A centroblast is a large, rapidly proliferating B lymphocyte located in the dark zone of germinal centers within secondary lymphoid organs, such as lymph nodes and spleen, where it undergoes clonal expansion and somatic hypermutation of immunoglobulin genes to generate antibody diversity during the humoral immune response.[1][2]Centroblasts originate from antigen-activated follicular B cells that have entered the germinal center following T cell-dependent activation, migrating to the dark zone under the influence of the chemokine receptor CXCR4 and its ligand CXCL12.[3] In this proliferative phase, centroblasts exhibit high mitotic activity, with each cell dividing approximately every 6-8 hours, and they downregulate surface immunoglobulin expression while upregulating activation-induced cytidine deaminase (AID) to facilitate hypermutation.[4] This process introduces point mutations into the variable regions of immunoglobulin genes at a rate of about 10^{-3} mutations per base pair per generation, enabling the selection of B cells producing higher-affinity antibodies against the inciting antigen.[2]Upon accumulating sufficient mutations, typically after 4-5 divisions, centroblasts transition to centrocytes by exiting the cell cycle, re-expressing surface immunoglobulin, and migrating to the light zone of the germinal center, where they undergo affinity-based selection by follicular dendritic cells and T follicular helper cells.[3] Successful centrocytes may differentiate into memory B cells or long-lived plasma cells, contributing to immunological memory and sustained antibody production, while unsuccessful ones undergo apoptosis.[5] This dynamic shuttling between dark and light zones, regulated by chemokine gradients and signaling pathways like those involving CD40 and BCR, ensures efficient affinity maturation and is critical for effective adaptive immunity.[4]
Definition and Morphology
Definition
A centroblast is an activated B lymphocyte at a specific stage in the germinal center reaction, characterized by rapid proliferation following antigen stimulation in the presence of T follicular helper cells.[1] These cells arise during the humoral immune response and are essential for expanding B cell clones with high-affinity antibodies.[6]Centroblasts measure 12-18 micrometers in diameter, significantly larger than naive or resting B cells, which typically range from 6-10 micrometers.[5] This size distinction reflects their activated state and metabolic demands for proliferation. They are primarily located within germinal centers of secondary lymphoid tissues.The term "centroblast" was introduced by pathologist Karl Lennert in 1969 amid studies on lymphoid follicle morphology and lymphoma classification, building on earlier 19th- and early 20th-century observations of germinal centers as proliferative sites in lymphoid organs.[7] Modern immunological understanding of centroblasts as key players in affinity maturation solidified in the decades following the 1960s, with advancements in B cell biology elucidating their role in adaptive immunity.[6]
Cellular Characteristics
Centroblasts are large lymphoid cells distinguished by their blastic morphology, featuring vesicular nuclei with one to three basophilic nucleoli typically located peripherally near the nuclear membrane, and a scant amount of amphophilic cytoplasm.[8] These morphological traits are observable under light microscopy and reflect their active proliferative state within germinal centers.[3]At the molecular level, centroblasts express high levels of key markers including the surface proteins CD38 and CD10, as well as the transcription factor BCL6, which are characteristic of germinal center B cells.[1] In contrast, they display low expression of surface immunoglobulin (sIg) and lack CD27, distinguishing them from naive and memory B cell populations.[3] These phenotypic markers facilitate identification via flow cytometry and immunohistochemistry in both normal and pathological contexts.[9]Proliferative activity in centroblasts is marked by high expression of Ki-67, indicating their engagement in the active cell division cycle.[10] This high proliferation rate underscores their role in rapid expansion within the dark zone of germinal centers.[1]Ultrastructurally, electron microscopy reveals prominent nucleoli and a predominantly euchromatic nucleus in centroblasts, consistent with their transcriptional and replicative hyperactivity.[3] The euchromatic configuration supports ongoing gene expression essential for their function, with minimal heterochromatin condensation.[11]
Development and Location
Origin from Naive B Cells
Naive B cells, residing in the follicles of secondary lymphoid organs such as lymph nodes and spleen, initiate their transformation into centroblasts upon encountering antigen presented on follicular dendritic cells or subcapsular macrophages. This activation begins with binding of the antigen to the B cell receptor (BCR), triggering intracellular signaling cascades that promote cell survival, proliferation, and upregulation of co-stimulatory molecules like CD86.30278-8) Concurrently, these activated B cells migrate from the follicle to the T-B cell border, where they interact with pre-follicular helper T cells that provide essential co-stimulatory signals, including CD40 ligand (CD40L) engagement with CD40 on B cells and cytokines such as interleukin-21 (IL-21).