaction between CD40 and CD40L, as well as B7-1/B7-2 and CD28/CTLA-4 (36), and appears to operate in a dose-dependent fashion (37).

Recently, many mutant mice generated by gene targeting have been found to lack GC. These include MHC class II-deficient (38), human CTLA-4 Ig transgenic (39), CD28-deficient (40, 41), B7-1/B7-2-double deficient (42), CD40-deficient (43), CD40L-deficient (44, 45), TNFR I-deficient (46), TNF-{alpha}-deficient (47), OCA-B deficient (48, 49), and LT-{alpha}-deficient mice (50, 51, 52). Ag-specific IgG1 titers after TD Ag immunization were decreased or absent in many of the mutant mice, such as in MHC class II-deficient, human CTLA-4 Ig transgenic, CD28-deficient, B7-1/B7-2-deficient, CD40-deficient and CD40L-deficient mice (38, 39, 40, 41, 42, 43, 44, 45), which are considered to have a defect in T cell-B cell (T-B) interaction. Furthermore, in human CTLA-4 Ig transgenic mice and CD28-deficient mice, not only isotype switching but also affinity maturation following somatic hypermutation were impaired (39, 40, 41). In other mice described above, affinity maturation was not examined, although isotype switching was considered to be impaired. In contrast, LT-{alpha}-deficient mice, which had no lymph nodes (LNs) or Peyer’s patches and failed to form GCs in the spleen, exhibited normal Ag-specific IgG1 responses and affinity maturation after TD Ag immunization (52).

The present study was designed to investigate whether TD Ag-specific IgG1 responses and affinity maturation could occur in lyn-/- mice, which are similar to LT-{alpha}-deficient mice in having a defect in GC formation but which differ in several B cell abnormalities. The lyn-/- mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken {gamma}-globulin (NP-CG). The present study shows that NP-specific IgG1 Abs were slightly reduced but easily detectable and that the affinity of Abs produced was comparable to that in wild-type (WT) mice. Since somatic hypermutation also occurred in the heavy-chain variable region at the same level as in WT mice, we conclude that isotype switching and affinity maturation occur normally in lyn-/- mice despite the defect in ability to form GCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Lyn-deficient (lyn-/-) mice (26) of a C57BL/6 background and normal C57BL/6 mice (Clea Japan, Tokoy, Japan) were maintained in our animal facility on a 12-h light/dark cycle.

Immunization of mice and FACS analysis

Mice (4–7 wk old) were immunized i.p. with 100 µg NP-CG absorbed to alum and boosted twice with the same Ag dose at day 21 and 28. NP-CG was a generous gift of Drs. T. Kaisho and K. Rajewsky (Institute for Genetics, University of Cologne, Cologne, Germany). Spleens and LNs were collected 7 days after the final boost (day 35), and cell suspensions derived from both organs were stained with biotin-labeled peanut agglutinin (PNA; Sigma Chemical, St. Louis, MO) and phycoerythrin (PE)-labeled anti-B220 Ab (PharMingen, San Diego, CA). Biotin-labeled PNA was visualized with streptavidin-Red670 (Life Technologies, Gaithersburg, MD). Cells were analyzed using a FACScan (Becton Dickinson, Mountain View, CA) with CELLQuest software.

Measurement of NP-specific Ab

Mice (4–7 wk old) were immunized i.p. with 100 µg NP-CG absorbed to alum and boosted with the same dose of Ag in PBS at day 21. Sera were collected 10 days after primary immunization (day 10) and 7 days after the second immunization (day 28). NP-specific Ab titers were determined by ELISA using microtiter plates coated with NP4-BSA and NP14-BSA (gifts of Drs. T. Kaisho and K. Rajewsky, University of Cologne). NP4-BSA- or NP14-BSA-coated plates were blocked with 1% BSA in PBS, and serially diluted serum samples were added to individual wells. Bound Abs were revealed by a goat anti-mouse IgG1 conjugated with horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL). NP-specific IgG1 titers were expressed in relative units compared with the hyperimmune mouse serum. (Anti-NP Ab titer of the hyperimmune serum was defined as 100 U.) Both high and low affinity Abs bound to the plate coated with NP14-BSA, whereas only high affinity Abs bound to the plate coated with NP4-BSA. Therefore, the ratio of NP4/NP14-binding Ab titers was used as an index for affinity maturation (52, 53).

RT-PCR for amplification of the VH186.2 gene and sequencing

Mice (4–7 wk old) were immunized i.p. with 100 µg NP-CG absorbed to alum and boosted twice, using the same Ag dose, at day 21 and day 28. Total RNA was purified from spleens 7 days after the final boost (day 35) using Isogen (Nippongene, Toyama, Japan). cDNA, synthesized from 3 µg of total RNA using oligo(dT)-primed reverse transcription, was used as a template in PCR. The upstream PCR primer was 5'-CATGCTCTTCTTGGCAGCAACAGC-3'(for VH 186.2), and the downstream primer was 5'-GTGCACACCGCTGGACAGG GATCC- 3' (for C{gamma}1) (52). PCR was performed with Pfu DNA polymerase (Strategene, La Jolla, CA) for 30 cycles of 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C. The PCR product was cloned into pCR2.1 (Invitrogen, San Diego, CA) and transformed into DH-5{alpha} bacteria. Resultant colonies were randomly picked, and the VH186.2 gene sequences were obtained by direct plasmid sequencing using an ABI Prism Sequencing Kit (Perkin-Elmer, Foster City, CA).

