Sublethal DNA Damage Switches Off B-Cell Effector Programs in an RA-FLS- PBMC Co-culture

Sublethal DNA Damage Switches Off B-Cell Effector Programs in an RA-FLS- PBMC Co-culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sublethal DNA Damage Switches Off B-Cell Effector Programs in an RA-FLS- PBMC Co-culture Denada Bruci, Torsten Lowin, Gerhard Fritz, Georg Pongratz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7912272/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2026 Read the published version in Cell Death Discovery → Version 1 posted 9 You are reading this latest preprint version Abstract Rheumatoid arthritis (RA) features lymphocyte-driven inflammation in which B cells, alongside T cells, play key effector roles (autoantibody production, antigen presentation, cytokines, or chemokines). Within B cells, during normal diversification, activation-induced cytidine deaminase (AID) introduces targeted DNA lesions in immunoglobulin loci (class-switch recombination/somatic hypermutation), creating a potential vulnerability to sublethal genotoxic stress. T cells also contribute to RA pathogenesis through cytokine production and cell-mediated responses, and are exposed to similar genotoxic stressors in the inflamed joint environment. Given this, we asked whether a single, sublethal insult can modulate lymphocyte effector function without overt cytotoxicity. Peripheral blood mononuclear cells from healthy donors were co-cultured with RA fibroblast-like synoviocytes and exposed once to an IC₂₀ or IC₅₀ concentration of γ-irradiation (γ-IR), hydrogen peroxide (H₂O₂), or the oxazaphosphorine metabolite 4-hydroperoxyifosfamide (4-OOH IFA). Viability, γ-H2AX kinetics, cell cycle status, cytokine and immunoglobulin secretion, and a 28-gene damage response/differentiation panel were quantified at either 24 hours or 5 days. Together, the data indicate that a single, carefully titrated low-concentration genotoxic hit can selectively suppress lymphocyte effector programs, with B cells being more durably affected than T cells. At 2 Gy, overall cell viability remained above 80%, whereas IL-10 expression declined by approximately 70%, demonstrating functional silencing in the absence of substantial cytotoxicity. Targeting this vulnerability may selectively dampen pathogenic B cell activity in RA while sparing overall immune viability and T-cell competence. Biological sciences/Immunology/Cell death and immune response Biological sciences/Drug discovery/Toxicology Health sciences/Diseases/Rheumatic diseases/Rheumatoid arthritis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease that afflicts ~ 0.5–1% of the world’s population and ranks among the leading causes of disability. ( 1 ). Pathologically, it is characterised by synovial hyperplasia, dense leukocyte infiltration, and progressive destruction of cartilage and bone ( 2 ). Although T cell help is indispensable for disease induction, longitudinal histology, serum biomarker studies, and treatment responses now suggest that B cells are pivotal amplifiers of chronic inflammation ( 3 , 4 ). Mature B cells accumulate in ectopic germinal centre-like clusters within RA synovium, secrete rheumatoid factor and anti-citrullinated protein antibodies, present antigen to T cells, and elaborate cytokines such as IL-6 and TNF-α ( 5 – 7 ). Depletion with the anti-CD20 antibody rituximab validates their clinical relevance; yet, one-third of patients remain refractory, and the risk of infection remains a concern ( 8 , 9 ). Consequently, strategies that functionally silence rather than eliminate pathogenic B cells have become an attractive research avenue. B cells are genetically unique because they engineer their genome; activation-induced cytidine deaminase (AID) deaminates cytosine during somatic hypermutation and class switch recombination, creating programmed double-strand breaks (DSBs) ( 10 , 11 ). This inherent genotoxic burden sensitizes B cells to DNA damage, as assessed by the Ser139-phosphorylated form of the histone variant H2AX (γ-H2AX), a marker of DNA damage ( 12 ). Indeed, γ-H2AX-positive B cell clusters are abundant in RA synovia and correlate with disease activity ( 13 , 14 ). Moreover, the inflamed joint milieu is a rich source of reactive oxygen species (ROS), reactive nitrogen intermediates, and is often exposed to low-dose ionizing radiation from diagnostic imaging, all of which are potential amplifiers of DNA damage ( 15 – 17 ). However, whether sublethal lesions alone can recalibrate B cell effector programmes without wholescale cytotoxicity remains unresolved. To explore that question, we employed a human co-culture model of fibroblast-like synoviocytes (FLS) from RA patients and peripheral blood mononuclear cells (PBMCs) from healthy donors. FLS provide contact-dependent and soluble factors that mimic the RA synovial microenvironment ( 18 , 19 ). We delivered a single, well-defined concentration (IC 20 or IC 50 ) of three mechanistically distinct genotoxins that are clinically or pathophysiologically relevant to RA: γ-irradiation (γ-IR) used in radiosynoviorthesis and encountered in diagnostic imaging, induces clustered DSBs repaired mainly by Ku-dependent non-homologous end joining (NHEJ) ( 20 , 21 ). Secondly, we used hydrogen peroxide (H₂O₂), a diffusible reactive oxygen species (ROS) generated not only by activated neutrophils and macrophages in RA joints but also endogenously by many cell types in inflammatory microenvironments ( 22 ). It causes base oxidation, single-strand breaks, and secondary DSBs requiring mixed base excision repair (BER), NHEJ, and homologous recombination (HR) repair ( 23 ). Lastly, 4-hydroperoxyifosfamide (4-OOH IFA), the active metabolite of the alkylator ifosfamide, structurally related to cyclophosphamide, which may be a necessary therapeutic approach in RA patients with interstitial lung disease or other B-cell dependent autoimmune disease, like systemic lupus erythematosus, forms among other DNA damages, interstrand cross-links that demand time-consuming homologous recombination (HR) ( 24 , 25 ). Functional, molecular, and viability endpoints were analysed to determine how genotoxic stress modulates B cell and T cell effector programs within an RA-relevant microenvironment. These insights enabled us to determine the extent to which sublethal genotoxic stress may attenuate B and T-cell effector programs while preserving overall viability. 2. Materials and Methods 1. Cell Culture and Co-culture Setup Fibroblast-like synoviocytes (FLS) were isolated from synovial tissue of patients with RA after informed consent and ethics approval (Study-Nr.: 2022–2189_7) ( 26 ). Cells were expanded to passage 3–5 and seeded at 1 × 10⁵ cells per well in 48-well plates. Cultures were maintained in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% GlutaMAX (Thermo Fisher Scientific), 1% sodium pyruvate (Thermo Fisher Scientific), 1% penicillin–streptomycin (Thermo Fisher Scientific), and 10 mM HEPES (Thermo Fisher Scientific) at 37°C, 5% CO₂. After 24 hours, peripheral blood mononuclear cells (PBMCs) from healthy donors were added. PBMCs were isolated by Lymphoprep density-gradient centrifugation (Progen), washed, counted, and added to RA-FLS at a 1:5 ratio (1 RA-FLS:5 PBMCs) in the same medium. 2. Genotoxic challenge and stimulation scheme Co-cultures or only PBMCs were treated with the following reagents: PBMC only with γ-IR (0–4 Gy) delivered using the Gammacell 1000 Elite radiation machine (Nordion International, and the PBMC/FLS co-cultures with 4-OOH IFA (0–34 µM) obtained from Niomech IIT GmbH, and H₂O₂ (0-160 µM) purchased from Sigma-Aldrich. After 24 hours, half the wells were harvested (acute end-point). The remaining wells were washed and stimulated with CpG ODN 2006 (5 µg/ mL; Invivogen) before further incubation until day 8. The complete treatment scheme is shown below in Supplementary Figure S1 . All compounds in this study are listed in Supplementary Table S1 . 3. Cell viability Assay Viability was assessed by Annexin V–FITC/PI staining (Miltenyi Biotec) per the manufacturer’s protocol and analysed on a CytoFLEX LX flow cytometer (Beckman Coulter) at 24 h and 8 d post-treatment (Supplementary Fig. S2 for gating). To determine sublethal working concentrations, co-cultures received graded γ-IR (0–4 Gy), 4-OOH IFA (0–34 µM), or H₂O₂ (0–160 µM). Four-parameter logistic regression yielded relative IC₅₀ values (Supplementary Table S2 ). From the same fits, IC₂₀ values at 24 h were approximately 0.1 Gy (γ-IR), 3.5 µM (4-OOH IFA), and 6 µM (H₂O₂). Unless stated otherwise, IC₅₀/IC₂₀ refer to these relative values (concentrations reducing viability to 50%/80% of vehicle-treated controls), i.e., sublethal benchmarks. All compounds and antibodies used in this study are listed in Supplementary Table S1 . 4. Cytokine and Immunoglobulin Quantification by ELISA ELISA kits were purchased from R&D Systems for cytokines and chemokines (e.g., IL-10, IFN-γ, APRIL, CD25/IL-2 Ra, IL-2) and StemCell Technologies for immunoglobulins (IgM, IgG, IgA, IgE). Assays were performed according to the manufacturers' protocols. Absorbance was measured at 450 nm using a Tecan Infinite M200 Pro microplate reader (Tecan), and the data were analysed using Microplate Manager software (Bio-Rad). Cytokine and immunoglobulin values are expressed as a percentage of the CpG-stimulated, but otherwise untreated control and are plotted against the corresponding day 8 viability obtained from the same wells. All used kits are found in Supplementary Table S1 . 