[12] These T cell-derived signals are critical for committing the B cells to the germinal center pathway, enhancing their expression of transcription factors like Bcl6 while suppressing alternative differentiation routes.[13]Following T cell help at the T-B border, the activated B cells undergo chemokine receptor remodeling to facilitate re-entry into the B cell follicle and seeding of nascent germinal centers. Specifically, they upregulate the receptor CXCR5 in response to CXCL13 gradients produced within follicles, while downregulating CCR7 to reduce retention in T cell zones.00114-9) This migratory shift, driven by CD40 and IL-21 signaling, positions the B cells for clustering and proliferation within the early germinal center structure.[12] The entire process from initial antigen encounter to centroblast formation typically unfolds over 3-7 days post-immunization, allowing time for selection of antigen-specific clones from a diverse naive repertoire.[14]Key molecular events preceding full centroblast identity include the initiation of signals that prime for activation-induced cytidine deaminase (AID) expression, such as elevated levels of IRF8 and early Bcl6 activity, though substantial AID protein levels and enzymatic function emerge only after germinal center entry.[15] These precursor changes support limited class-switch recombination in pre-germinal center cells but primarily prepare the B cells for the hypermutagenic environment ahead.00301-0) Once established as centroblasts, these cells rapidly proliferate within the germinal centers.[10]
Location in Germinal Centers
Centroblasts are primarily located within secondary lymphoid organs, including lymph nodes, spleen, and Peyer's patches, where they form part of the germinal centers that develop in response to antigenic stimulation.[3][16] They can also appear in tertiary lymphoid structures, which arise in non-lymphoid tissues during chronic inflammation or in association with tumors, mimicking the organization of secondary lymphoid organs.[17]Within germinal centers, centroblasts are concentrated in the darkzone, a region characterized by rapid cellular activity and a distinct compartmentalization from the lightzone.[3] This zonal distribution positions centroblasts amidst a supportive network of stromal cells, including CXCL12-producing fibroblasts that maintain the darkzone architecture, and scattered follicular helper T (Tfh) cells that contribute to the germinal center microenvironment.[18][19]Centroblasts exhibit limited direct proximity to follicular dendritic cells (FDCs), which are predominantly localized in the light zone and play a key role in antigen retention through immune complexes; this isolation contrasts with centrocytes, which interact more closely with FDCs for selection processes.[20][1] Their positioning is dynamically regulated by chemokine gradients, with high levels of CXCL12 in the dark zone promoting retention via CXCR4 expression on centroblasts, while CXCL13 gradients facilitate periodic migration toward the light zone.[21][22] This cyclic reorientation, originating from activated naive B cells, ensures ongoing adaptation within the germinal center.[18]
Function in Immune Response
Proliferation in the Dark Zone
Centroblasts in the dark zone of germinal centers undergo rapid proliferation, characterized by cell cycle times of approximately 6-8 hours, allowing one division per cycle and facilitating rapid clonal expansion.[23] This high division rate supports substantial clonal expansion, often reaching 100- to 1000-fold increases in selected clones over the course of the germinal center reaction, amplifying B cell populations for subsequent selection processes.[24] The process is confined primarily to the dark zone, where centroblasts spend most of their time in active cell cycling.Proliferation is driven by interactions between CD40 on centroblasts and CD40L expressed by follicular helper T cells, which provide essential survival and expansion signals. Cytokines such as IL-21 and BAFF further enhance this process by promoting cellsurvival and division, with IL-21 particularly critical for maintaining darkzone residency and proliferationdynamics.[25] In the darkzone, FOXO1 transcription factor activity is high, instructing a geneprogram that supports the proliferative state, including upregulation of chemokine receptor CXCR4 for zone retention; suppression of FOXO1 via PI3K signaling in the light zone inhibits proliferation to facilitate selection.[26]At the cellular level, centroblasts are predominantly in S, G2, and M phases of the cell cycle, reflecting their commitment to division.01236-5) Key regulators include cyclin D3, which drives "inertial" cycling in the dark zone to sustain rapid divisions, and E2F transcription factors, which are stabilized during mitosis to shorten G1 and accelerate S phase progression.[27] This cell cycle configuration allows concurrent somatic hypermutation during proliferation rounds.Clonal dynamics in the dark zone involve stochastic elements, where initial divisions occur without stringent affinity-based selection, permitting diversification before high-affinity clones are preferentially expanded upon return from the light zone.[24] This permissive proliferation sets the stage for affinity maturation by generating diverse progeny, with survival biases emerging later in the response.[28]
Somatic Hypermutation