Histology and immunostaining

Mice (6 wk old) were immunized i.p. with 100 µg of NP-CG absorbed to alum. Fourteen days after immunization, spleens were fixed in formalin for 24 h, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. For immunostaining of the tissue sections, mice were immunized i.p. with 100 µg of NP-CG absorbed to alum and boosted twice, using the same Ag dose, at day 21 and day 28. Seven days after the final boost (at day 35), spleens were isolated for frozen sections. The sections were fixed in ethanol containing 5% acetic acid at -20°C for 20 min, blocked in 0.1 M Tris, 0.1 M NaCl, 0.1% Tween 20, 0.1% BSA, and 30% normal rat serum with 200 µg/ml 2.4G2 (a gift of Dr. T. Takemori), and incubated with FITC-labeled anti-B220 Ab (PharMingen) and PE-labeled [(4-hydroxy-5-iodonitrophenyl)acetyl]21-BSA (a kind gift from Dr. T. Takemori, National Institute of Infectious Diseases, Tokyo, Japan) for 2 h at room temperature.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Defective GC formation in lyn-/- mice

To confirm the previous report that GCs formed very poorly in lyn-/- mice (25), we immunized mice (6 wk old) with NP-CG, a well-characterized TD immunogen (52, 53, 54, 55). In WT mice, secondary follicles with GC structure were observed in the white pulp areas after immunization (Fig. 1Go, A and C). In contrast, neither lymphoid follicles nor GCs could be detected in lyn-/- mice (Fig. 1Go, B and D). In addition, similar results were obtained when mice were immunized with SRBC (data not shown). Therefore, we concluded that lyn-/- mice were defective in GC formation. Since affinity maturation following somatic hypermutation has been considered to occur within GCs (29, 30, 31, 32), we then investigated whether this phenomenon could occur in lyn-/- mice.



View larger version (137K):
[in this window]
[in a new window]
  		FIGURE 1. GC formation was not observed in lyn-/- mice. Mice (6 wk old) were immunized with 100 µg of NP-CG absorbed to alum. Fourteen days after immunization, spleens were stained with hematoxylin and eosin. A, WT spleen at x100 magnification; B, lyn-/- spleen at x100 magnification; C, WT spleen at x400 magnification; D, lyn-/- spleen at x400 magnification. GC, secondary follicle with germinal center; WP, white pulp; LZ, light zone; DZ, dark zone.

 
PNAhigh B cells were not induced even after multiple immunizations of lyn-/- mice with NP-CG

Before investigating affinity maturation in lyn-/- mice, we examined whether PNA-positive B cells were induced after NP immunization, because PNA binds strongly to B cells in GCs (29). In WT mice, PNAhigh B cells in spleen were clearly induced after multiple immunizations. Before immunization, the PNAhigh B cells in total spleen cells was 0.9% (Fig. 2GoA). After multiple immunizations with NP-CG, the number of PNAhigh B cells increased up to 4.3% of total spleen cells (Fig. 2GoC). In contrast, even after multiple immunizations with NP-CG, PNAhigh B cells were not significantly increased in lyn-/- mice, being 0.2% before immunization and 0.4% after immunization (Fig. 2Go, B and D). As described previously, there were 40 to 60% fewer B220+ cells in the spleen in lyn-/- mice than in WT mice (25, 26). The percentage of PNAhigh cells among B220+ B cells was 0.8% in lyn-/- mice before immunization, which was comparable to 1.7% in WT mice (Fig. 2AGo, B). However, while the PNAhigh B220+ spleen cells increased in WT mice up to 5.6% after NP immunizations, there was no increase in lyn-/- mice (1.6%) (Fig. 2Go, C and D). Similarly, the mean numbers of PNAhigh splenic B cells from three WT mice increased after immunization, whereas the numbers of B cells from three lyn-/- mice were not increased even after immunization (Table IGo). Similar results were obtained with LN cells (Fig. 3Go). The PNAhigh cells among LN B cells also increased from 1.9 to 4.1% in WT mice, whereas there was no increase in lyn-/- mice (0.9 and 0.7% before and after NP immunization, respectively). The mean cell numbers of PNAhigh cells in LN cells from three mice were also similar to those in spleen cells (Table IGo). Furthermore, PNAhigh B cells were not induced 7 days after the single immunization with SRBC in lyn-/- mice, although they were strongly induced in WT mice (data not shown). These results indicated that PNAhigh B cells are not induced in lyn-/- mice even after multiple immunizations with NP-CG. The findings are consistent with the results shown in Figure 1Go and with the previous study demonstrating the defect in ability to form GCs in lyn-/- mice (25).



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 2. Immunizations with NP-CG induced PNAhigh B cells in spleen of WT mice, but not of lyn-/- mice. Cells were stained with PE-labeled anti-B220 and biotin-labeled PNA and streptavidin-Red670. A, WT spleen cells before immunization; B, lyn-/- spleen cells before immunization; C, WT cells after third immunization; D, lyn-/- cells after third immunization; shown in contour plots representative of three experiments. Percentages of PNAhigh B220+ cells relative to total spleen cells are shown on each contour plot. Numbers in parentheses indicate percentages of PNAhigh cells among total B220+ B cells.