5. Flow-cytometric assays Common preparation. Cells were detached with Accutase (10 min, 37°C), washed in FACS buffer (PBS, 3% FBS, 2 mM EDTA), blocked with FcR reagent (1:5, Miltenyi; 15 min,4 o C), and stained with surface markers (20 min, RT). Lineages were defined using: CD19 eFluor 405 (Invitrogen; 1:20), CD4-PerCP/Vio700 (1:50), CD8-VioGreen (1:50), CD27-VioBright R720 (1:50), and CD3-PE/Vio770 (1:50). Cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific; 15 min, RT) and permeabilised in 0.1% Triton X-100 (Thermo Fisher Scientific; 10 min, RT), then stained for intracellular targets in PBS with 0.05% Tween-20 and 3% FBS. Data were acquired on a CytoFLEX LX (Beckman Coulter) and analysed in FlowJo v10.8.1. All compounds and antibodies used in this study are listed in Supplementary Table S1 . 5.1 γ-H2AX Assay for DNA Damage After surface staining, fixation, and permeabilization, cells were then incubated with anti-γ-H2AX-Alexa Fluor 488 (BD Biosciences, 1:20) for 1 hour, RT. It was followed by a washing step and then resuspended in FACS buffer. Gating strategy is outlined in Supplementary Figure S3 . 5.2. Ki-67 and PI Cell Cycle Assay After permeabilization, cells were incubated with Ki-67-FITC (Miltenyi Biotec, 1:50) for 20 minutes at room temperature in the dark. They were then counterstained with PI (propidium iodide, Miltenyi Biotec, 1:100) for 20 minutes at room temperature in the dark. Gating strategy is outlined in Supplementary Figure S4 . All compounds and antibodies used in this study are listed in Supplementary Table S1 6. Real-Time Quantitative PCR (RT-qPCR) Total RNA was extracted from co-cultured cells or RA-FLS only, at 24 hours or 5 days post-treatment using the NucleoSpin RNA isolation kit (Macherey-Nagel) following the manufacturer’s instructions. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed with SYBR Green Master Mix (Applied Biosystems) to quantify the expression of BCL-6, BAFF, AICDA, and other target genes. 18S rRNA was used as the reference gene, and relative gene expression was calculated using the ΔΔCt method. The primer sequences are given in Supplementary Table S2 . Note: RA-FLS are not antibody-producing cells; immunoglobulin and AICDA/GC-factor panels are included to document baseline/indirect transcriptional effects in the stromal compartment. 7. Statistical Analysis Data are expressed as mean ± standard error of the mean (SEM). For viability, DNA damage, cell cycle, cytokine/chemokine/immunoglobulin (day 8), and RT-qPCR analyses, we used repeated measures one-way ANOVA with Dunnett’s post hoc test to account for the paired and matched nature of samples derived from the same donors across multiple conditions. Significance thresholds: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). Analyses were performed in GraphPad Prism 8. All experiments used independent donor material. The language and grammar of this manuscript were checked using AI language editing assistance, with all content, data interpretation, and conclusions generated solely by the authors. 3. Results Differential short and sustained loss of viability in RA-FLS/healthy PBMC co-cultures after a single γ-IR, 4-OOH IFA, or H₂O₂ challenge. To characterize exposure response and temporal effects, we quantified viability at 24 hours and day 8 in RA-FLS/healthy PBMC co-cultures following a single graded exposure to γ-IR (Gy), 4-OOH IFA, or H₂O₂. γ-IR induced a monotonic decline in viability up to 2 Gy at 24 hours, with an estimated IC₅₀ of 0.4 Gy; viability stabilized at higher doses (Fig. 1 A, Supplementary Table S1 ). Despite inter-donor variability limiting post hoc significance, overall ANOVA confirmed a treatment effect. By day 8, both variance and viability further declined (IC₅₀ ≈ 0.9 Gy), but the concentration-response did not change beyond 2 Gy, suggesting stable cytotoxicity among surviving cells. RA-FLS monocultures remained fully viable as they were not irradiated. 4-OOH IFA exposure resulted in the most pronounced loss of viability. Statistically significant reductions began at 10 µM (IC₅₀, 14 µM), and were amplified by day 8 with IC₅₀ ≈ 7 µM (Fig. 1 B, Supplementary Table S1 ). High concentrations also impaired fibroblast viability (RA-FLS IC₅₀ = 7.7 µM), though FLS were generally more resistant than PBMCs. H₂O₂ yielded intermediate effects: a concentration-dependent viability loss was observed from 10 µM upward at 24 hours (IC₅₀, 23 µM), but viability partially recovered by day 8 (IC₅₀, 60 µM). RA-FLS monocultures were unaffected by H₂O₂ (Fig. 1 C, Supplementary Table S1 ). Overall, γ-IR and H₂O₂ caused moderate, delayed cytotoxicity in PBMC/FLS co-cultures, whereas 4-OOH IFA induced rapid and sustained cell loss, including partial toxicity to FLS at high concentrations. These distinct profiles supported the selection of IC₂₀ and IC₅₀ concentrations for functional and mechanistic assays. Day 8 was prioritized for effector readouts to capture RA-FLS/PBMC crosstalk and stable mediator secretion, minimizing the influence of transient early effects. Single hit genotoxic stress uncouples lymphokine and immunoglobulin production from cell survival. To evaluate functional consequences independent of early time points, we measured day-8 concentration–response production of IL-10, IFN-γ, IL-2, sCD25/IL-2Rα, APRIL, and IgG/IgA/IgM across γ-IR, 4-OOH IFA, and H₂O₂ treatment. We then used Spearman correlation analyses to relate these effector outputs to corresponding viability measurements, clarifying the relationship between cell survival and functional changes. Following γ-IR exposure, IL-10 and IFN-γ secretion were substantially reduced at concentrations that preserved cell viability, with both cytokines suppressed by approximately 70% at 2 Gy, where 8-day viability remained ≥ 80% (Fig. 2 A, D). IgG and IgA levels were diminished to 40–50%, while APRIL, sCD25, and IgM decreased to 50–60% of CpG-stimulated controls at 2 Gy, (Fig. 3 A, D, G; Supplementary Fig. 5A, G). IL-2 was reduced by 60% at 1.5 Gy recovering at higher doses. (Supplementary Fig. 5D). Spearman correlation analysis revealed positive associations between cell viability and IL-10, IgG, IgM, and sCD25. In contrast, IgA, IL-2, and APRIL secretion did not correlate with viability, indicating partial decoupling of specific mediators from cell survival (Supplementary Table S4 ). Treatment 4-OOH IFA elicited an abrupt shutdown of cytokine production, with IL-10 and IFN-γ reduced by over 50% at 5 µM, despite ∼70% viable cells (Fig. 2 B, E). At 10 µM, IgG and IgA concentrations were reduced to 30% of control, accompanied by ∼60% viability (Fig. 3 E, H). Reductions in APRIL and IgM approached 40%, CD25 declined to 70%, and IL-2 to 20% of control (Fig. 3 B, Supplementary Fig. 5B, E, H). All immune effectors determined under 4-OOH IFA treatment were positively correlated with cell viability (Supplementary Table S4 ). H₂O₂ induced a gradual suppression of function, with cell viability maintained above 90% up to 50 µM. IL-10 decreased by about 30% at 25 µM and transiently converged with the viability curve at 20 µM before separating at higher doses (Fig. 2 C). IFN-γ remained consistently below the viability trajectory for the entire concentration range (Fig. 2 F). IgG and IgA were first inhibited at 25 µM without concurrent viability loss; parallel declines in both antibody secretion and viability emerged only at concentrations ≥ 75 µM (Fig. 3 F, I). In contrast, IgM and CD25 each dropped to 40–50% of control at 5 µM, APRIL declined by 60% at 40 µM, and IL-2 was suppressed by 20% at the lowest concentration tested (Fig. 3 C, Supplementary Fig. S5 C, F, I). Correlations between cell viability and IgG, IgA, and IgM weakened under H₂O₂, whereas CD25 and APRIL retained a positive association; IL-2 secretion was uncoupled from viability (Supplementary Table S4 ). In summary, sublethal γ-IR, 4-OOH IFA, or H₂O₂ exposures consistently suppressed immunoglobulin output and accessory factor release most notably the release of IL-2, a cytokine mainly secreted by T cells that promotes their proliferation and survival. This pattern indicates a DNA damage checkpoint silencing immune effector programs at non-cytotoxic concentrations, prompting mechanistic investigation of γ-H2AX accrual in distinct lymphocyte subsets. Kinetics of γ-H2AX accumulation reveal lineage-specific DNA damage responses at sublethal and half-lethal concentrations After establishing IC₂₀ and IC₅₀ concentrations from viability assays and observing durable suppression of cytokine and immunoglobulin secretion, we next investigated DNA damage responses in selected lymphocyte subsets by assessing γ-H2AX accumulation following exposure to γ-IR, 4-OOH IFA, or H₂O₂. At [IC₂₀] (sublethal), γ-IR produced a rapid increase in γ-H2AX across all investigated cell populations, peaking at 2 hours and remaining elevated above control at 24 hours, indicating incomplete repair (Fig. 4 A, Supplementary Figure S6 -A). CD4⁺, CD8⁺ T cells, and memory B cells reached 6-7-fold above baseline, while naïve B cells increased to 10-fold at 24 hours. 4-OOH IFA produced a gradual increase in y-H2AX, reaching up to 6-fold by 24 hours (Fig. 4 B, Supplementary Figure S6 -B), and H₂O₂ elicited a modest early increase with a secondary peak at 8–16 hours, stabilizing at ~ 4-fold by 24 hours (Fig. 4 C, Supplementary Figure S6 -C). At [IC₅₀], similar cell specific patterns were observed but with higher amplitudes. γ-IR triggered 2-hour γ-H2AX peaks of 25-fold in CD4⁺, 15-fold in CD8⁺, 20-fold in memory B cells, and 9-10-fold in naïve B cells, decreasing by half at later timepoints except in naïve B cells, where the signal persisted (Fig. 4 D, Supplementary Figure S6 -D). 