Abstract
Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3−/− mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM− pool is more severely reduced. Interestingly, despite a compensatory increase in cyclin D2 expression, a significant number of Ccnd3−/− GC B cells accumulate in quiescent G0 state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.
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Abbreviations
- CSR:
-
(class switch recombination)
- Cdk:
-
(cyclin dependent kinase)
- DAPI:
-
(diamidino-2-phenylindole)
- DLBCL:
-
(diffuse large B cell lymphoma)
- DN:
-
(double negative)
- FDC:
-
(follicular dendritic cell)
- Fo:
-
(follicular B cells)
- GC:
-
(germinal center)
- IHC:
-
(immunohistochemistry)
- KO:
-
(knock-out)
- LN:
-
(lymph node)
- MZ:
-
(marginal zone)
- NP:
-
(nitrophenyl)
- PNA:
-
(peanut agglutinin)
- PI:
-
(propidium iodide)
- SHM:
-
(somatic hypermutation)
- Tfh:
-
(T follicular helper cells)
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Acknowledgements
We thank Dr Riccardo Dalla-Favera (Columbia University, USA) for sharing the Iμ-HA-BCL6 mouse strain, Drs Ulf Klein (Columbia University, USA) and Liang Zhu (Albert Einstein College of Medicine (AECOM), USA) for insightful suggestions, Dr Mimi Kim (AECOM) for expert statistical analysis, Drs Sergio Roa, Lydia Zhao and Susan Buhl (AECOM), and Lucas Tsikitas (Weill Cornell College of Medicine, USA) for their invaluable technical support. We are also indebted to Drs Arthur Skoultchi, Liang Zhu and Ganjam Kalpana (AECOM) for reagents. The AECOM Flow Cytometry Facility and Histopathology Facility are gratefully acknowledged. This work was supported by the Medical Scientist Training Program T32GM007288 (J.U.P.) and grants from the National Institutes of Health R01 CA85573 (B.H.Y), R01 CA72649 and CA102705 (M.D.S.), K08 CA127353 (R.S.), R01 AR049126 and AI051392 (B.D.), and P01 CA109901 (P.S). Support also came from the Harry Eagle Chair provided by the National Women's Division to M.D.S.
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(Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Figure S1
Histological validation of Cyclin D3 antibody and characterization of B cell splenic architecture in KO animals. (PDF 138 kb)
Figure S2
Schematic illustration of the immunization and bleeding schedule used to elicit T-cell dependent anti-NP response in Figure 4. (PDF 86 kb)
Figure S3
Class Switch Recombination to IgG1 is moderately impaired in the absence of cyclin D3. (PDF 26 kb)
Table S1
Antibodies used for IHC and Western Blot analyses. (PDF 52 kb)
Table S2
Sequences of qRT-PCR primers. (PDF 58 kb)
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Peled, J., Yu, J., Venkatesh, J. et al. Requirement for cyclin D3 in germinal center formation and function. Cell Res 20, 631–646 (2010). https://doi.org/10.1038/cr.2010.55
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DOI: https://doi.org/10.1038/cr.2010.55
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