 

View this table:
[in this window]
[in a new window]
  		Table I. PNAhigh cells in spleen and LN before and after immunization with NP-CG

 


View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 3. PNAhigh B cells were not induced in LN of lyn-/- mice even after multiple immunizations with NP-CG. Cells were stained with PE-labeled anti-B220 Ab and biotin-labeled PNA and streptavidin-Red670. A, WT LN cells before immunization; B, lyn-/- LN cells before immunization; C, WT cells after three immunizations; and D, lyn-/- cells after three immunizations; shown in contour plots representative of three experiments. Percentages of PNAhigh B220+ cells relative to total LN cells are shown on each contour plot. Numbers in parentheses indicate percentage of PNAhigh cells among total B220+ B cells.

 
No impairment of NP-specific IgG1 responses and affinity maturation in lyn-/- mice

Since lyn-/- mice have a defect in the ability to form GCs, we investigated NP-specific IgG1 responses and affinity maturation, which is considered to occur within GCs (29, 30, 31, 32). Both WT and lyn-/- mice showed similar serum IgG1 titers before (25, 26) and after immunization (25), although lyn-/- mice showed a much higher level of serum IgM than WT mice (25, 26). The titers of NP-specific IgG1 Abs were low at day 10 in both WT and lyn-/- mice (Fig. 4Go, A and B), and by day 28, titers of NP14-binding IgG1 Abs were elevated in WT mice (21.6 ± 22.5 U) (Fig. 4GoA). In lyn-/- mice, NP14-binding IgG1 Abs (15.7 ± 15.4 U) were slightly reduced but readily detectable, indicating that isotype switching occurred despite the defect in ability to form GCs. The appearance of NP4-binding Abs indicates affinity maturation. In fact, by day 28, NP4-binding IgG1 Abs were detectable in WT mice (24.2 ± 20.3 U) (Fig. 4GoB). In lyn-/- mice, NP4-binding Abs (14.4 ± 18.7 U) were also detectable. Furthermore, the ratio of NP4-binding/NP14-binding Abs was increased by day 28 in both WT and lyn-/- mice (0.84 ± 0.42 and 0.68 ± 0.32 in WT and lyn-/- mice, respectively) (Fig. 4GoC). In addition, immunostaining using NIP21-BSA showed that NP-specific B cells did exist in the spleens of both WT and lyn-/- mice after NP immunizations. As shown in Figure 5Go, comparable numbers of NP-binding B cells were observed in the spleens of lyn-/- mice and in WT mice, although GC formation was missing in the lyn-/- mice. We therefore concluded that Ag-specific IgG1 responses and affinity maturation occur in lyn-/- mice. The appearance of high affinity Abs in lyn-/- mice after boosting demonstrated that generation of memory B cells was not affected in the absence of Lyn kinase. Thus, affinity maturation, isotype switching, and generation of memory B cells occur in lyn-/- mice even without GC formation.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. NP-specific IgG1 Ab responses in lyn-/- mice. Filled circles show the titers of WT mice, and open circles show those of lyn-/- mice. A, Titers of NP-specific IgG1 Abs with low and high affinity using NP14-BSA-coated plates. B, Titers of NP-specific IgG1 Abs with high affinity using NP4-BSA-coated plates. C, The ratio of NP4/NP14-binding Abs calculated with the values from A and B. Horizontal bars in each graph indicate the average of the values from nine mice. There were no statistical differences between WT and lyn-/- mice on all the graphs (unpaired Student’s t test, >0.5).

 


View larger version (110K):
[in this window]
[in a new window]
  		FIGURE 5. Immunostaining of NP-specific B cells in spleen of lyn-/- mice after NP-CG immunizations. Mice were i.p. immunized with 100 µg NP-CG absorbed to alum, then boosted twice using the same Ag dose. Seven days after the final boost, spleens were collected for frozen sections. The sections were stained with FITC-labeled anti-B220 Ab (green) and NIP21-BSA-PE (red). A, WT spleen; B, lyn-/- spleen. Original magnification at x200.

 
The frequency of somatic hypermutation of the VH186.2 gene in lyn-/- mice was comparable to that in WT mice