4-OOH IFA reached comparable levels at 24 hours (Fig. 4 E, Supplementary Figure S6 -E). H₂O₂ showed biphasic kinetics, y-H2AX peaking at 4-6-fold at 8 hours and declining to 2-3-fold by 16 hours (Fig. 4 F, Supplementary Figure S6 -F). Adjusted p-values for all cell types/time points are reported in supplementary table S5 . Basal γ-H2AX MFI levels were higher in naïve and memory B cells compared to CD4⁺ and CD8⁺ T cells (Supplementary Figure S7 ), with significant fluctuations in B cell subsets over time even without treatment. All treated sample data were normalized to time-matched controls to enable accurate intra-lineage comparisons. Comparisons of γ-H2AX MFI across treatments at both [IC₂₀] and [IC₅₀] (Supplementary Figure S8 ) confirmed strongest early signals with γ-IR, especially at [IC₅₀]. These analyses demonstrate that DNA damage signalling is shaped by both lesion-specific chemistry and cell-intrinsic factors, influencing the dynamics and magnitude of repair responses. DNA damage checkpoints translate into lineage-specific cell cycle blockade in B cells Distinct γ-H2AX signalling among lymphoid lineages suggests differential checkpoint engagement with functional consequences. To assess whether these lineage-specific signals affect cell-cycle progression, we analysed distributions in CD4⁺ and CD8⁺ T cells, as well as naïve and memory B cells, 24 and 48 hours following a single sublethal (IC₂₀) or IC 50 challenge with γ-IR, 4-OOH IFA, or H₂O₂. This links γ-H2AX burden to cell-cycle inhibition and proliferation arrest. Ki-67-FITC/PI staining showed that at 24 hours post-IC₂₀ γ-IR, memory B cells shifted out of quiescence into G₁, increasing from 18% ± 2% to 64% ± 6%; naïve B cells similarly accumulated in G₁ increasing from 4% ± 1% to 21% ± 4% (Fig. 5 A), while IC₅₀ γ-IR produced no significant change. For 4-OOH IFA at [IC₅₀], memory B cells showed G₁ increase from 14% ± 3% to 46% ± 5% and S-G₂-M transition from 0% to 5% ± 1%, with naïve B cells demonstrating modest G₁ and S-G₂-M enrichment (Fig. 5 B). H₂O₂ at [IC₂₀] induced the strongest arrest: memory B cells redistributed into G₁ from 15% ± 2% to 55% ± 5% and S-G₂-M from 0% to 10% ± 2%, while naïve B cells increased G₁ to 33% ± 4% at [IC₂₀] and 23% ± 3% at [IC₅₀] (Fig. 5 C). After 24 hours of CpG-ODN2006 stimulation (48 h total treatment), naïve B cells in all treatment groups reverted to quiescent status (G₀ >85%, G₁ < 10%). Memory B cells retained treatment-specific blocks: γ-IR IC₂₀ concentration G₁ stayed at 75% ± 6% versus controls: 15% ± 2%, S-G₂-M dropped from 3% ± 1% to 0% (Fig. 5 D). 4-OOH IFA [IC₅₀] memory G₁ persisted at 65% ± 5%, S–G₂–M at 7% ± 1%. H₂O₂ [IC₂₀] memory G₁ was 62% ± 4%, S-G₂-M at 9% ± 2%, and [IC₅₀] memory G₁ at 34% ± 3%, despite maintained viability. CD4⁺ and CD8⁺ T cells showed only transient G₁ increases (γ-IR IC₂₀) or S-phase spikes (H₂O₂ [IC₅₀]) at 24 hours, both resolving by 48 hours (Supplementary Figure S9 ). These findings demonstrate that DNA damage-induced checkpoints establish durable, lineage-specific cell cycle arrest, especially in memory B cells, likely contributing to selective impairment in immune cell proliferation and function. Transcriptional Responses to DNA Damage in PBMC/RA-FLS Co-cultures and RA-FLS Monocultures To associate γ-H2AX and cell-cycle checkpoint data with the functional readouts, we profiled 28 transcripts in PBMC/RA-FLS co-cultures and RA-FLS monocultures (supplementary figure S10 ) by RT-qPCR, collecting RNA at 24 hours after [IC₂₀] or [IC₅₀] genotoxic challenge, and again on day 5 for differentiation markers. At [IC₂₀], all stressors triggered a robust sensor burst in co-culture: ATM, APEX1, XRCC6, and XRCC5 increased by 20-30-fold after γ-IR and 4-OOH IFA, and 15-20-fold after H₂O₂ (Fig. 6 A-C). At [IC₅₀], for γ-IR, the four sensors returned to baseline, whereas 4-OOH IFA and H₂O₂ maintained strong sensor upregulation (10-25-fold). RAD51 but not RAD50 was upregulated 4-6-fold at [IC₂₀] and [IC₅₀] for 4-OOH IFA and at IC₂₀ for γ-IR and H₂O₂ (Supplementary Figure S10 ). RA-FLS monocultures showed limited transcriptional changes: H₂O₂ selectively increased XRCC5 at 24 hours, and 4-OOH IFA elevated XRCC5, RAD50, and RAD51 levels (Supplementary Figure S11 ). TP53 transcript was only slightly reduced at 4-OOH IFA [IC₂₀] and H 2 O 2 [IC 50 ], while CDKN1A/P21 did not change. BAX increased 3-fold at γ-IR [IC₅₀], 2-fold at 4-OOH IFA [IC₅₀], but decreased at H₂O₂ [IC₅₀] (Fig. 6 D-F). FAS did not change, while FASLG showed specific upregulation with each agent. BCL-2 was enhanced 2-fold at γ-IR IC₅₀ and H₂O₂ IC₂₀ doses but declined at H₂O₂ [IC₅₀]. BRCA1, BRCA2, and PDCD1 were unchanged; BCL-6 increased 600-700-fold under all treatments at IC 20 and IC 50 doses/concentrations. BAFF was upregulated by 10-fold at both doses of γ-IR and at 4-OOH IFA by 4-fold at [IC 20 ] and [IC 50 ]with a more limited increase for H₂O₂ at [IC₂₀] (Supplementary Figure S10 ). In RA-FLS, H₂O₂ [IC₂₀] significantly induced BCL-6, BAX, and TP53; 4-OOH IFA [IC₅₀] elevated FAS, FASLG, and PDCD1 levels, while BCL-6 and BAX were upregulated at [IC₂₀]. BRCA2 and TP53 increased after [IC₂₀] (Supplementary Figure S10 ). These data reveal treatment-specific transcriptomic responses in DNA damage sensors, repair genes, and apoptotic regulators, underlying the observed functional impairments under genotoxic stress. Early blockade and late persistence of the B cell differentiation program To relate the functional loss of Ig secretion to transcriptional checkpoints, we quantified canonical differentiation and plasma cell genes in PBMC/RA-FLS co-cultures at 24 hours and day 5 post-IC₂₀ or IC₅₀ pulse, focusing on B cell genes due to the clear impact on Ig secretion, and did not profile T cell differentiation genes in this study. Twenty-four hours after exposure, all three stressors triggered distinct but overlapping bursts in B cell program genes. γ-IR induced > 200-fold rises in XBP1 and IRF4 (both at [IC₂₀] and [IC₅₀]), and BACH2, PAX5 (at [IC₂₀]), with AICDA strongly repressed under both concentrations (Fig. 7 A-C). 4-OOH IFA drove all five factors above 200-fold at [IC₂₀] and maintained high levels except PRDM1 at [IC₅₀]; AICDA was unchanged. H₂O₂ largely mirrored γ-IR for XBP1, IRF4, and PAX5, with BACH2 steeply elevated only at [IC₂₀]. PRDM1 was repressed at [IC₅₀], and AICDA was only reduced by γ-IR (Fig. 7 A-C). Five days after the single stressor exposure, expression profiles diverged. After γ-IR, XBP1, IRF4, and PAX5 remained ≥ 200-fold [(IC₂₀]) and ~ 300-fold ([IC₅₀]); PRDM1 persisted at [IC₂₀]; BACH2 returned to baseline, AICDA remained high to > 200-fold. With 4-OOH IFA, only PRDM1 and AICDA stayed elevated; others were reduced to almost baseline levels. For H₂O₂, PRDM1 remained high at [IC₂₀], IRF4 showed modest increase ([IC₅₀]), AICDA surpassed 500-fold, others stayed near baseline (Fig. 7 D-F). In RA-FLS alone (24h), B-cell differentiation genes showed modest, limited changes (Supplementary Fig. S11 ). Gene panels five days after challenge linked germinal center survival signals and class switch recombination with heavy chain transcription (Fig. 7 G-I). γ-IR (at IC₂₀/IC₅₀ doses) increased BCL-6, BAFF, and µ heavy chain ~ 200–300-fold. IgG and IgA rose sharply only at IC₂₀, not at IC₅₀. For 4-OOH IFA, BAFF transcripts increased at both concentrations, IgM dropped at [IC₂₀] but rebounded at [IC₅₀], and IgG and IgA increased five-fold. H₂O₂ enhanced transcription of BAFF and all heavy chains (4-200-fold), with BCL-6 unchanged. This suggests differentiation blockade and lingering transcriptional shifts, rather than full germinal centre output. These late timepoint data reveal that different genotoxins and y-IR uniquely reactivate B cell effector gene expression, suggesting varied impacts on functional recovery after damage. 4. Discussion This study delineates how a single genotoxic stressor from DNA damage to checkpoint activation impacts immune cell function, especially memory B cells, as shown by y-H2AX and cell cycle analysis in RA-FLS/healthy PBMC co-cultures. By integrating viability, γ-H2AX kinetics, cell cycle, and transcriptomic analyses, we show that γ-IR, 4-OOH IFA, and H₂O₂ exert distinct effects across immune lineages, especially memory B cells, which seem uniquely sensitive to sustained DNA damage signalling and functional shutdown. While classical DNA damage signalling in lymphocytes usually involves ATM and p53 activation, our co-culture system induced ATM but not p53, suggesting established DNA damage models may not fully apply in mixed cultures. ( 27 , 28 ). 4.1 Early versus sustained cytotoxicity: how stressor type sets the tone Each genotoxic stressor induced a distinct viability trajectory in the RA-FLS/healthy PBMC co-culture. γ-IR produced only modest early death, yet induces a delayed attrition. In contrast, 4-OOH IFA caused the steepest and most sustained loss of PBMC viability. H₂O₂ had an intermediate effect between these extremes. Early viability dropped, but partial recovery by day 8, which might suggest the selection or adaptation of ROS-tolerant cells, consistent with the transcriptional upregulation of antioxidant programs seen in other models ( 29 – 31 ). The stromal compartment behaved differently. In our design, RA-FLS were not irradiated, so no inference about radioresistance can be drawn. Under 4-OOH IFA, RA-FLS monocultures retained substantially higher viability than PBMC-rich co-cultures at matched concentrations but did show impairment at the higher concentrations, indicating relative but not absolute resilience. Using H₂O₂, RA-FLS were largely unaffected across the tested concentrations. Therefore, PBMCs are the primary cytotoxic target of all three stressors, whereas FLS are comparatively spared by H₂O₂ and only partly affected by high-concentration 4-OOH IFA, consistent with previous studies demonstrating the resilience of fibroblast-like synoviocytes to oxidative damage ( 32 ). 