700

Cell 159, October 23, 2014 ©2014 Elsevier Inc.

DOI http://dx.doi.org/10.1016/j.cell.2014.10.012

See online version for legend and references.

Medulla

)))

Tingible-body macrophage (TBM)

Conventional dendritic cell

Plasma cell

Antigen

Antigen-specific T cell

Affinity

Dark Zone (DZ)

DZ B cells with deleterious mutations die and are cleared by TBM.

During proliferation, DZ cells upregulate AID and Polη, leading to somatic hypermutation of immunoglobulin variable regions and generation of mutants with altered antibody affinity.

DZ B cells divide rapidly for a number of cycles proportional to the strength of the Tfh signal received in LZ.

TBM

↓CXCR4

Dysfunctional surface Ig

Triggered in most or all B cells based on a cell-intrinsic timer (~50% of DZ cells transition to LZ over a 6 hr period)

DZ→LZ transition

Lower-affinity DZ B cells die by apoptosis and are cleared by TBM.

Higher-affinity cells survive and either re-enter DZ or are exported as PC or BMEM.

↑cMyc

CD40L

BDZ

BDZ

Dark Zone

Light Zone

CD83hi CD86hi G1/early S

BLZ

BDZ

↑CXCR4

TFH

CD4+ CXCR5hi PD-1hi

AIDhi Polηhi= SHM G1/S/G2/M

Triggered in the 10-30% B cells with higher affinity for antigen by Tfh cell help. Takes place during S phase.

LZ→DZ transition

TBM

Low affinity

)))

B cells with higher affinity (?); trigger largely unknown.

Export as BMEM

BMEM

PC

Export as PC

V. Dissolution (Days >21)

B cells with highest affinity. Triggered by signals from Tfh.

FDC

To other GCs

DZ

LZ

IV. Mature GC: selection (Days 8-21)

Light Zone (LZ)

)))

III. Early GC: expansion (Days 5-7)

LZ B cells capture antigen from follicular dendritic cells (FDCs) and compete for limiting T follicular helper (Tfh) cells.

Short-lived PC

Early Bmem

GC B

II. T:B border phase (Days 2-4)

Tfh ↑CXCR5

Cortex (B cell follicle)

Antigen-specific B cell

Lymph node

Paracortex (T cell zone)