It has been previously reported that the combination of the heavy chain variable region gene VH186.2 and the {lambda}1 light chain predominates during the NP-specific response, and somatic hypermutation is induced in the VH186.2 gene in the NP-specific secondary response (31, 34, 52, 54, 55). Since the mutated VH186.2 gene is highly associated with acquisition of high affinity to NP, we investigated whether or not VH186.2 IgG1 transcripts expressed in spleen cells from lyn-/- mice mutated after multiple immunizations with NP-CG. In WT mice, mutation did not lead to stop codons and showed a preference for transitions rather than transversions and a tendency of mutations at AGC/T sequences (Fig. 6Go and Table IIGo). These results are consistent with those previously described (56, 57). In lyn-/- mice, mutations in the VH186.2 gene showed a similar pattern, although these mutations did not necessarily prefer transitions to transversions (Table IIGo). Change of Trp to Leu at codon 33 in the first complementarity-determining region (CDR1) of the VH186.2 gene results in the acquisition of high affinity for NP (52, 55). This type of mutation was observed in 14 of 15 independent clones derived from WT mice and in 13 of 15 independent clones derived from lyn-/- mice (Fig. 6Go). Mutation frequency in lyn-/- mice was comparable to that in WT mice, 5.7 ± 2.3 per clone in WT mice and 5.6 ± 1.8 per clone in lyn-/- mice, respectively (Table IIIGo). If somatic mutation occurred randomly, the expected R/S (ratio of replacement (R) to silent (S) mutations) in the CDRs (CDR1 and CDR2) would be 4.9, and that ratio in the framework regions would be 2.6, as previously described (54). Elevated ratios of R/S in the CDRs, but not the framework regions, suggested Ag-driven somatic hypermutation. The R/S ratio in the framework region was almost equal to what was expected (3.1 in WT mice and 3.2 in lyn-/- mice) (Table IIIGo). In contrast, the ratio in the CDRs was much higher than expected in both WT and lyn-/- mice (8.0 in WT mice and 11.7 in lyn-/- mice). We concluded, therefore, that Ag-induced somatic hypermutation occurs in lyn-/- mice and that lyn-/- mice are able to undergo affinity maturation and memory B cell generation.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Somatic hypermutation occurred in both WT and lyn-/- mice. Selected codons of the germline VH186.2 gene are shown on the top line with the amino acid translation shown as a single-letter code. CDR1 and CDR2 domains are overlined. Each of 15 expressed sequences from WT (A) and lyn-/- (B) mice is shown. These sequences were obtained from three mice of both WT and lyn-/-. Dashes indicate nucleotides that are identical with the germline sequence.

 

View this table:
[in this window]
[in a new window]
  		Table II. Distribution of base substitutions in VH 186.2 sequences from WT and lyn-/-mice

 

View this table:
[in this window]
[in a new window]
  		Table III. Pattern of somatic mutations in VH186.2 sequences from WT and lyn-/-mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, Ag-specific IgG1 responses and affinity maturation following somatic hypermutation in lyn-/- mice were investigated. When lyn-/- mice were immunized with NP-CG, NP-specific IgG1 Abs were produced, and the affinity of IgG1 Abs matured with NP secondary responses to the same level as in WT mice. Mutations in the VH186.2 gene of lyn-/- mice were found at a frequency similar to that in WT mice. In addition, a mutation from Trp to Leu at codon 33 in CDR1 of the VH186.2 gene and an elevated R/S ratio in CDRs but not in framework regions suggested that Ag-induced selection also occurred in both WT mice and lyn-/- mice. The results indicate that isotype switching and affinity maturation occur normally in lyn-/- mice depite the defect in formation of GCs.

Several possibilities might be considered to explain the defect in GC formation in lyn-/- mice. 1) Lyn-/- mice may be disrupted in T-B interaction. 2) Lyn-/- mice may have some abnormalities in cells supporting GC formation such as interdigitating dendritic cells (28) or follicular dendritic cells (FDCs) (58, 59). 3) Lyn-/- mice may have difficulty in forming B cell follicles because of reduced numbers of circulating B cells and insufficient expansion in response to CD40 signal. 4) Lyn kinase may play a role in the homing capacity of B cells to secondary lymphoid organs. In contrast to mutant mice with a disruption in T-B interaction, such as CD40-deficient or B7-1/B7-2-deficient mice (42, 43), which fail to form GCs, lyn-/- mice responded normally to TD Ags, indicating that T-B interaction may not be impaired by the Lyn defect. Th cells and interdigitating dendritic cells play an important role in the activation of B cells in T cell-rich zones of the periarteriolar lymphatic sheath (PALS) in the splenic white pulp (28). Lyn kinase may be associated with the function of interdigitating dendritic cells, although Lyn has never been reported to play any role in dendritic cells. FDCs in primary and secondary B cell follicles can retain unprocessed Ag on their surface in the form of immune complexes for several months, and may play an important role in positive selection of the centrocytes with high affinity for Ag (28). FDCs are reported to form the clusters through interaction between ICAM-1 on FDCs and LFA-1 on B cells. VLA-4 on activated B cells supports B cell-FDC cluster formation by binding to VCAM on FDCs (52). Organized FDC structure, such as FDC-B cell clusters, was reportedly absent in LT-{alpha}-deficient mice in which GCs were absent but affinity maturation occurred normally (52). Lyn-/- mice may also exhibit some abnormalities in the function or structure of FDCs, although Lyn kinase has not been reported to be associated with the function of FDCs or interdigitating dendritic cells. The numbers of peripheral B cells in lyn-/- mice are 40 to 60% less than that in WT mice (25, 26). In addition, splenic B cells in lyn-/- mice show a reduced proliferative response to CD40L (26, 27). The combination of small numbers of B cells and the defect in ability to expand in response to CD40 signaling might cause a lack of B cell follicles in lyn-/- mice. Therefore, PNAhigh B cells may not be induced and GCs may not be formed in lyn-/- mice. Since splenic B cells of lyn-/- mice could not make complexes nor form follicles in the white pulp, Lyn kinase may be associated with homing or adhesion to B cell follicles. However, Lyn has not been demonstrated to be downstream of adhesion molecule-mediated signaling, and homing abnormalities of B cells have not been detected in lyn-/- mice. Thus, although lyn-/- mice showed defective formation of B cell follicles and GCs, the molecular mechanism for the deficiency is still unclear.

