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. 2013 Jun 27;8(6):e67587.
doi: 10.1371/journal.pone.0067587. Print 2013.

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen-Specific IgG and IgM, in Response to T-dependent Antigens

Sreemanti Basu  1 , Avijit Ray, Bonnie N Dittel
Affiliations

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen-Specific IgG and IgM, in Response to T-dependent Antigens

Sreemanti Basu et al. PLoS One. .

Abstract

The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2(-/-) mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2(-/-) mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2(-/-) mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. GC B cells are reduced, but antigen-specific plasma B cell formation and antigen-specific serum IgM and IgG1 levels are unaltered in CB2−/− mice upon i.p.
immunization with a T-dependent antigen. WT (open bars) and CB2−/− (closed bars) mice were i.p. immunized with 30 µg alum-precipitated NP-CGG. (A–D) Spleens and BM were collected before (day 0) or 7, 11, 14, and 28 days after immunization for the analysis of GC B cells in the spleen by flow cytometry (A) and NP-specific IgM and IgG1-secreting plasma cells in the spleen (B, C, respectively) and BM (D) by ELISPOT. (A) The percentage of Fas+GL7+ GC B cells within the B220+ population is shown. (B–D) Frequencies of NP-specific IgM+ (B) and IgG1 + spots (C) per 106 splenocytes and NP-specific IgG1 + spots per 106 BM cells (D) on the indicated days are shown. (E, F) Serum was collected from NP-CGG-immunized WT and CB2−/− mice on days 0, 7, 14 and 28 and NP-specific IgM and IgG1 titers were measured from two-fold serially diluted serum (1/400 to 1/3200 for IgM and 1/20000 to 1/160000 for IgG1) by ELISA. The O.D. value for NP-specific IgM at the 1/1600 dilution (E) and NP-specific IgG1 at the 1/40000 dilution (F) are shown. Data shown are the mean ± SEM from two independent experiments each with 3–4 mice per group. **p<0.01.
Figure 2
Figure 2. Upon s.c. immunization with NP-CGG GC formation occurs primarily in the draining LN.
WT and CB2−/− mice were s.c. immunized with 30 µg alum-precipitated NP-CGG s.c. on the shoulders. On day seven, the draining brachial and non-draining axillary LN and the spleen were collected and GC B cells were analyzed by flow cytometry. Representative dot plots show relative frequencies of Fas+GL7+ GC B cells within the B220+ population in the brachial LN (top row), axillary LN (middle row), and the spleen (bottom row) from unimmunized controls (left panels), immunized WT (middle panels) and CB2−/− (right panels) mice. Numbers on the plots represent the percentage of cells in the corresponding gate. Data shown are one representative experiment of three.
Figure 3
Figure 3. Upon s.c. NP-CGG immunization CB2−/− mice elicit normal GC B cell and antigen-specific IgM and IgG1 responses.
WT (open bars) and CB2−/− (closed bars) mice were s.c. immunized with 30 µg alum-precipitated NP-CGG. (A–C) On day 0 or days 5, 7, 10, 14, and 28 post-immunization, the draining LN were collected and GC B cells were analyzed by flow cytometry (A) and NP-specific IgM and IgG1 secreting plasma cells were determined by ELISPOT (B, C, respectively). (A) The percentage of Fas+GL7+ GC B cells within the B220+ population is shown. (B, C) Frequencies of NP-specific IgM+ (B) and IgG1 + spots (C) per 106 LN cells are shown. (D, E) NP-specific IgM and IgG1 titers were measured from two-fold serially diluted serum (1/200 to 1/1600 for IgM and 1/10000 to 1/80000 for IgG1) from NP-CGG-immunized WT and CB2−/− mice on days 0, 7, 14, and 28 by ELISA. The O.D. values for NP-specific IgM at the 1/800 dilution (E) and NP-specific IgG1 at the 1/40000 dilution (F) are shown. Data shown are the mean ± SEM from two independent experiments each with 3 mice per group (n = 6).
Figure 4
Figure 4. CB2-deficiency does not affect affinity maturation.
WT (open bars) and CB2−/− (closed bars) mice were immunized with 30 µg NP-CGG in alum (i.p.) and rechallenged with 30 µg of NP-CGG in PBS (i.p.) on day 42 post-primary immunization. BM and serum were collected on day 28 after the primary and day seven after the secondary immunization for the analysis of affinity maturation. (A) Frequencies of high affinity (NP4-specific) and total (NP25-specific) antigen-specific IgG1-secreting plasma cells per 106 BM cells were determined by ELISPOT. Affinity maturation calculated as a ratio of high affinity to total antigen-specific IgG1-secreting cells (NP4:NP25) is shown. (B) The titers of high affinity (NP4-specific) and total (NP25-specific) antigen-specific IgG1 were determined from two-fold serially diluted serum (1/20000 to 1/160000) by ELISA and the O.D. values at the 1/40000 dilution were used to calculate affinity maturation. Affinity maturation calculated as an O.D. ratio of high affinity to total antigen-specific IgG1 (NP4:NP25) is shown. Data shown are the mean ± SEM from two independent experiments each with 2–4 mice per group (n = 6–7).
Figure 5
Figure 5. Memory B cell formation is reduced while the secondary humoral response after NP-CGG immunization is unaffected in CB2−/− mice.
WT and CB2−/− mice were immunized (i.p.) with 30 µg alum-precipitated NP-CGG and rechallenged with 30 µg of NP-CGG in PBS (i.p.) on day 42 post-primary immunization. (A,B) BM and spleen were collected on day 28 after the primary and day seven after the secondary immunization for the analysis of memory B cells by flow cytometry. (A) Representative flow cytometric plots show the gating strategy for memory B cells. B220+ cells were analyzed for NP8 and IgG1 in unimmunized (left panel) and in immunized (middle panel) mice and NP8 +IgG1 + cells were further analyzed for the expression of IgD and CD38 (right panel). The B220+NP8 +IgG1 +IgD−CD38+ population represented antigen-specific high-affinity class-switched memory B cells. (B) The cumulative percentage of memory B cells within the lymphocyte population of the BM and spleen is shown. (C, D) On day seven after the secondary immunization the frequency of NP-specific IgG1-secreting plasma cells in the BM was determined by ELISPOT (C) and the NP-specific serum IgG1 titer was measured from two-fold serially diluted serum (1/40000–1/320000; O.D. at 1/160000 the dilution is shown) by ELISA (D). (B–D) Data are represented as the mean ± SEM from two independent experiments each with 2–4 mice per group (n = 6–7). *p<0.05.
Figure 6
Figure 6. WT and CB2−/− mice elicit comparable antigen-specific IgM and IgG responses in the serum after sRBC immunization (i.p.).
WT and CB2−/− mice were immunized (i.p.) with 1×108 sRBC and serum was collected on days 0 (pre-immunization), 7, 14, and 28. Antigen-specific IgM and IgG titers were measured from three-fold serially diluted (1/50 to 1/1350 for IgM and 1/150 to 1/4050 for IgG) serum samples by ELISA. The O.D. values for sRBC-specific IgM at the 1/50 dilution (A) and sRBC-specific IgG at the 1/450 dilution (B) are shown. Data shown are the mean ± SEM from two independent experiments each with 3–4 mice per group (n = 6–8).
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