Somatic hypermutation (SHM) in centroblasts is initiated by the enzyme activation-induced cytidine deaminase (AID), which deaminates cytosines to uracils in the DNA of immunoglobulin variable (IgV) regions, primarily on single-stranded DNA exposed during transcription.[29] This deamination creates a uracil:guanine mismatch that is processed by error-prone DNA repair pathways, leading to point mutations at a rate of approximately 10^{-3} to 10^{-4} mutations per base pair per generation, which is about a million times higher than the spontaneous mutation rate in B cells.[30] The process occurs exclusively in proliferating centroblasts within the germinal center dark zone, introducing nucleotide substitutions that diversify the antibody repertoire.[29]Targeting of AID is preferential to IgV regions due to their active transcription and specific sequence motifs like WRC (where W is A or T, and R is A or G), which account for 80-90% of deamination hotspots.[29] Following deamination, base excision repair (BER) or mismatch repair (MMR) pathways engage: in BER, uracil-DNA glycosylase (UNG) removes the uracil, creating an abasic site that is bypassed by error-prone translesion synthesis polymerases; in MMR, MSH2-MSH6 recognizes the mismatch and recruits error-prone polymerases such as polymerase eta (Pol η), which preferentially introduces mutations at A:T base pairs.[31] These pathways amplify mutations beyond the initial C:G sites, with Pol η playing a dominant role in A:T mutagenesis.[32]Over the proliferative phase in centroblasts, SHM introduces 10-20 mutations per IgV gene on average, resulting in both beneficial changes that enhance antibody affinity for the antigen and deleterious mutations that may impair function or contribute to off-target effects like lymphomagenesis.[30] This variability drives affinity maturation but requires subsequent selection to favor high-affinity clones. The process is tightly regulated to ensure specificity: SHM is transcription-dependent, with stalled RNA polymerase II at IgV regions facilitating AID access to single-stranded DNA; the transcription elongation factor Spt5 enhances this stalling, promoting deamination at target sites while limiting off-target activity.[33]
Differentiation and Outcomes
Transition to Centrocytes
The transition from centroblasts to centrocytes is initiated by specific molecular triggers following the proliferation phase in the germinal center dark zone. Key changes include the downregulation of the chemokine receptor CXCR4, which is essential for maintaining the centroblast phenotype and dark zone positioning. Concurrently, there is an upregulation of activation markers such as CD83 and CD86, as well as surface immunoglobulin (sIg), marking the shift toward a more antigen-responsive state.[34]This phenotypic reprogramming is coupled with altered migratory cues that direct the cells from the dark zone to the light zone. Centroblasts downregulate CXCR4 responsiveness to CXCL12, which retains them in the dark zone, and migrate toward CXCL13 via CXCR5, which is abundant in the light zone and facilitates chemotaxis toward follicular dendritic cells and T follicular helper cells. This chemokine-driven migration ensures that the post-proliferative cells relocate for antigen selection.[34][25]Accompanying these changes is a distinct phenotypic shift that defines centrocytes. These cells exhibit heightened sensitivity to apoptosis, preparing them for selection based on affinity. This transition typically occurs within 1-2 days after the proliferationphase, as part of a timed cellular program that integrates prior somatic hypermutations accumulated during the centroblast stage.[34]
Generation of Plasma and Memory Cells
Following selection in the germinal center light zone, surviving centrocytes derived from centroblasts differentiate into either plasmablasts, which mature into long-lived plasma cells, or memory B cells, thereby contributing to sustained humoral immunity. This bifurcation is governed by a transcriptional network where the balance between BLIMP1 (PRDM1) and the BCL6-BACH2 axis determines cell fate. High-affinity centrocytes receiving strong T follicular helper cell signals, including IL-21, upregulate BLIMP1, which represses BCL6 and Pax5 to promote plasmablast differentiation and antibody secretion, while BCL6 sustains germinal center identity and, in conjunction with BACH2, favors memory B cell formation by inhibiting BLIMP1 expression.[35][15][36]Plasmablasts generated through this pathway exhibit high-affinity, class-switched antibodies, predominantly IgG, IgA, or IgE, enabling effective pathogen neutralization and mucosal immunity. These cells migrate from secondary lymphoid organs to survival niches in the bone marrow, where they terminally differentiate into long-lived plasma cells supported by APRIL and BAFF, producing antibodies for years without further division. In contrast, memory B cells enter a quiescent, recirculating state, retaining somatically hypermutated B cell receptors (BCRs) that confer enhanced affinity upon re-encountering antigen, allowing rapid reactivation into plasmablasts during secondary responses.[36][15][35]Only a small fraction of the original centroblast clones—estimated at less than 10%—successfully differentiate into these effector lineages, as most undergo apoptosis during iterative selection to refine the antibody repertoire toward high affinity and diversity. This stringent efficiency ensures that the humoral response prioritizes potent, specific immunity while minimizing autoreactivity.[14][37]
Clinical and Pathological Aspects
Role in B-Cell Lymphomas