lyn-/- mice displayed normal isotype switching and affinity maturation despite the defect in GC formation. It has been reported that Lyn kinase is activated by CD40 ligation (15), and splenic B cells of lyn-/- mice showed reduced proliferative responses to CD40L and reduced susceptibility to Fas-mediated apoptosis through CD40 (26, 27). These results suggested that Lyn might be involved in signal transduction from CD40. Previous studies reported that CD40-deficient and CD40L-deficient mice failed to form GCs and did not produce Ag-specific IgG1, indicating a crucial role of CD40 signaling in isotype switching(43, 44, 45). Thus, there is a discrepancy between the results in lyn-/- mice and those in CD40-deficient or CD40L-deficient mice concerning isotype switching despite the absence of GC formation in both cases. In addition, although CD40-CD40L signal was considered essential for affinity maturation following somatic hypermutation (36), lyn-/- mice showed a normal response. The explanation for the discrepancy may be that CD40-mediated signaling consist of two pathways, only one of which is Lyn dependent (26). It is possible that isotype switching and affinity maturation following somatic hypermutation are induced by the Lyn-independent CD40-mediated signal pathway. This possibility explains why lyn-/- mice showed normal Ag-specific IgG1 isotype switching and affinity maturation. Alternatively, other Src family kinases may compensate for the lack of functional Lyn, but this is unlikely because Lyn/Fyn doubly deficient mice also showed a defect similar to that of lyn-/- mice (Nishizumi et al., unpublished observations).

Why do isotype switching and affinity maturation occur in lyn-/- mice despite the lack of GC formation? One explanation may be that although typical GCs were absent in both spleen and LN, small numbers of abnormally located PNA+ B cells were present in either or both organs, and those cells could induce isotype switching and affinity maturation without formation of any GC structure. This explanation is consistent with previous studies demonstrating that PNA+ B cells themselves can internalize immunogen and present it to T cells in vivo, while a bulk population of B cells containing few PNA+ cells and a majority of PNA- cells could not present the immunizing Ag to T cells (58, 60). This possibility, however, is unlikely, since the number of PNA+ B cells greatly decreased in the spleens and LNs in lyn-/- mice. Another explanation may be that Lyn kinase-deficient B cells could not express receptors for PNA, but these PNA-negative B cells still function in the GC reaction by internalizing immunogen and presenting it to T cells. Thus, PNA expression may have nothing to do with the function of B cells in GCs. Alternatively, isotype switching and affinity maturation may be independent of GC formation or the appearance of PNA+ B cells, but these functions may be dependent on establishing an environment in which cross-interaction between T cells, B cell and dendritic cells can take place. At least, the significance of the GC architecture itself should be reconsidered, since isotype switching, affinity maturation, and differentiation to B cell memory clearly can occur without GC structure in both LT-{alpha}-deficient mice and Lyn-deficient mice. Lyn-/- mice may provide a good model for elucidating the physiologic significance of GCs.


   Acknowledgments
 
The authors thank Drs. T. Kaisho and K. Rajewsky for providing NP-CG, NP4-BSA, and NP14-BSA and Dr. Y. Nakayama for NP-CG. We also thank Dr. T. Takemori for providing 2.4G2 and PE-labeled NIP21-BSA, Dr. P. Burrows for helpful discussion and critical reading of the manuscript, Dr. T. Iwaki for technical help in histologic studies, and Dr. M. Matsumoto for helpful comments.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Takeshi Watanabe, Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-0054, Japan. E-mail address: Back

2 Abbreviations used in this paper: BCR, B cell antigen receptor; sIgM, surface IgM; PTK, protein tyrosine kinase; PLC, phospholipase C; PI-3, phosphatidylinositol-3; CD40L, CD40 ligand; GC, germinal center; TD, thymus-dependent; T-B, T cell-B cell; NP-CG, (4-hydroxy-3-nitrophenyl)acetyl-chicken {gamma}-globulin; WT, wild-type; LN, lymph node; PNA, peanut agglutinin; CDR, complementarity-determining region; FDC, follicular dendritic cell; PE, phycoerythrin. Back

