When human tonsil-derived fibroblasts were pushed into a senescent state—the arrested, inflammatory condition that accumulates with age—they lost the ability to support B cell proliferation and instead actively suppressed it, even while the B cells displayed the molecular hallmarks of activation. The research, conducted entirely in vitro using human cells, builds on earlier work showing that the ageing immune system is undermined not only by intrinsic defects in lymphocytes but by the microenvironment that surrounds them. Previous heterochronic parabiosis experiments in mice, in which young and old animals share a circulatory system, revealed that aged lymph nodes fail to support germinal centre formation even when populated by young T and B cells.

Follicular dendritic cells, a specialised fibroblast type that coordinates the movement and localisation of T and B cells within germinal centres and acts as an antigen library to sustain the response, showed impaired activation and expansion in aged mice, alongside reduced expression of the B cell-attracting chemokine CXCL13 and of adhesion molecules and Fc and complement receptors essential for B cell communication. What remained unknown was whether senescence within lymphoid fibroblasts could directly impair B cell function. To address this, the team used YK6 fibroblasts, an immortalised human tonsil-derived cell line engineered to express the B cell-supporting molecules CD40L and BAFF.

The researchers first confirmed that these cells resemble the B zone-supportive fibroblasts, follicular dendritic cells and marginal reticular cells found in human lymph nodes rather than T zone fibroblastic reticular cells, although they lack several canonical follicular dendritic cell markers such as CR1, L1CAM, CD32b and CD23. In co-culture experiments, unmodified YK6 fibroblasts enhanced B cell activation: naive B cells cultured alongside them proliferated and upregulated the activation markers CD69 and ICAM-1 even without additional stimulation. When the co-cultures were embedded in a three-dimensional Matrigel matrix, which prevented the fibroblasts from overgrowing the culture, the supportive effect became clearer still, with significant increases in the proportion of ICAM-1-positive B cells under both low- and high-stimulation conditions.

Five days of treatment with bleomycin, a DNA-damaging agent, induced a robust senescence programme in YK6 cells: they stained positive for senescence-associated beta-galactosidase, enlarged and became more granular, lost Ki67 expression indicating cell cycle arrest, and upregulated the senescence genes CDKN1A and IL6. Bulk RNA sequencing confirmed the shift, with more than 18,000 genes differentially expressed, strong downregulation of cell cycle pathways such as E2F targets and the G2M checkpoint, and enrichment of the SenMayo senescence gene set alongside multiple immune and inflammatory pathways. Notably, when the researchers compared these data with existing sequencing of marginal reticular cells from young and aged mouse lymph nodes, the aged mouse cells were also enriched for the SenMayo gene set and showed upregulation of key senescence-associated genes including Il6, Cdkn1a and Igfbp7, suggesting the bleomycin model captures at least some features of aged lymphoid fibroblasts.

When B cells were co-cultured with these senescent fibroblasts, the effect was dramatic. In three-dimensional cultures, B cell proliferation was essentially abrogated under both low- and high-stimulation conditions, despite the B cells showing largely normal activation. CD69 and ICAM-1 expression indicated that the cells had responded to their stimulatory cocktail, and measurements taken 48 hours after stimulation, before any cell division, confirmed comparable activation between control and senescent co-cultures. The B cells were not failing to activate; they were failing to divide. The authors noted that CD69 expression is division-linked, being highest at early divisions, which helped reconcile the subtle differences in activation marker patterns observed after four days of culture.

Dissecting the mechanism, the researchers collected conditioned media from senescent fibroblasts and cultured B cells in it. The conditioned media produced only a modest reduction in proliferation—B cells typically lagged one division peak behind controls—far short of the complete abrogation seen in direct co-culture. The team also ruled out a senescence bystander effect, showing that exposure to senescent conditioned media induced only subtle senescence-like changes in fresh fibroblasts, much smaller than those caused by direct bleomycin treatment. Together, these findings indicate that surface-expressed proteins on senescent fibroblasts are the major driver of the B cell blockade, with secreted factors making a secondary contribution.

Transcriptomic and flow cytometric analysis of the senescent fibroblasts revealed a telling shift in their surface repertoire. Expression of the engineered CD40L and BAFF transgenes fell, and BAFF secretion into the culture media declined. At the same time, the senescent cells upregulated transcripts for ICAM1, CD274 (PD-L1), CD74, TNFRSF14 (HVEM) and PDCD1LG2 (PD-L2), while the ectonucleotidase CD73, encoded by NT5E, was slightly reduced. ICAM-1 protein increased at the cell surface, detectable as early as 24 hours after bleomycin treatment, suggesting it is a direct response to DNA damage. Several of these molecules—PD-L1, PD-L2, HVEM—are ligands for receptors known to restrain B cell proliferation, including PD-1 and BTLA. The authors speculate that heightened ICAM-1 may physically tether B cells to the fibroblasts, prolonging their exposure to these negative regulators; in senescent co-cultures, B cells were observed sitting along fibroblast processes rather than forming the classic proliferative clusters typical of responding lymphocytes.

The metabolic consequences for the B cells were equally revealing. B cells co-cultured with control fibroblasts readily upregulated the glucose importer GLUT1 and the amino acid transporter CD98, hallmarks of the metabolic rewiring that fuels proliferation. Those cultured with senescent fibroblasts were impaired in their GLUT1 upregulation, although CD98 induction was preserved. Using the SCENITH assay, which measures protein synthesis through puromycin incorporation while pharmacologically separating dependence on glycolysis and oxidative phosphorylation, the researchers found that activated, undivided B cells in both conditions were almost entirely reliant on glycolysis. Yet protein synthesis was significantly lower in the senescent co-cultures across all metabolic conditions. The result is a portrait of failed activation: B cells that have committed to glycolysis but cannot translate proteins or progress through the cell cycle—a state the authors liken to the proliferative unresponsiveness seen in chronic lymphocytic leukaemia.

The authors are careful to delineate the limits of the model. YK6 fibroblasts lack key follicular dendritic cell markers such as CR1 and FcγRIIB, and the overlap between bleomycin-induced senescence and aged mouse lymph node fibroblasts was limited outside the shared SenMayo enrichment, partly reflecting low cell numbers and species differences. Because no aged human lymph node fibroblast sequencing dataset exists, the relevance to human ageing remains an open question. Even so, the study establishes a hitherto unexpected principle: fibroblast-intrinsic senescence can directly suppress human B cell proliferation through contact-dependent mechanisms, offering a plausible route by which the ageing lymphoid microenvironment contributes to weakened vaccine responses.

Subject of Research: Contact-dependent inhibition of human B cell proliferation by senescent lymphoid fibroblasts

Article Title: Senescent Fibroblasts Impair B Cell Proliferation via Cell–Cell Contact

Article References: Vezyrgianni, K., MacLeod, E., Nutley, A., Zhu, Y., & Denton, A. E. (2026). Senescent Fibroblasts Impair B Cell Proliferation via Cell–Cell Contact. Aging Cell, 25(10), Article e70766. https://doi.org/10.1111/acel.70766

Image Credits: AI Generated

DOI: 10.1111/acel.70766

Keywords: B cells, cellular senescence, fibroblasts, follicular dendritic cells, germinal centre, ageing, immunosenescence, vaccine response, glycolysis, ICAM-1, PD-L1, in vitro co-culture