4.2 Functional silencing precedes cell death: A transcriptional checkpoint Across all three stressors, functional silencing of IL-10, IFN-γ, IgG, and IgA routinely occurred before overt cytotoxicity, suggesting regulation at a transcriptional checkpoint while cells remained viable. This pattern fits an established DNA damage response hierarchy, in which ATM sensor activation triggers chromatin tightening and transient repression of differentiation programs (AICDA-PRDM1-XBP1 axis), prioritizing repair over specialized immune functions ( 33 , 34 ). Our findings support this mechanism, as robust induction of ATM and associated sensors occurred rapidly with only modest changes in apoptotic genes and cell cycle effectors ( 33 , 34 ). However, positive correlations between immune effector production and viability also indicate a relationship with cell death, and thus cannot, on their own, exclude a model in which protein loss simply results from cytotoxicity. Indeed, the inverse correlation between cell survival and effector loss could reflect either checkpoint-regulated suppression or progressive cell death. The most rigorous demonstration of independent checkpoint regulation would require finding a factor positively associated with cell death or specifically upregulated in surviving cells post-stress a criterion that DNA repair factors could fulfill if de novo synthesis coincides with increased cell death. Our transcriptomic data show that ATM, APEX1, XRCC6, and XRCC5 are rapidly upregulated after stress, matching this expectation ( 35 – 37 ). Agent-specific chemistries further shape the depth and breadth of functional silencing. γ-IR activates ATM signalling, leading to moderate and sustained suppression of gene expression, while 4-OOH IFA likely induces robust ATR-CHK1 response and uniform transcriptional slowdown, and H₂O₂ evokes mixed base damage mechanisms. Notably, APRIL and IL-2 demonstrate regulation independent of viability, implicating additional pathways (e.g., NF-κB, AP-1, STATs) beyond ATM-driven checkpoints ( 38 – 40 ). Antibody isotypes also showed distinct regulation. IgG and IgA were more sensitive to early shutdown than IgM, consistent with the metabolic demands of class-switch recombination (CSR), dependent on AID and ER/UPR programs tightly monitored by the DNA damage response. Basal IgM production, being less energetically demanding, persisted under stress, while CSR and high-level antibody output were selectively suppressed. These patterns corroborate prior findings and reinforce a model of stress-dependent transcriptional throttling ( 41 – 45 ). Given that these class-switched isotypes predominantly arise from memory B cells, and our γ-H2AX and cell cycle data substantiate vulnerability in this subset, we infer that the observed isotype suppression under genotoxic stress might be driven primarily by memory B cell checkpoint engagement. However, as naïve B cells can also become activated and undergo class-switch recombination under CpG stimulation, we cannot fully exclude their contribution to the observed effects. Quantifying the ratio of reactivated memory versus newly activated naïve B cells would be required to substantiate this inference, which therefore remains interpretive but central to our model ( 46 , 47 ). Overall, robust sensor induction and selective effector suppression at sublethal exposures, in the absence of strong apoptotic signals, argue most convincingly for early checkpoint regulation as the principal mechanism, rather than cell death per se. This aligns with increasing evidence that DDR pathways can directly downregulate immune effector gene expression via ATM/ATR signalling before apoptosis is initiated ( 48 ). While our study cannot disambiguate single-cell checkpoint effects from population-level coordination, the observed association between repair factor induction and cell death strengthens the case for differential regulation in this coculture setting, mimicking the situation in inflamed RA joints. 4.3 Lesion kinetics and cell cycle checkpoint responses Our kinetic analyses revealed stressor-specific γ-H2AX and cell cycle checkpoint engagement, with prolonged and synchronous activation specifically demonstrated in memory B cells. γ-IR drove a rapid and synchronous γ-H2AX peak across lymphoid lineages, with T cells displaying the highest early activation and swift resolution, whereas naïve B cells exhibited sustained γ-H2AX at late timepoints, suggesting slower repair capacity. In contrast, 4-OOH IFA produced a delayed, monotonic γ-H2AX increase, consistent with interstrand crosslink damage and prolonged checkpoint activation. H₂O₂ triggered a biphasic γ-H2AX response, resulting in mixed and transient cell cycle checkpoint patterns ( 49 , 50 ). Notably, 4-OOH IFA's prolonged checkpoint engagement, reflected by delayed RAD51 induction and persistent γ-H2AX in memory B cells, coincided with selective loss of antibody outputs and differentiation markers ( 51 , 52 ). H₂O₂, by contrast, allowed functional recovery following checkpoint resolution. These findings highlight a nuanced picture: memory B cells are uniquely vulnerable to prolonged checkpoint activation and functional suppression under genotoxic conditions, a central finding of our study. Although an initial DNA damage checkpoint attenuates antibody secretion, oxidative lesions appear to be repaired or tolerated sufficiently allowing CpG stimulation to activate the secretory program, including class-switch output at later time points ( 53 , 54 ). This transient, stress-adapted phenotype aligns with prior studies showing that efficient repair of oxidative DNA damage enables rapid B-cell recovery and renewed antibody production (Mori et al., 2009). Similar observations have also been reported by Valverde et al. (2018), who demonstrated that cells exposed to sublethal oxidative stress upregulate DNA repair pathways and undergo adaptive responses enabling genomic protection and functional recovery ( 53 , 54 ). Some limitations need to be discussed. Direct measurement of DNA lesion spectra, repair kinetics, and protein-level validation in disease-relevant, single-cell assays would strengthen mechanistic conclusions. Our approach, leveraging population-level analyses in pathophysiologically relevant co-cultures, nonetheless demonstrates coordinated DNA damage checkpoint engagement and immune suppression. The inclusion of PBMC co-cultures introduces the potential for paracrine effects but more closely reflects in vivo immune-stromal interactions, an essential consideration for RA pathogenesis. In summary, our findings show that sublethal, type-dependent genotoxic stress can selectively engage DNA damage checkpoints in memory B cells while largely preserving overall cell viability in a physiologically relevant PBMC co-culture. By characterizing these coordinated responses, this study contributes to a clearer understanding of how genotoxic stress affects immune function, and establishes a foundation for future investigations in disease contexts and more refined cell populations. 5. Conclusion In summary, we delineate a coherent sequence linking DNA damage response dynamics, measured by γ-H2AX kinetics and associated sensor gene expression, to a cell cycle checkpoint engagement and subsequent transcriptional alterations that explain how DNA damage can selectively recalibrate B cell function. These findings might provide a rationale for precision, low-concentration genotoxic modulation of target cell in autoimmune diseases, suggesting that a brief exposure to an alkylator or radiotherapeutic agent might silence pathogenic B cells while leaving stromal fibroblasts and most T cells intact. Mapping this cascade provides a rational basis for coupling genotoxic agents with targeted immune modulation in rheumatoid arthritis and related B-cell-driven disorders. Abbreviations RA- Rheumatoid Arthritis RA-FLS — rheumatoid-arthritis fibroblast-like synoviocytes PBMC(s) — peripheral blood mononuclear cell(s) γ-IR — gamma irradiation 4-OOH IFA — 4-hydroperoxyifosfamide H₂O₂ — hydrogen peroxide IC₂₀ / IC₅₀ — concentration causing 20%/50% reduction in viability or function γ-H2AX — phosphorylated histone H2AX (Ser139) MFI — median fluorescence intensity CpG-ODN2006 — CpG oligodeoxynucleotide 2006 NHEJ / HR / BER — non-homologous end joining / homologous recombination / base-excision repair Ser139-Serine 139 Declarations Ethics statements: Studies involving animal subjects No animal studies are presented in this manuscript. Studies involving human subjects: The studies involving humans were approved by the local ethics committee and conducted according to the ethical principles outlined in the Declaration of Helsinki. Written informed consent has been obtained from the patients to publish this paper. The studies were conducted in accordance with local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Author Contributions : Conceptualization, D.B., G.P., T.L., and G.F., methodology, D.B. G.P., T.L., and G.F, validation, D.B., investigation, data curation, D.B., writing original draft preparation, D.B., G.P., T.L., and G.F., . All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—417677437/GRK2578. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare that they have no conflicts of interest. 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On “day 0,” freshly isolated healthy-donor PBMCs were added to RA-FLS at a 1:5 (FLS:PBMC) ratio, and genotoxic stress was applied as follows: for the alkylator and oxidant arms, 4-OOH ifosfamide or H₂O₂ (at concentrations spanning the empirically determined [IC₂₀] and [IC₅₀]) was added directly to the whole co-culture; for the irradiation arm, PBMCs were γ-irradiated separately and then immediately transferred onto RA-FLS (RA-FLS were not irradiated). DNA-damage signalling (γ-H2AX, flow cytometry) was profiled between 0 and 24 hours post-treatment (2, 8, 16, 24 hours). At 24 hours, cells were harvested for the first RT-qPCR panel (damage sensors/repair and early B-cell differentiation genes) and for Ki-67-FITC/PI cell-cycle analysis. Immediately after the 24 hours read-outs, cultures were restimulated with CpG-ODN2006; a second Ki-67-FITC/PI measurement was acquired 24 hours later (i.e., 48 hours from treatment). A delayed RT-qPCR harvest was performed on day 5 to capture slower transcriptional programmes, and supernatants were collected on day 8 for ELISA (cytokines and immunoglobulins). All measurements were normalised to time-matched untreated controls; additional details (exact concentrations, replicates, and statistics) are provided in the Methods. SupplementaryFigureS2.tif Supplementary Figure S2: Flow cytometry gating strategy for viability assays (illustrative, no statistics). (i) FSCA × SSCA to select the total leukocyte population and exclude debris, (ii) FSCH × FSCA doublet discrimination gate, (iii) Annexin V-FITC /PI gates were used to quantify live (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), and late apoptotic (Annexin V+/PI+), and necrotic cells (Annexin V-/PI+), in RA-FLS/healthy PBMC co-cultures. The “Viable” population (green gate) provided the percentage values plotted in central Figure 1. SupplementaryFigureS3.tif Supplementary Figure S3: Flow cytometry gating strategies used in this study: Panel A γ-H2AX assay. Left-to-right: (i) FSCA × SSCA to select the total leukocyte population and exclude debris, (ii) FSCH × FSCA doublet discrimination gate, (iii) lineage cocktail gate separating CD4⁺ T cells, CD8⁺ T cells, naïve B cells (CD19⁺ CD27⁻), and memory B-cells (CD19⁺ CD27⁺), (iv) histogram overlay of γ-H2AX- Alexa 488 median fluorescence intensity (MFI) for each lineage at the indicated concentration and time. SupplementaryFigureS4.tif Supplementary Figure S4: Panel B Ki-67-FITC / propidium iodide (PI) cell cycle assay (i) FSC/SSC and doublet exclusion as in Panel A, (ii) FSCH × FSCA doublet discrimination gate, (iii) lineage cocktail gate separating CD4⁺ T cells, CD8⁺ T cells, Naïve B cells (CD19⁺ CD27⁻), and Memory B cells (CD19⁺ CD27⁺), (iv) bivariate plot of Ki-67-FITC versus PI fluorescence with quadrant gates defining G₀ (Ki67⁻ / 2N DNA), G₁ (Ki67⁺ / 2N), and S–G₂/M (Ki-67 variable / >2N) fractions. A debris exclusion polygon was drawn on the PI area versus the PI width to remove subG₀ events. Gates were applied identically to all treatment groups and timepoints. Data were acquired on a CytoFLEX LX (Beckman Coulter) and analysed in FlowJo v10.9. SupplementaryFigureS5.tif Supplementary Figure S5: Extended cytokine/chemokine panel concentration–response curves.Eight-day post-treatment supernatant concentrations (red) APRIL ( A-C), IL-2 (D-F), and soluble CD25/IL-2 Ra (G-I), outputs, plotted against the matched day 8 viability (black) from Fig. 1 for γ-IR (A, D, G, ), 4-OOH IFA (B, E, H, ), and H₂O₂ (C, F, I, ). Values are expressed as % of CpG-stimulated untreated control. Spearman correlations with viability are summarized in Supplementary Table S2. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. n (biological replicates): PBMC/RA-FLS, N=3, n = 2 SupplementaryFigureS6.tif Supplementary Figure S6: Log-scaled γ-H2AX kinetics for each lineage and stressor. IC₂₀ (A-C) and IC₅₀ (D-F) time courses (2, 8, 16, 24 h) of log (γ-H2AX median fluorescence intensity (MFI) in gated CD4⁺ T (red), CD8⁺ T (blue), Memory B (CD19⁺CD27⁺) (green), and Naïve B (CD19⁺CD27⁻) (black) cells after (A, D) γ-IR, (B, E) 4-OOH IFA, or (C, F) H₂O₂. MFI is normalized to the time-matched untreated control. These panels complement central Figure 4 by displaying the same data on a log 10 axis to emphasise peak spreading and convergence. Gating strategy: Supplementary Fig. S4; Statistics (given in Supplementary Table S3): repeated -measures one-way ANOVA with Dunnett’s post-hoc vs time-matched control at each timepoint. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. SupplementaryFigureS7.tif Supplementary Figure S7: Absolute y-H2AX MFI values for control untreated samples.(A) time courses (2, 8, 16, 24 h) of absolute γ-H2AX median fluorescence intensity (MFI) in gated CD4⁺ T (red), CD8⁺ T (blue), Memory B (CD19⁺CD27⁺) (green), and Naïve B (CD19⁺CD27⁻)(black) control untreated cells. (B) Bar graphs absolute show median fluorescence intensity (MFI) of phosphorylated histone H2AX (Ser139) in CD4⁺ T cells, CD8⁺ T cells, memory (CD19⁺ CD27⁺) B cells, and naïve (CD19⁺ CD27⁻) B cells 0-24hours after treatment. Data are mean ± SEM. Statistics: repeated -measures one-way ANOVA within each lineage followed by Dunnett’s post-hoc test versus control ( * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns, not significant). SupplementaryFigureS8.tif Supplementary Figure S8: Comparison of γH2AX DNA-damage signal for each treatment within immune subsets (0-24 hours) . Bar graphs show median fluorescence intensity (MFI) of phosphorylated histone H2AX (Ser139) in CD4⁺ T cells, CD8⁺ T cells, memory (CD19⁺ CD27⁺) B cells, and naïve (CD19⁺ CD27⁻) B cells 0-24hours after treatment, normalised to the time-matched untreated control. For each lineage, bars represent the indicated treatment(s) at the viability-matched concentrations ([IC₂₀] and/or [IC₅₀]) defined in Fig. 1, with values normalized to the time-matched untreated control. Data are mean ± SEM. Statistics: repeated -measures one-way ANOVA within each lineage followed by Dunnett’s post-hoc test versus control (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns, not significant). SupplementaryFigureS9.tif Supplementary Figure S9: CD4⁺ and CD8⁺ T-cell Ki-67-FITC /PI profiles. Analogous to Figure 5, the transient and fully reversible checkpoints in T-cell subsets are shown. Ki-67-FITC/Propidium iodide (PI) profiles at 24 hours after the single [IC₂₀] or [IC₅₀] pulse (A-C) and 48 hours (i.e., 24 hours after CpG restimulation, D-F). For each time point, the three stressors are shown side by side: γ-IR (A, D), 4-OOH IFA (B, E), and H₂O₂ (C-F). Within each stressor, the left-hand bars represent CD4 T cells, and the right-hand bars represent CD8 T cells. Stacked columns display mean ± SEM percentages of cells in G₀ (blue), G₁ (green) and S–G₂–M (red) phases. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. SupplementaryFigureS10.tif Supplementary Figure S10: DNA-damage/ checkpoint regulators and B cell survival factors at 24 h by RT-qPCR. Bar charts show fold change (ΔΔCt) mRNA relative to time-matched untreated control, measured 24 hour after a single IC₂₀ or IC₅₀ concentration of γ-IR (A, D) , 4-OOH IFA (B,E) , or H₂O₂ (D,F) (mean ± SEM). Upper row (A–C): RAD50, RAD51, TP53, CDKN1A (p21)Lower row (D–F): BRCA1, BRCA2, PDCD1, BCL-6, and BAFF. It complements the regulator panel in Figure 6. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS :N=3, n = 2. SupplementaryFigureS11.tif Supplementary Figure S11: RA-FLS monocultures: 24 h RT-qPCR profile across gene modules after oxidative or alkylating stress . RA-FLS monocultures (“FLS only”, no PBMCs) were exposed once to H₂O₂ (A–D) or 4-OOH ifosfamide (4-OOH IFA) (E–H) at the indicated concentrations (matching the nominal [IC₂₀]/[IC₅₀] levels defined in Fig. 1 for the co-culture). Bars show mean ± SEM, N = 3 independent RA-FLS donors. Panel map (gene grouping by pathway): A, E): DNA damage sensors/repair: ATM, APEX1, XRCC6 (Ku70), XRCC5 (Ku80), RAD50, RAD51. B, F) Late cell differentiation effector readout/apoptosis (survival/BAFF axis and heavy-chain transcripts): BCL6, BAFF, FAS,FASL,PCDC1,IgM,IgG,IgA,IgE (no results of the Igs). C) G-cell cycle checkpoint/apoptosis/co-inhibitory: TP53, CDKN1A (p21), BAX, BCL2 BRCA1, BRCA2.D, H) B-cell differentiation cassette (reported here in FLS as off-target/trans-effects controls): BACH2, PAX5, IRF4, PRDM1, XBP1, AICDA. Statistics were performed within each panel using repeated -measures one-way ANOVA with Dunnett’s post-hoc test versus the untreated control: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ***p ≤ 0.0001; ns, not significant. 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1","display":"","copyAsset":false,"role":"figure","size":2400085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-concentration genotoxic stress produces distinct short- and long-term viability phenotypes in PBMC/RA-FLS co-culture. \u003c/strong\u003eConcentration response of viable cells (Annexin V-FITC/PI) at 24 h (black) and day 8 (red) after a single exposure to (A) γ-irradiation (0–4 Gy), (B) 4-OOH IFA (0–34 µM), or (C) H₂O₂ (0–160 µM). For (A–C), RA-FLS- untreated (A) and treated (B-C) monoculture controls are shown (blue). Dots are mean ± SEM; each donor contributed one measurement per concentration. Values are expressed as % of the untreated control. Four-parameter logistic fits defined IC₂₀ and IC₅₀ concentrations. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant. PBMC/RA-FLS, N=9, n = 1, RA-FLS monoculture, N = 3, n=1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/c627a78cf5d86eebcf251cc7.png"},{"id":96249415,"identity":"e85f2704-1d2e-4a1b-be39-00b1388d7d0a","added_by":"auto","created_at":"2025-11-19 07:33:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1249873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSublethal DNA damage suppresses IL-10 and IFN-γ secretion more strongly than it reduces cell survival. \u003c/strong\u003eEight-day post-treatment supernatant concentrations (red) of IL-10 (A-C) and IFN-γ (D-F) plotted against the matched day 8 viability (black) from Fig. 1 for γ-IR (A, D), 4-OOH IFA (B, E), and H₂O₂ (C, F). Values are expressed as % of CpG-stimulated untreated control. Spearman correlations with viability are summarized in Supplementary Table S2. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS: N=3, n = 3 .\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/4f979d6407767d53602ddcad.png"},{"id":96110760,"identity":"07df46e2-a0a7-428b-a0d2-93a8f4f4113d","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1373323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of sublethal genotoxic stress on antibody secretion and cell viability.\u003c/strong\u003e Eight-day post-treatment supernatant concentrations (red) vs IgM (A-C), IgG (D-F) and IgA (G-I) plotted against the matched day 8 viability (black) from Fig. 1 for γ-IR (A, D,G), 4-OOH IFA (B, E,H), and H₂O₂ (C, F, I). Values are expressed as % of CpG-stimulated untreated control. Spearman correlations with viability are summarized in Supplementary Table S2. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS : N=3, n = 3.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/ae1d62d5cb5e35b39cfee4e3.png"},{"id":96250348,"identity":"80cc7882-7e0e-4c2d-8e13-b08e28185614","added_by":"auto","created_at":"2025-11-19 07:38:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1378381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eγ-H2AX kinetics reveal lineage-specific DNA damage burdens at IC₂₀ versus IC₅₀.\u003cbr\u003e\n\u003c/strong\u003eIC₂₀ (A-C) and IC₅₀ (D-F) time courses (2, 8, 16, 24 hours) of γ-H2AX median fluorescence intensity (MFI) in gated CD4⁺ T, CD8⁺ T, Memory B (CD19⁺CD27⁺), and Naïve B (CD19⁺CD27⁻) cells after (A, D) γ-IR, (B, E) 4-OOH IFA, or (C, F) H₂O₂. MFI is normalized to the time-matched untreated control. Gating strategy: Supplementary Fig. S4; Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs time-matched control at each timepoint. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/fb834b60a9379bfe07f377af.png"},{"id":96250221,"identity":"5595fcc2-45dd-4a08-87ac-1947d92a5693","added_by":"auto","created_at":"2025-11-19 07:37:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1339058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSublethal genotoxic stress imposes distinct cell cycle checkpoints in memory versus naïve B cells.\u003c/strong\u003e Ki-67-FITC/Propidium iodide (PI) profiles at 24 hours after the single IC₂₀ or IC₅₀ pulse (A-C) and 48 hours (i.e., 24 hours after CpG stimulation, D-F). For each time point, the three stressors are shown side by side: γ-IR (A, D), 4-OOH IFA (B,E), and H₂O₂ (C-F). Within each stressor, the left-hand bars represent memory B cells, and the right-hand bars represent naïve B cells. Stacked columns display mean ± SEM percentages of cells in G₀ (blue), G₁ (green) and S–G₂–M (red) phases . Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/885051ddad48cb5128e57c26.png"},{"id":96247195,"identity":"32a3a076-b8e9-4626-8498-a138ebed81d3","added_by":"auto","created_at":"2025-11-19 07:27:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1418241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly transcriptional wiring of the damage response at 24 hours.\u003c/strong\u003eBar charts show fold change (ΔΔCt) mRNA relative to time-matched untreated control, measured 24 hours after a single IC₂₀ or IC₅₀ concentration of γ-IR (A, D) , 4-OOH IFA (B, E) , or H₂O₂ (D, F) (mean ± SEM). Upper row (A–C): DNA damage sensors and repair genes (ATM, APEX1, XRCC6, XRCC5). Lower row (D–F): apoptosis regulators (FAS/FASLG, (BAX, BCL-2). Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS : N=3, n = 2.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/c6ca70b87e28d6b877f078d0.png"},{"id":96110767,"identity":"f28678da-5952-45fa-8a6c-8c7f6cfbfe4e","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1563688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of B cell differentiation, survival genes and heavy chain transcripts\u003c/strong\u003e.Bar charts show fold change (ΔΔCt) mRNA relative to time-matched untreated control, measured 24 hour or 5 days after a single IC₂₀ or IC₅₀ concentration of γ-IR (A, D, G), 4-OOH IFA (B, E, H) , or H₂O₂ (D, F, I) (mean ± SEM). First and second row: BACH2, XBP1, IRF4, PAX5, PRDM1, and AICDA at 24 hour (A-C) and 5 days (D-F) after IC₂₀ or IC₅₀ concentrations of γ-IR, 4-OOH IFA, or H₂O₂. Third row: \u0026nbsp;BCL-6, BAFF, IgM, IgG, and IgA at day 5 (G-I) following IC₂₀ or IC₅₀ of γ-IR, 4-OOH IFA, or H₂O₂. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS : N=3, n = 2.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/ee46c3ffa5e2f0cb0ff8acda.png"},{"id":96110792,"identity":"0e81db74-92bb-4d50-9de3-459f982cc968","added_by":"auto","created_at":"2025-11-17 17:08:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":924754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of the study: Panels A–C trace the time-ordered molecular and cellular responses to (A) γ-irradiation (γ-IR), (B) 4-hydroperoxyifosfamide (4-OOH IFA), and (C) hydrogen peroxide (H₂O₂).\u003c/strong\u003e Each pathway begins with damage sensing, marked by γ-H2AX and ATM induction, followed by engagement of repair programs example (APEX1, XRCC5/6). By day 2, cells activate cell cycle checkpoints and arrest in G₁. Transcriptional programs rebound by day 5, with upregulation of differentiation regulators (IRF4, XBP1, PAX5) and immunoglobulin genes. By day 8, functional silencing emerges, evidenced by reduced cytokine secretion (e.g., IL-10, IFN-γ). M.B cells stands for Memory B cells. The inset panel illustrates a proposed clinical application. It suggests that a precisely titrated, low-dose genotoxic exposure might dampen pathogenic B-cell activity while sparing stromal fibroblasts and T-cell compartments, suggesting a therapeutic avenue for rheumatoid arthritis and other B cell–driven conditions. Together, these data outline a testable framework for optimizing genotoxic immunomodulation. Figure 8 was created using BioRender , used with permission.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/8df41adc2c6e21a3a2f62090.png"},{"id":105884277,"identity":"7f6d6a97-aba9-4a83-b4b6-a7aba22b989e","added_by":"auto","created_at":"2026-04-01 07:12:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13119960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/e64b5758-04d9-43f3-8428-8ecaa52e42fa.pdf"},{"id":96248820,"identity":"9f3a2ff3-07ff-4175-8a4b-df639cae61d7","added_by":"auto","created_at":"2025-11-19 07:29:23","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":597842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. Experimental timeline and sampling scheme. \u003c/strong\u003eRA-FLS were seeded 24 h before treatment (“day −1”). On “day 0,” freshly isolated healthy-donor PBMCs were added to RA-FLS at a 1:5 (FLS:PBMC) ratio, and genotoxic stress was applied as follows: for the alkylator and oxidant arms, 4-OOH ifosfamide or H₂O₂ (at concentrations spanning the empirically determined [IC₂₀] and [IC₅₀]) was added directly to the whole co-culture; for the irradiation arm, PBMCs were γ-irradiated separately and then immediately transferred onto RA-FLS (RA-FLS were not irradiated). DNA-damage signalling (γ-H2AX, flow cytometry) was profiled between 0 and 24 hours post-treatment (2, 8, 16, 24 hours). At 24 hours, cells were harvested for the first RT-qPCR panel (damage sensors/repair and early B-cell differentiation genes) and for Ki-67-FITC/PI cell-cycle analysis. Immediately after the 24 hours read-outs, cultures were restimulated with CpG-ODN2006; a second Ki-67-FITC/PI measurement was acquired 24 hours later (i.e., 48 hours from treatment). A delayed RT-qPCR harvest was performed on day 5 to capture slower transcriptional programmes, and supernatants were collected on day 8 for ELISA (cytokines and immunoglobulins). All measurements were normalised to time-matched untreated controls; additional details (exact concentrations, replicates, and statistics) are provided in the Methods.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/2cf269f769b1e34e09ff2905.tif"},{"id":96110781,"identity":"157cc933-f2eb-48f7-8c0d-57986ccc8b77","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2451592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S2: Flow cytometry gating strategy for viability assays (illustrative, no statistics).\u003c/strong\u003e (i) FSCA × SSCA to select the total leukocyte population and exclude debris, (ii) FSCH × FSCA doublet discrimination gate, (iii) Annexin V-FITC /PI gates were used to quantify live (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), and late apoptotic (Annexin V+/PI+), and necrotic cells (Annexin V-/PI+), in RA-FLS/healthy PBMC co-cultures. The “Viable” population (green gate) provided the percentage values plotted in central Figure 1.