Received for publication August 18, 1997. Accepted for publication January 21, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reth, M.. 1992. Antigen receptors on B lymphocytes. Annu. Rev. Cell Biol. 10:673.
  2. Yamanashi, Y., T. Kakiuchi, J. Mizuguchi, T. Yamamoto, K. Toyoshima. 1991. Association of B cell antigen receptor with protein tyrosine kinase Lyn. Science 251:192.[Abstract/Free Full Text]
  3. Burkhardt, A. L., M. Brunamick, J. B. Bolen, J. J. Mond. 1991. Anti- immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88:7410.[Abstract/Free Full Text]
  4. Campbell, M.-A., B. M. Sefton. 1992. Association between B-lymphocytes membrane immunoglobulin and multiple members of the src family of protein tyrosine kinases. Mol. Cell. Biol. 12:2315.[Abstract/Free Full Text]
  5. Gold, M. R., L. Matsuuchi, R. B. Kelly, A. L. DeFranco. 1991. Tyrosine phosphorylation of the B-cell antigen receptor following crosslinking. Proc. Natl. Acad. Sci. USA 88:3436.[Abstract/Free Full Text]
  6. Coggeshall, K. M., J. C. McHagh, A. Altman. 1992. Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-{gamma}2 in B lymphocytes. Proc. Natl. Acad. Sci. USA 89:5660.[Abstract/Free Full Text]
  7. Yamanashi, Y., Y. Fukui, B. Wongsasant, Y. Kinoshita, Y. Ichimori, K. Toyoshima, T. Yamamoto. 1992. Activation of src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 89:1118.[Abstract/Free Full Text]
  8. Bustelo, X. R., M. Barbacid. 1992. Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells. Science 256:1196.[Abstract/Free Full Text]
  9. Gold, M. R., M. T. Crowley, G. A. Martin, F. McCorrmick, A. L. DeFranco. 1993. Targets of B lymphocyte antigen receptor signal transduction include the p21rasGTPase-activating protein (GAP) and two GAP-associated proteins. J. Immunol. 150:377.[Abstract]
  10. Saxton, T. M., I. von Oostveen, D. Bowtell, R. Aebersold, M. R. Gold. 1994. B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activation SHC, GRB2, mSOS1 and a 145-kDa tyrosine- phosphorylated protein. J. Immunol. 153:623.[Abstract]
  11. Yamanashi, Y., M. Okada, T. Semda, T. Yamori, H. Umemori, S. Tsunasawa, K. Toyoshima, D. Kitamura, T. Watanabe, and T. Yamamoto. Identification of HS1 proteins as a major substrate of protein-tyrosine kinase(s) upon B cell antigen receptor-mediated signaling. Proc. Natl. Sci. USA 90:3631.
  12. Aoki, Y., Y.-T. Kim, R. Stillwell, T. J. Kim, S. Pillai. 1995. The SH2 domain of src family kinases associate with Syk. J. Biol. Chem. 270:15658.[Abstract/Free Full Text]
  13. Yamanashi, Y., S. Mori, M. Yoshida, T. Kishimoto, K. Inoue, T. Yamamoto, K. Toyoshima. 1989. Selective expression of protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T cell lymphotropic virus type I. Proc. Natl. Acad. Sci. USA 86:6538.[Abstract/Free Full Text]
  14. Umemori, H., A. Wanaka, H. kata, M. Takeuchi, M. Tohyama, T. Yamamoto. 1992. Specific expression of a fyn and lyn, lymphocyte antigen receptor-associated tyrosine kinases in central nervous system. Mol. Brain Res. 16:303.[Medline]
  15. Ren, C. L., T. Morio, S. M. Fu, R. S. Geha. 1994. Signal transduction via CD40 involves activation of lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C{gamma}2. J. Exp. Med. 179:673.[Abstract/Free Full Text]
  16. Stefanova, I., M. L. Corcoran, E. M. Horak, L. M. Wahl, J. B. Bolen, I. D. Horak. 1993. Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase p53/56lyn. J. Biol. Chem. 268:20725.[Abstract/Free Full Text]
  17. Eiseman, E., J. B. Bolen. 1992. Engagement of high affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355:78.[Medline]
  18. Kobayashi, N. H., T. Kono, M. Hatakeyama, Y. Minami, T. Miyazaki, R. M. Perlmutter, T. Taniguchi. 1993. Functional coupling of the src-family protein tyrosine kinases p59lyn and p53/p56lyn with the interleukin 2 receptor: implication for redundancy and pleiotropism in cytokine signal transduction. Proc. Natl. Acad. Sci. USA 90:4201.[Abstract/Free Full Text]
  19. Corey, S. J., A. L. Burkhardt, J. B. Bolen, R. L. Geahlen, L. S. Tkatch, D. J. Tweady. 1994. Granulocyte colony-stimulating factor receptor signaling involves the formation of a three-component complex with Lyn and Syk protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 91:4683.[Abstract/Free Full Text]
  20. Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor coupled Ca2+ mobilization through distinct pathways. EMBO J. 13:1341.[Medline]
  21. Scheuermann, R. H., E. Racila, T. Tucker, E. Yefenof, N. E. Street, E. S. Vitetta, L. J. Picker, J. W. Uhr. 1994. Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells. Proc. Natl. Acad. Sci. USA 91:4048.[Abstract/Free Full Text]
  22. Faris, M., F. Gaskin, J. T. Parsons, S. M. Fu. 1994. CD40 signaling pathway induces rapid dephosphorylation and phosphorylation of tyrosine-phosphorylated proteins including protein tyrosine kinase Lyn, Fyn, and Syk phosphorylated protein. J. Exp. Med. 179:1923.[Abstract/Free Full Text]
  23. Banchereau, T., P. D. Paoli, A. Valle, E. Garcia, F. Rousset. 1991. Long-term function B cell lines dependent on interleukin-4 and antibody to CD40. Science 251:70.[Abstract/Free Full Text]