↑CXCR5

↑CCR7

I. Antigen encounter (Days 0-1)

Gabriel D. Victora Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA

SnapShot: The Germinal Center Reaction

SnapShot: The Germinal Center Reaction Gabriel D. Victora Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA A prominent feature of the humoral immune response is the progressive increase in antibody affinity over time. This process takes place in the germinal center (GC), a temporary structure that arises in lymphoid organs upon antigenic exposure. Within this structure, B cells undergo a Darwinian process consisting of iterative cycles of random somatic hypermutation (SHM) of immunoglobulin variable regions, driven by the enzyme activation-induced cytidine deaminase (AID), and selection of progressively higher-affinity mutants. Differentiation of some of these higher-affinity mutants into plasma cells or memory B cells leads to the increasing affinity of serum antibodies and the high affinity of the recall response. Temporal Evolution of the GC Reaction GC duration can vary greatly, depending on the experimental system, from a few weeks to several months or longer. The kinetics detailed below are typical for subcutaneous immunization of mice with hapten-carrier conjugates in alum adjuvant. Antigen Encounter (0–1 Days after Immunization). Antigen arrives at lymphoid organs either via blood or lymphatics or is transported by conventional dendritic cells (cDCs). B cells whose surface immunoglobulins (sIg) bind the antigen will be triggered to upregulate chemokine receptor CCR7 and migrate toward the T zone. Antigen-specific CD4+ helper T cells encounter antigen presented on the surface of cDCs as peptides in the context of major histocompatibility complex class II (pMHC), which kick starts T cell proliferation. A subpopulation of these T cells upregulates chemokine receptor CXCR5 and moves toward the B cell follicle. T:B Border Phase (2–4 Days after Immunization). Antigen-activated B and T cells meet at the border of the T cell zone and B cell follicle. B cells have internalized and processed antigen and now present pMHC on their surface. Cognate interaction with T cells delivers signals that promote B cell proliferation and differentiation. This is a competitive step for B cells, and only those with higher affinity will successfully obtain access to cognate help from T cells and progress beyond this stage. Differentiation can occur along three paths: (1) GC B cells migrate to the follicle center to seed early GCs; (2) early plasmablasts/plasma cells migrate to the outer follicle and begin early antibody production; and (3) early memory cells are generated. Interaction with B cells promotes T cell differentiation into the T follicular helper (Tfh) phenotype (CXCR5hiPD-1hi) and migration into the B cell follicle. Early GC (Days 5–7). B cells migrate toward the center of the follicle, where they proliferate intensely and initiate AID-driven SHM. This takes place amidst a network of follicular dendritic cells (FDCs), differentiated stromal cells that retain immune complexes containing the immunizing antigen. B cell proliferation results in progressively larger clusters of B cells, which, at this stage, contain only few Tfh cells. Mature GC (Days 8–21). Early GCs split into two zones; a light zone (LZ) containing FDCs, LZ B cells (also referred to as “centrocytes”), and a high density of Tfh cells; and a dark zone (DZ), which consists mostly of rapidly proliferating DZ B cells (also referred to as “centroblasts”). Tingible body macrophages (TBM) that engulf dying B cells can be found throughout the GC at this stage. Cyclic migration of B cells between LZ and DZ leads to affinity maturation, as described below. Tfh cells at this stage can migrate between neighboring GCs in the same lymph node. GC Dissolution (Days 21 onward). GCs begin to dissipate; B cells are fewer in number and less concentrated in the central follicle, and TBMs become more evident. The onset of GC dissolution can vary substantially (from a few days to several months after GC maturation), depending on the mode of antigen encounter. Little is known about the factors that trigger GC dissolution. Mechanism of Selection in the GC B cells arrive in the LZ from the DZ as a panel of AID-generated mutants bearing sIgs of different affinities for antigen. LZ B cells can be identified by high levels of expression of activation markers CD83 and CD86 and low levels of chemokine receptor CXCR4 (the latter allows them to escape the pull of chemokine CXCL12, highly expressed in the DZ). LZ B cells use their newly minted sIgs to retrieve immune complexes from FDCs and present the retrieved antigen as pMHC. Mutants with higher-affinity sIgs present higher densities of pMHC, thus competing successfully for signals from a limiting number of GC-resident Tfh cells that trigger positive selection. Tfh-derived signals, such as CD40L binding to CD40, trigger positive selection. This involves upregulation of the transcription factor cMyc in selected B cells. B cells that fail to obtain sufficient T cell help die by apoptosis and are cleared by TBM. Like their T:B border counterparts, positively selected LZ B cells can follow along one of three fates. In the most studied of these, 10%–30% of all LZ B cells re-enter the DZ for additional round(s) of SHM. DZ re-entry occurs during S phase, which progresses concomitantly with upregulation of chemokine receptor CXCR4. This leads to mitosis taking place almost exclusively in the DZ. CD83lowCD86lowCXCR4hi DZ cells then undergo a number of divisions proportional to the strength of the signal delivered by Tfh cells in the LZ. Upregulation of AID and error-prone DNA polymerase η (Polη) triggers further SHM. Loss of sIg expression due to crippling mutations leads to cell death and clearance by TBM. Surviving B cells can either re-enter the LZ (which happens at a rate of 50% of cells per 4–6 hr period) or differentiate into late plasmablasts/ plasma cells or memory B cells. Plasma cell differentiation appears to be reserved to the highest-affinity B cells and can be triggered by signals from Tfh cells. Little is known about the signals that promote the differentiation of memory B cells. Acknowledgments I thank H. Ploegh, A. Gitlin, and members of the Victora lab for helpful comments. I apologize to my colleagues whose work was not directly cited due to space constraints. References Allen, C.D., Ansel, K.M., Low, C., Lesley, R., Tamamura, H., Fujii, N., and Cyster, J.G. (2004). Nat. Immunol. 5, 943–952. Berek, C., Berger, A., and Apel, M. (1991). Cell 67, 1121–1129. Crotty, S. (2011). Annu. Rev. Immunol. 29, 621–663. Gitlin, A.D., Shulman, Z., and Nussenzweig, M.C. (2014). Nature 509, 637–640. Jacob, J., Kelsoe, G., Rajewsky, K., and Weiss, U. (1991). Nature 354, 389–392. MacLennan, I.C. (1994). Annu. Rev. Immunol. 12, 117–139. Okada, T., Miller, M.J., Parker, I., Krummel, M.F., Neighbors, M., Hartley, S.B., O’Garra, A., Cahalan, M.D., and Cyster, J.G. (2005). PLoS Biol. 3, e150. Shulman, Z., Gitlin, A.D., Targ, S., Jankovic, M., Pasqual, G., Nussenzweig, M.C., and Victora, G.D. (2013). Science 341, 673–677. Victora, G.D., and Nussenzweig, M.C. (2012). Annu. Rev. Immunol. 30, 429–457. Victora, G.D., Schwickert, T.A., Fooksman, D.R., Kamphorst, A.O., Meyer-Hermann, M., Dustin, M.L., and Nussenzweig, M.C. (2010). Cell 143, 592–605.

700.e1 Cell 159, October 23, 2014 ©2014 Elsevier Inc.  DOI http://dx.doi.org/10.1016/j.cell.2014.10.012