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/c7917cc24e8858caf803a255.tif"},{"id":96248909,"identity":"642e1d49-73ff-4abc-8b0b-1d8646d9dab5","added_by":"auto","created_at":"2025-11-19 07:29:39","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1432552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S3: Flow cytometry gating strategies used in this study:\u003c/strong\u003e Panel A γ-H2AX assay. Left-to-right: (i) FSCA × SSCA to select the total leukocyte population and exclude debris, (ii) FSCH × FSCA doublet discrimination gate, (iii) lineage cocktail gate separating CD4⁺ T cells, CD8⁺ T cells, naïve B cells (CD19⁺ CD27⁻), and memory B-cells (CD19⁺ CD27⁺), (iv) histogram overlay of γ-H2AX- Alexa 488 median fluorescence intensity (MFI) for each lineage at the indicated concentration and time.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/df39e3ac30e66a5a8a9f1521.tif"},{"id":96249365,"identity":"66026a62-d74d-4b43-a6e9-622d6fe0dd5d","added_by":"auto","created_at":"2025-11-19 07:33:12","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1805680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S4: Panel B Ki-67-FITC / propidium iodide (PI) cell cycle assay \u003c/strong\u003e(i) FSC/SSC and doublet exclusion as in Panel A, (ii) FSCH × FSCA doublet discrimination gate, (iii) lineage cocktail gate separating CD4⁺ T cells, CD8⁺ T cells, Naïve B cells (CD19⁺ CD27⁻), and Memory B cells (CD19⁺ CD27⁺), (iv) bivariate plot of Ki-67-FITC versus PI fluorescence with quadrant gates defining G₀ (Ki67⁻ / 2N DNA), G₁ (Ki67⁺ / 2N), and S–G₂/M (Ki-67 variable / \u0026gt;2N) fractions. A debris exclusion polygon was drawn on the PI area versus the PI width to remove subG₀ events. Gates were applied identically to all treatment groups and timepoints. Data were acquired on a CytoFLEX LX (Beckman Coulter) and analysed in FlowJo v10.9.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/0e421be17b20d8bf4de6df14.tif"},{"id":96110776,"identity":"5d2fc134-9f0c-4f99-b862-d00799e9acc8","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1011444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S5: Extended cytokine/chemokine panel concentration–response curves\u003c/strong\u003e.Eight-day post-treatment supernatant concentrations (red) APRIL ( A-C), IL-2 (D-F), and soluble CD25/IL-2 Ra (G-I), outputs, plotted against the matched day 8 viability (black) from Fig. 1 for γ-IR (A, D, G, ), 4-OOH IFA (B, E, H, ), and H₂O₂ (C, F, I, ). Values are expressed as % of CpG-stimulated untreated control. Spearman correlations with viability are summarized in Supplementary Table S2. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. n (biological replicates): PBMC/RA-FLS, N=3, n = 2\u003c/p\u003e","description":"","filename":"SupplementaryFigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/45669ead39213a57dba58644.tif"},{"id":96247202,"identity":"c15ad6e0-2860-4d45-9e27-82041e0054bb","added_by":"auto","created_at":"2025-11-19 07:27:14","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":888218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S6: Log-scaled γ-H2AX kinetics for each lineage and stressor.\u003c/strong\u003e IC₂₀ (A-C) and IC₅₀ (D-F) time courses (2, 8, 16, 24 h) of log (γ-H2AX median fluorescence intensity (MFI) in gated CD4⁺ T (red), CD8⁺ T (blue), Memory B (CD19⁺CD27⁺) (green), and Naïve B (CD19⁺CD27⁻) (black) cells after (A, D) γ-IR, (B, E) 4-OOH IFA, or (C, F) H₂O₂. MFI is normalized to the time-matched untreated control. These panels complement central Figure 4 by displaying the same data on a log\u003csub\u003e10\u003c/sub\u003e axis to emphasise peak spreading and convergence. Gating strategy: Supplementary Fig. S4; Statistics (given in Supplementary Table S3): repeated -measures one-way ANOVA with Dunnett’s post-hoc vs time-matched control at each timepoint. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/747542d22b27ab6952601b8c.tif"},{"id":96248495,"identity":"e56821a7-e029-4fd0-827b-deb518d8b812","added_by":"auto","created_at":"2025-11-19 07:28:31","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":767814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S7: Absolute y-H2AX MFI values for control untreated samples\u003c/strong\u003e.(A) time courses (2, 8, 16, 24 h) of absolute γ-H2AX median fluorescence intensity (MFI) in gated CD4⁺ T (red), CD8⁺ T (blue), Memory B (CD19⁺CD27⁺) (green), and Naïve B (CD19⁺CD27⁻)(black) control untreated cells. (B) Bar graphs absolute show median fluorescence intensity (MFI) of phosphorylated histone H2AX (Ser139) in CD4⁺ T cells, CD8⁺ T cells, memory (CD19⁺ CD27⁺) B cells, and naïve (CD19⁺ CD27⁻) B cells 0-24hours after treatment. Data are mean ± SEM. Statistics: repeated -measures one-way ANOVA within each lineage followed by Dunnett’s post-hoc test versus control ( * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"SupplementaryFigureS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/2c8febbdc2994d962b0a4147.tif"},{"id":96248534,"identity":"21c1b4b9-3c14-43d9-9e2b-f7fe4818f806","added_by":"auto","created_at":"2025-11-19 07:28:35","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":736420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S8: Comparison of γH2AX DNA-damage signal for each treatment within immune subsets (0-24 hours) .\u003c/strong\u003e Bar graphs show median fluorescence intensity (MFI) of phosphorylated histone H2AX (Ser139) in CD4⁺ T cells, CD8⁺ T cells, memory (CD19⁺ CD27⁺) B cells, and naïve (CD19⁺ CD27⁻) B cells 0-24hours after treatment, normalised to the time-matched untreated control. For each lineage, bars represent the indicated treatment(s) at the viability-matched concentrations ([IC₂₀] and/or [IC₅₀]) defined in Fig. 1, with values normalized to the time-matched untreated control. Data are mean ± SEM. Statistics: repeated -measures one-way ANOVA within each lineage followed by Dunnett’s post-hoc test versus control (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns, not significant).\u003c/p\u003e","description":"","filename":"SupplementaryFigureS8.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/13928dd567795e49ec9acd70.tif"},{"id":96110774,"identity":"99355022-b931-43f9-8ceb-300192f07a87","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":813334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S9: CD4⁺ and CD8⁺ T-cell Ki-67-FITC /PI profiles.\u003cbr\u003e\n \u003c/strong\u003eAnalogous to Figure 5, the transient and fully reversible checkpoints in T-cell subsets are shown. Ki-67-FITC/Propidium iodide (PI) profiles at 24 hours after the single [IC₂₀] or [IC₅₀] pulse (A-C) and 48 hours (i.e., 24 hours after CpG restimulation, D-F). For each time point, the three stressors are shown side by side: γ-IR (A, D), 4-OOH IFA (B, E), and H₂O₂ (C-F). Within each stressor, the left-hand bars represent CD4 T cells, and the right-hand bars represent CD8 T cells. Stacked columns display mean ± SEM percentages of cells in G₀ (blue), G₁ (green) and S–G₂–M (red) phases. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs untreated control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS9.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/4b42e0e2f53561260d074b33.tif"},{"id":96110777,"identity":"93c80f21-0c35-4aba-84a7-b37e2315f5be","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":902134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S10: DNA-damage/ checkpoint regulators and B cell survival factors at 24 h by RT-qPCR.\u003c/strong\u003e Bar charts show fold change (ΔΔCt) mRNA relative to time-matched untreated control, measured 24 hour after a single IC₂₀ or IC₅₀ concentration of γ-IR (A, D) , 4-OOH IFA (B,E) , or H₂O₂ (D,F) (mean ± SEM). Upper row (A–C): RAD50, RAD51, TP53, CDKN1A (p21)Lower row (D–F): BRCA1, BRCA2, PDCD1, BCL-6, and BAFF. It complements the regulator panel in Figure 6. Statistics: repeated -measures one-way ANOVA with Dunnett’s post-hoc vs control. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. PBMC/RA-FLS :N=3, n = 2.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS10.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/da1895f2d3ca6c077e622c52.tif"},{"id":96110783,"identity":"b8064375-e570-4ed2-a8ce-55eb931cbd92","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":917360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S11: RA-FLS monocultures: 24 h RT-qPCR profile across gene modules after oxidative or alkylating stress\u003c/strong\u003e . RA-FLS monocultures (“FLS only”, no PBMCs) were exposed once to H₂O₂ (A–D) or 4-OOH ifosfamide (4-OOH IFA) (E–H) at the indicated concentrations (matching the nominal [IC₂₀]/[IC₅₀] levels defined in Fig. 1 for the co-culture). Bars show mean ± SEM, N = 3 independent RA-FLS donors. Panel map (gene grouping by pathway): A, E): DNA damage sensors/repair: ATM, APEX1, XRCC6 (Ku70), XRCC5 (Ku80), RAD50, RAD51. B, F) Late cell differentiation effector readout/apoptosis (survival/BAFF axis and heavy-chain transcripts): BCL6, BAFF, FAS,FASL,PCDC1,IgM,IgG,IgA,IgE (no results of the Igs). C) G-cell cycle checkpoint/apoptosis/co-inhibitory: TP53, CDKN1A (p21), BAX, BCL2 BRCA1, BRCA2.D, H) B-cell differentiation cassette (reported here in FLS as off-target/trans-effects controls): BACH2, PAX5, IRF4, PRDM1, XBP1, AICDA.