  24. Kehry, M. R.. 1996. CD40-mediated signaling in B cells: balancing cell survival, growth, and death. J. Immunol. 156:2345.[Abstract]
  25. Hibbs, M. L., D. M. Tarlinton, J. Armes, D. Grail, G. Hodgson, R. Maglitto, S. A. Stacher, A. R. Dum. 1995. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83:301.[Medline]
  26. Nishizumi, H., I. Taniuchi, Y. Yamanashi, D. Kitamura, D. Ilic, S. Mori, T. Watanabe, T. Yamamoto. 1995. Impaired proliferation of peripheral B cells and indication of autoimmune disease in Lyn-deficient mice. Immunity 3:549.[Medline]
  27. Wang, J., T. Koizumi, T. Watanabe. 1996. Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in lyn-deficient mice. J. Exp. Med. 184:831.[Abstract/Free Full Text]
  28. Dulendram, B., V. D. Rosemary, G. J. V. Nossal. 1997. Unological tolerance in germinal centres. Immunol. Today 18:27.[Medline]
  29. Jacob, J., G. Kelsoe, K. Rajewsky, U. Weiss. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354:389.[Medline]
  30. Küppers, R., R. Zhao, M.-L. Hansmann, K. Rajewsky. 1993. Tracing B cell development in human germline centres by molecular analysis of single cells picked from histological sections. EMBO J. 12:4955.[Medline]
  31. Jacob, J., J. Przylepa, C. Miller, G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The Kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178:1293.[Abstract/Free Full Text]
  32. Berek, C., A. Berger, M. Apel. 1991. Maturation of the immune response in germinal centers. Cell 67:1121.[Medline]
  33. Liu, Y. J., F. Malisan, O. De Bouteiller, C. Guret, S. Lebeque, J. Banchereau, F. C. Mills, E. E. Max, H. Martinez-Valdes. 1996. Within germinal center, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation. Immunity 4:241.[Medline]
  34. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173:1165.[Abstract/Free Full Text]
  35. Toellner, K.-M., A. Gulbranson-Judge, D. R. Taylor, D. M.-Y. Sze, I. C. M. MacLennan. 1996. Immunoglobulin switch transcript production in vivo related to the site and time of antigen-specific B cell activation. J. Exp. Med. 183:2303.[Abstract/Free Full Text]
  36. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, G. Kelsoe. 1995. Cellular interaction in germinal centers: role of CD40 ligand and B7-2 in established germinal centers. J. Immunol. 155:556.[Abstract]
  37. Miller, C., J. Stedra, G. Kelsoe, J. Cerny. 1995. Facultative role of germinal centers and T cells in the somatic diversification of IgVH genes. J. Exp. Med. 181:1319.[Abstract/Free Full Text]
  38. Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.[Medline]
  39. Lane, P., C. Burdet, S. Hubele, D. Scheidegger, U. Müller, F. McConnell, M. H. Kosco-Vilbois. 1994. B cell function in mice transgenic for mCTLA4-H{gamma}1: lack of germinal centers correlated with poor affinity maturation and class switching. J. Exp. Med. 179:819.[Abstract/Free Full Text]
  40. Ferguson, S. E., S. Han, G. Kelsoe, C. B. Thompson. 1996. CD28 is required for germinal center formation. J. Immunol. 156:4576.[Abstract]
  41. Shakiman, A., K. Pfeffer, K. P. Lee, T. M. Kündig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak. 1993. Differential T cell costimulatory requirement in CD28-deficient mice. Science 261:609.[Abstract/Free Full Text]
  42. Borriello, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Tacoly, T. B. Strom, E. M. Simpson, G. J. Freedam, A. H. Sharpe. 1997. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6:303.[Medline]
  43. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune response in CD40- deficient mice impaired immunoglobulin class switching and germinal center formation. Immunity 1:167.[Medline]
  44. Renshow, B. R., III W. C. Fanslow, R. J. Armitage, K. A. Campbell, D. Liggritt, B. Wright, B. L. Darison, C. R. Malisyewski. 1994. Humoral immune response in CD40 ligand-deficient mice. J. Exp. Med. 180:1889.[Abstract/Free Full Text]
  45. Xu, J. C., T. M. Foy, J. D. Laman, E. A. Elliott, J. J. Bum, T. J. Waldschmidt, J. Elsemore, R. J. Noell, R. A. Flavell. 1994. Mice deficient for CD40 ligand. Immunity 1:423.[Medline]
  46. Le Hir, M., H. Bluethmann, M. H. Kosco-Vilbois, M. Müller, F. D. Pardova, M. Moore, B. Ryffel, H.-P. Eugster. 1996. Differentiation of follicular dendritic cells and full antibody responses require tumor necrosis factor receptor-1 signaling. J. Exp. Med. 183:2367.[Abstract/Free Full Text]
  47. Pasparakis, M., L. Alexopoulou, V. Episkopou, G. Kollias. 1996. Immune and inflammatory responses in TNF{alpha}-deficient mice: a critical requirement for TNF{alpha} in the formation of primary B cell follicles, follicular dendritic cell network and germinal centers and in the maturation of the humoral immune response. J. Exp. Med. 184:1397.[Abstract/Free Full Text]
  48. Schubart, D. B., A. Rolink, M. H. Kosco-Vilbois, F. Botteri, P. Matthias. 1996. B-cell-specific coactivator OBF-1/OCA-B/Bob 1 required for immune response and germinal center formation. Nature 383:538.[Medline]
  49. Kim, U., X.-F. Qin, S. Gong, S. Stevens, Y. Luo, M. Nussenzweig, R. G. Roeder. 1996. The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes. Nature 383:542.[Medline]
