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Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.

Takahashi Y, Cerasoli DM, Dal Porto JM, Shimoda M, Freund R, Fang W, Telander DG, Malvey EN, Mueller DL, Behrens TW, Kelsoe G - J. Exp. Med. (1999)

Bottom Line: Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs.Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses.Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, USA.

ABSTRACT
The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

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bcl-xL transgenic mice produce GC and IgG1 antibody responses that are similar to control animals. Splenocytes were recovered from transgenic mice or wild-type controls at various times after immunization with NP-CG. (A) The numbers of λ1+ GCs were determined by histological staining with anti-λ1 antibody and PNA. Each point represents the average number of λ1+ GCs per histologic section (≥3 sections representing ≈2/3 splenic area) in single transgenic (open circles) or wild-type (filled circles) mice. (B) The percentage of GC B cells in live lymphocytes was assessed by flow cytometry using anti-B220 and anti–GL-7 antibodies. Each point represents the frequency mean (± SD) of GC B cells (percentage of total lymphoid gate) in single transgenic (open circles) and control (filled circles) mice 8, 12, and 35 d after immunization. (C) Numbers of NP-specific AFCs from BM (circles) and spleen (squares) of bcl-xL transgenic (open) or littermate control mice (filled) were determined by ELISPOT using NP23-BSA as the capture antigen. Frequencies of AFCs in naive mice (day 0) from both groups were <0.2 × 10−5. (D) NP-specific serum antibody from transgenic (open) or wild-type control mice (filled) was determined by ELISA using NP23-BSA. The average values (± SD) for serum antibody concentrations from five to seven individual mice per time point are presented.
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Figure 3: bcl-xL transgenic mice produce GC and IgG1 antibody responses that are similar to control animals. Splenocytes were recovered from transgenic mice or wild-type controls at various times after immunization with NP-CG. (A) The numbers of λ1+ GCs were determined by histological staining with anti-λ1 antibody and PNA. Each point represents the average number of λ1+ GCs per histologic section (≥3 sections representing ≈2/3 splenic area) in single transgenic (open circles) or wild-type (filled circles) mice. (B) The percentage of GC B cells in live lymphocytes was assessed by flow cytometry using anti-B220 and anti–GL-7 antibodies. Each point represents the frequency mean (± SD) of GC B cells (percentage of total lymphoid gate) in single transgenic (open circles) and control (filled circles) mice 8, 12, and 35 d after immunization. (C) Numbers of NP-specific AFCs from BM (circles) and spleen (squares) of bcl-xL transgenic (open) or littermate control mice (filled) were determined by ELISPOT using NP23-BSA as the capture antigen. Frequencies of AFCs in naive mice (day 0) from both groups were <0.2 × 10−5. (D) NP-specific serum antibody from transgenic (open) or wild-type control mice (filled) was determined by ELISA using NP23-BSA. The average values (± SD) for serum antibody concentrations from five to seven individual mice per time point are presented.

Mentions: To assess the effects of the bcl-xL transgene on GC development, we compared the GC reaction of transgenic and control mice at day 12 after immunization with NP-CG. This antigen elicits a characteristic hapten-specific antibody that bears a λ1 L chain and an H chain encoded by a canonical VDJ gene rearrangement 32. We identified λ1+ GCs in spleen sections by labeling with PNA and anti-λ1 antibody 32 and determined the average number of λ1+ GCs per section from groups of transgenic and control mice (Fig. 3 A). Differences in the number or size (data not shown) of GCs were not observed between the groups, nor did the mean frequency of λ1+ GCs significantly differ between transgenic (35.4%) and wild-type mice (41.7%). When transgenic mice were immunized with carrier protein alone, the average frequency of λ1+ GCs was 7.6%. Thus, frequent λ1+ GCs in transgenic mice result from immunization with NP rather than altered λ1 L chain expression.


Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.