Centroblasts are implicated in the pathogenesis of B-cell lymphomas through aberrant proliferation and genetic alterations that mimic or dysregulate their normal germinal center functions. In diffuse large B-cell lymphoma (DLBCL), the most common aggressive non-Hodgkin lymphoma, the centroblastic subtype is characterized by a diffuse proliferation of large neoplastic B cells resembling centroblasts, defined morphologically as cases with fewer than 90% immunoblastic cells.[38] This subtype accounts for approximately 80% of DLBCL cases and is prevalent in the germinal center B-cell-like (GCB) molecular subtype, which constitutes about 50-60% of all DLBCL.[39][38]Pathogenic mechanisms in centroblast-like lymphomas often involve dysregulation of key transcription factors such as BCL6 and MYC. BCL6 translocations, occurring in 20-30% of GCB-DLBCL, sustain uncontrolled proliferation by repressing DNA damage response genes, mimicking the role of BCL6 in normal centroblast survival.[40] Similarly, MYC rearrangements, present in 8-14% of DLBCL, drive hyperproliferation akin to dark zone centroblasts and are frequently co-occurring with BCL2 or BCL6 alterations in double-hit lymphomas, conferring aggressive behavior.[41] Additionally, hyperactivity of activation-induced cytidine deaminase (AID), normally restricted to immunoglobulin loci in centroblasts, leads to off-target mutations in tumor suppressor genes like TP53, which are found in over 20% of DLBCL cases and associated with oncogenic transformation through double-strand breaks.[42][43]Beyond DLBCL, centroblast-like proliferation is evident in Burkitt lymphoma, where the tumor cells exhibit a transcriptional profile closely resembling that of dark zone centroblasts, with high MYC expression driving rapid division despite morphological differences in cell size.[44] In follicular lymphoma, transformation to DLBCL often results in a centroblastic morphology, with the aggressive component showing diffuse sheets of large centroblast-like cells emerging from the indolent follicular architecture.[45]The incidence of centroblast-dominant features is particularly high in GCB-DLBCL, where they correlate with the germinal center origin, but cases with prominent centroblastic morphology and concurrent MYC or TP53 alterations exhibit poorer prognosis, with 5-year survival rates dropping below 50% compared to standard GCB-DLBCL outcomes of 60-70%.[38][46]
Diagnostic and Research Implications
Centroblasts play a central role in the histopathological diagnosis of B-cell lymphomas, particularly in grading and subclassifying indolent and aggressive neoplasms originating from germinal center B cells. In follicular lymphoma (FL), the 2022 World Health Organization (WHO) 5th edition classification considers grading optional; if performed, it is based on the number of centroblasts per mm², with grades 1-2 defined by fewer than 90 centroblasts per mm² and grade 3 by more than 90 centroblasts per mm². Grade 3A features centrocytes alongside centroblasts, while grade 3B consists of sheets of centroblasts without centrocytes, influencing prognostic stratification and treatment decisions.[47] This morphological assessment, often supplemented by immunohistochemistry for markers such as CD10, BCL6, and BCL2, helps distinguish FL from reactive germinal centers and other low-grade lymphomas, though interobserver variability in centroblast counting can challenge reproducibility.[48] In diffuse large B-cell lymphoma (DLBCL), the centroblastic variant—characterized by medium-to-large cells with vesicular nuclei, fine chromatin, and multiple nucleoli resembling normal centroblasts—represents the most common subtype and is identified through routine histology and immunophenotyping, aiding in germinal center B-cell (GCB) versus activated B-cell (ABC) subtype assignment via algorithms like Hans or COO classifiers, which carry distinct prognostic implications.[49][50]Beyond morphology, centroblast identification informs molecular diagnostics in lymphomas, where high centroblast content correlates with somatic hypermutation patterns and genetic aberrations like BCL2 rearrangements in FL or MYC/BCL2 double-hit status in high-grade cases, guiding targeted therapies such as rituximab or BTK inhibitors.[51] Automated image analysis tools are emerging to standardize centroblast quantification, reducing subjectivity in FL grading and potentially improving diagnostic accuracy in resource-limited settings.[52]In research, centroblasts serve as a critical model for elucidating B-cell biology, particularly the mechanisms of affinity maturation and clonal selection within germinal centers. Studies using single-cell RNA sequencing have revealed transcriptional heterogeneity among centroblasts, highlighting dynamic shifts in proliferation, DNA repair, and apoptosis genes that underpin somatic hypermutation driven by activation-induced cytidine deaminase (AID), with implications for understanding vaccine-induced humoral responses.[53][10] CXCR4-based sorting enables isolation of pure centroblast populations from human tonsils, facilitating functional assays that dissect their role in dark zone proliferation and transition to centrocytes, as CXCR4 deficiency impairs dark zone access and clonal competition.[54] This has advanced insights into germinal center dynamics, including c-MYC regulation of survival and the impact of T follicular helper cells on centroblast maintenance, informing strategies to enhance memory B-cell generation in immunotherapy and autoimmunity models.[55][56]