  50. DeTogni, P., J. Goellner, N. H. Ruddle, P. R. Streeter, A. Fick, S. Mariathasan, S. C. Smith, R. Carlson, L. P. Shornick, J. Strauss-Schoenberger, J. H. Russel, R. Karr, D. D. Chaplin. 1994. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin-{alpha}. Science 264:703.[Abstract/Free Full Text]
  51. Banks, T. A., B. T. Rouse, M. K. Kerley, P. J. Blair, V. L. Godfrey, N. A. Kuklin, D. M. Bouley, J. Thomas, S. Kanangat, M. L. Mucenski. 1995. Lymphotoxin-{alpha}-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
  52. Matsumoto, M., S. F. Lo, C. J. L. Carruthers, J. Min, S. Mariathasan, G. Huang, D. R. Plas, S. M. Martin, R. S. Geha, M. H. Nahm, D. D. Chaplin. 1996. Affinity maturation without germinal cetres in lymphotoxin-{alpha}-deficient mice. Nature 382:462.[Medline]
  53. Rose, J., K. Rajewsky. 1993. Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells. J. Exp. Med. 177:45.[Abstract/Free Full Text]
  54. Weiss, U., R. Zoebelein, K. Rajewsky. 1992. Accumulation of somatic mutations in the B cell compartment after primary immunization with a T cell- dependent antigen. Eur. J. Immunol. 22:511.[Medline]
  55. Rajewsky, K., I. Förster, A. Cumano. 1987. Evolutionary and somatic selection of the antibody repertoire in the mouse. Science 238:1088.[Abstract/Free Full Text]
  56. Neuberger, M. S., C. Milstein. 1995. Somatic hypermutation. Curr. Opin. Immunol. 7:248.[Medline]
  57. Wangner, S. D., C. Milstein, N. S. Neuberger. 1995. Codon bias targets mutation. Nature 376:732.[Medline]
  58. Kosco-Vilbois, M. H., H. Zentgraf, J. Gerdes, J.-Y. Bonnefoy. 1997. To ‘B’ or not to ‘B’ a germinal center?. Immunol. Today 18:225.[Medline]
  59. Tew, J. G., R. P. Phipps, T. E. Mandel. 1980. The maintenance and the regulation of the humoral immune responses: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells. Immunol. Rev. 53:175.[Medline]
  60. Guéry, J. C., F. Ria, L. Adorini. 1996. Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant. J. Exp. Med. 183:751.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
L. Quemeneur, V. Angeli, M. Chopin, and R. Jessberger
SWAP-70 deficiency causes high-affinity plasma cell generation despite impaired germinal center formation
Blood, March 1, 2008; 111(5): 2714 - 2724.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Yang, G. C. Waldbieser, and C. J. Lobb
The Nucleotide Targets of Somatic Mutation and the Role of Selection in Immunoglobulin Heavy Chains of a Teleost Fish
J. Immunol., February 1, 2006; 176(3): 1655 - 1667.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Z. K. Mirnics, E. Caudell, Y. Gao, K. Kuwahara, N. Sakaguchi, T. Kurosaki, J. Burnside, K. Mirnics, and S. J. Corey
Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center
J. Immunol., April 1, 2004; 172(7): 4133 - 4141.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
S. Inui, K. Maeda, D. R. Hua, T. Yamashita, H. Yamamoto, E. Miyamoto, S. Aizawa, and N. Sakaguchi
BCR signal through {alpha}4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation
Int. Immunol., February 1, 2002; 14(2): 177 - 187.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Alfonso, J.-O. Han, G. S. Williams, and L. Karlsson
The Impact of H2-DM on Humoral Immune Responses
J. Immunol., December 1, 2001; 167(11): 6348 - 6355.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. J. Driver, L. J. McHeyzer-Williams, M. Cool, D. B. Stetson, and M. G. McHeyzer-Williams
Development and Maintenance of a B220- Memory B Cell Compartment
J. Immunol., August 1, 2001; 167(3): 1393 - 1405.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S.-j. Seo, J. Buckler, and J. Erikson
Novel Roles for Lyn in B Cell Migration and Lipopolysaccharide Responsiveness Revealed Using Anti-Double-Stranded DNA Ig Transgenic Mice
J. Immunol., March 15, 2001; 166(6): 3710 - 3716.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Brauninger, W. Yang, H.-H. Wacker, K. Rajewsky, R. Kuppers, and M.-L. Hansmann
B-cell development in progressively transformed germinal centers: similarities and differences compared with classical germinal centers and lymphocyte-predominant Hodgkin disease
Blood, February 1, 2001; 97(3): 714 - 719.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
X. Wu, N. Jiang, Y.-F. Fang, C. Xu, D. Mao, J. Singh, Y.-X. Fu, and H. Molina
Impaired Affinity Maturation in Cr2-/- Mice Is Rescued by Adjuvants Without Improvement in Germinal Center Development
J. Immunol., September 15, 2000; 165(6): 3119 - 3127.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
J. Uchida, T. Yasui, Y. Takaoka-Shichijo, M. Muraoka, W. Kulwichit, N. Raab-Traub, and H. Kikutani
Mimicry of CD40 Signals by Epstein-Barr Virus LMP1 in B Lymphocyte Responses
Science, October 8, 1999; 286(5438): 300 - 303.
[Abstract] [Full Text]

Home page
 BloodHome page
A. R. Thompsett, D. W. Ellison, F. K. Stevenson, and D. Zhu
VH Gene Sequences From Primary Central Nervous System Lymphomas Indicate Derivation From Highly Mutated Germinal Center B Cells With Ongoing Mutational Activity
Blood, September 1, 1999; 94(5): 1738 - 1746.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. HogenEsch, S. Janke, D. Boggess, and J. P. Sundberg
Absence of Peyer's Pat