Takahashi Y, Cerasoli DM, Dal Porto JM, Shimoda M, Freund R, Fang W, Telander DG, Malvey EN, Mueller DL, Behrens TW, Kelsoe G - J. Exp. Med. (1999)

bcl-xL transgenic mice produce GC and IgG1 antibody responses that are similar to control animals. Splenocytes were recovered from transgenic mice or wild-type controls at various times after immunization with NP-CG. (A) The numbers of λ1+ GCs were determined by histological staining with anti-λ1 antibody and PNA. Each point represents the average number of λ1+ GCs per histologic section (≥3 sections representing ≈2/3 splenic area) in single transgenic (open circles) or wild-type (filled circles) mice. (B) The percentage of GC B cells in live lymphocytes was assessed by flow cytometry using anti-B220 and anti–GL-7 antibodies. Each point represents the frequency mean (± SD) of GC B cells (percentage of total lymphoid gate) in single transgenic (open circles) and control (filled circles) mice 8, 12, and 35 d after immunization. (C) Numbers of NP-specific AFCs from BM (circles) and spleen (squares) of bcl-xL transgenic (open) or littermate control mice (filled) were determined by ELISPOT using NP23-BSA as the capture antigen. Frequencies of AFCs in naive mice (day 0) from both groups were <0.2 × 10−5. (D) NP-specific serum antibody from transgenic (open) or wild-type control mice (filled) was determined by ELISA using NP23-BSA. The average values (± SD) for serum antibody concentrations from five to seven individual mice per time point are presented.
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Related In: Results  -  Collection

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Figure 3: bcl-xL transgenic mice produce GC and IgG1 antibody responses that are similar to control animals. Splenocytes were recovered from transgenic mice or wild-type controls at various times after immunization with NP-CG. (A) The numbers of λ1+ GCs were determined by histological staining with anti-λ1 antibody and PNA. Each point represents the average number of λ1+ GCs per histologic section (≥3 sections representing ≈2/3 splenic area) in single transgenic (open circles) or wild-type (filled circles) mice. (B) The percentage of GC B cells in live lymphocytes was assessed by flow cytometry using anti-B220 and anti–GL-7 antibodies. Each point represents the frequency mean (± SD) of GC B cells (percentage of total lymphoid gate) in single transgenic (open circles) and control (filled circles) mice 8, 12, and 35 d after immunization. (C) Numbers of NP-specific AFCs from BM (circles) and spleen (squares) of bcl-xL transgenic (open) or littermate control mice (filled) were determined by ELISPOT using NP23-BSA as the capture antigen. Frequencies of AFCs in naive mice (day 0) from both groups were <0.2 × 10−5. (D) NP-specific serum antibody from transgenic (open) or wild-type control mice (filled) was determined by ELISA using NP23-BSA. The average values (± SD) for serum antibody concentrations from five to seven individual mice per time point are presented.
Mentions: To assess the effects of the bcl-xL transgene on GC development, we compared the GC reaction of transgenic and control mice at day 12 after immunization with NP-CG. This antigen elicits a characteristic hapten-specific antibody that bears a λ1 L chain and an H chain encoded by a canonical VDJ gene rearrangement 32. We identified λ1+ GCs in spleen sections by labeling with PNA and anti-λ1 antibody 32 and determined the average number of λ1+ GCs per section from groups of transgenic and control mice (Fig. 3 A). Differences in the number or size (data not shown) of GCs were not observed between the groups, nor did the mean frequency of λ1+ GCs significantly differ between transgenic (35.4%) and wild-type mice (41.7%). When transgenic mice were immunized with carrier protein alone, the average frequency of λ1+ GCs was 7.6%. Thus, frequent λ1+ GCs in transgenic mice result from immunization with NP rather than altered λ1 L chain expression.

Bottom Line: Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs.Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses.Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, USA.

ABSTRACT
The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.

Show MeSH
Related in: MedlinePlus