\u003cbr\u003e\nStatistics were performed within each panel using repeated -measures one-way ANOVA with Dunnett’s post-hoc test versus the untreated control: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ***p ≤ 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS11.tif","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/9c554b953e1c8316060ad99a.tif"},{"id":96110786,"identity":"f7ba8132-c575-470b-ad87-28a710400356","added_by":"auto","created_at":"2025-11-17 17:08:52","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":40637,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7912272/v1/140ed6143e35641328fcd5d1.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Sublethal DNA Damage Switches Off B-Cell Effector Programs in an RA-FLS- PBMC Co-culture","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is a systemic autoimmune disease that afflicts\u0026thinsp;~\u0026thinsp;0.5\u0026ndash;1% of the world\u0026rsquo;s population and ranks among the leading causes of disability. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Pathologically, it is characterised by synovial hyperplasia, dense leukocyte infiltration, and progressive destruction of cartilage and bone (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although T cell help is indispensable for disease induction, longitudinal histology, serum biomarker studies, and treatment responses now suggest that B cells are pivotal amplifiers of chronic inflammation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Mature B cells accumulate in ectopic germinal centre-like clusters within RA synovium, secrete rheumatoid factor and anti-citrullinated protein antibodies, present antigen to T cells, and elaborate cytokines such as IL-6 and TNF-α (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Depletion with the anti-CD20 antibody rituximab validates their clinical relevance; yet, one-third of patients remain refractory, and the risk of infection remains a concern (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Consequently, strategies that functionally silence rather than eliminate pathogenic B cells have become an attractive research avenue.\u003c/p\u003e\u003cp\u003eB cells are genetically unique because they engineer their genome; activation-induced cytidine deaminase (AID) deaminates cytosine during somatic hypermutation and class switch recombination, creating programmed double-strand breaks (DSBs) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This inherent genotoxic burden sensitizes B cells to DNA damage, as assessed by the Ser139-phosphorylated form of the histone variant H2AX (γ-H2AX), a marker of DNA damage (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Indeed, γ-H2AX-positive B cell clusters are abundant in RA synovia and correlate with disease activity (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Moreover, the inflamed joint milieu is a rich source of reactive oxygen species (ROS), reactive nitrogen intermediates, and is often exposed to low-dose ionizing radiation from diagnostic imaging, all of which are potential amplifiers of DNA damage (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, whether sublethal lesions alone can recalibrate B cell effector programmes without wholescale cytotoxicity remains unresolved.\u003c/p\u003e\u003cp\u003eTo explore that question, we employed a human co-culture model of fibroblast-like synoviocytes (FLS) from RA patients and peripheral blood mononuclear cells (PBMCs) from healthy donors. FLS provide contact-dependent and soluble factors that mimic the RA synovial microenvironment (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). We delivered a single, well-defined concentration (IC\u003csub\u003e20\u003c/sub\u003e or IC\u003csub\u003e50\u003c/sub\u003e) of three mechanistically distinct genotoxins that are clinically or pathophysiologically relevant to RA: γ-irradiation (γ-IR) used in radiosynoviorthesis and encountered in diagnostic imaging, induces clustered DSBs repaired mainly by Ku-dependent non-homologous end joining (NHEJ) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Secondly, we used hydrogen peroxide (H₂O₂), a diffusible reactive oxygen species (ROS) generated not only by activated neutrophils and macrophages in RA joints but also endogenously by many cell types in inflammatory microenvironments (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). It causes base oxidation, single-strand breaks, and secondary DSBs requiring mixed base excision repair (BER), NHEJ, and homologous recombination (HR) repair (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Lastly, 4-hydroperoxyifosfamide (4-OOH IFA), the active metabolite of the alkylator ifosfamide, structurally related to cyclophosphamide, which may be a necessary therapeutic approach in RA patients with interstitial lung disease or other B-cell dependent autoimmune disease, like systemic lupus erythematosus, forms among other DNA damages, interstrand cross-links that demand time-consuming homologous recombination (HR) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Functional, molecular, and viability endpoints were analysed to determine how genotoxic stress modulates B cell and T cell effector programs within an RA-relevant microenvironment. These insights enabled us to determine the extent to which sublethal genotoxic stress may attenuate B and T-cell effector programs while preserving overall viability.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\n\u003ch3\u003e1. Cell Culture and Co-culture Setup\u003c/h3\u003e\n\u003cp\u003eFibroblast-like synoviocytes (FLS) were isolated from synovial tissue of patients with RA after informed consent and ethics approval (Study-Nr.: 2022\u0026ndash;2189_7) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Cells were expanded to passage 3\u0026ndash;5 and seeded at 1 \u0026times; 10⁵ cells per well in 48-well plates. Cultures were maintained in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% GlutaMAX (Thermo Fisher Scientific), 1% sodium pyruvate (Thermo Fisher Scientific), 1% penicillin\u0026ndash;streptomycin (Thermo Fisher Scientific), and 10 mM HEPES (Thermo Fisher Scientific) at 37\u0026deg;C, 5% CO₂. After 24 hours, peripheral blood mononuclear cells (PBMCs) from healthy donors were added. PBMCs were isolated by Lymphoprep density-gradient centrifugation (Progen), washed, counted, and added to RA-FLS at a 1:5 ratio (1 RA-FLS:5 PBMCs) in the same medium.\u003c/p\u003e\n\u003ch3\u003e2. Genotoxic challenge and stimulation scheme\u003c/h3\u003e\n\u003cp\u003eCo-cultures or only PBMCs were treated with the following reagents: PBMC only with γ-IR (0\u0026ndash;4 Gy) delivered using the Gammacell 1000 Elite radiation machine (Nordion International, and the PBMC/FLS co-cultures with 4-OOH IFA (0\u0026ndash;34 \u0026micro;M) obtained from Niomech IIT GmbH, and H₂O₂ (0-160 \u0026micro;M) purchased from Sigma-Aldrich. After 24 hours, half the wells were harvested (acute end-point). The remaining wells were washed and stimulated with CpG ODN 2006 (5 \u0026micro;g/ mL; Invivogen) before further incubation until day 8. The complete treatment scheme is shown below in Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All compounds in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003e3. Cell viability Assay\u003c/h3\u003e\n\u003cp\u003eViability was assessed by Annexin V\u0026ndash;FITC/PI staining (Miltenyi Biotec) per the manufacturer\u0026rsquo;s protocol and analysed on a CytoFLEX LX flow cytometer (Beckman Coulter) at 24 h and 8 d post-treatment (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e for gating). To determine sublethal working concentrations, co-cultures received graded γ-IR (0\u0026ndash;4 Gy), 4-OOH IFA (0\u0026ndash;34 \u0026micro;M), or H₂O₂ (0\u0026ndash;160 \u0026micro;M). Four-parameter logistic regression yielded relative IC₅₀ values (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). From the same fits, IC₂₀ values at 24 h were approximately 0.1 Gy (γ-IR), 3.5 \u0026micro;M (4-OOH IFA), and 6 \u0026micro;M (H₂O₂). Unless stated otherwise, IC₅₀/IC₂₀ refer to these relative values (concentrations reducing viability to 50%/80% of vehicle-treated controls), i.e., sublethal benchmarks. All compounds and antibodies used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003e4. Cytokine and Immunoglobulin Quantification by ELISA\u003c/h3\u003e\n\u003cp\u003eELISA kits were purchased from R\u0026amp;D Systems for cytokines and chemokines (e.g., IL-10, IFN-γ, APRIL, CD25/IL-2 Ra, IL-2) and StemCell Technologies for immunoglobulins (IgM, IgG, IgA, IgE). Assays were performed according to the manufacturers' protocols. Absorbance was measured at 450 nm using a Tecan Infinite M200 Pro microplate reader (Tecan), and the data were analysed using Microplate Manager software (Bio-Rad). Cytokine and immunoglobulin values are expressed as a percentage of the CpG-stimulated, but otherwise untreated control and are plotted against the corresponding day 8 viability obtained from the same wells. All used kits are found in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003e5. Flow-cytometric assays\u003c/h3\u003e\n\u003cp\u003eCommon preparation. Cells were detached with Accutase (10 min, 37\u0026deg;C), washed in FACS buffer (PBS, 3% FBS, 2 mM EDTA), blocked with FcR reagent (1:5, Miltenyi; 15 min,4\u003csup\u003eo\u003c/sup\u003eC), and stained with surface markers (20 min, RT). Lineages were defined using: CD19 eFluor 405 (Invitrogen; 1:20), CD4-PerCP/Vio700 (1:50), CD8-VioGreen (1:50), CD27-VioBright R720 (1:50), and CD3-PE/Vio770 (1:50). Cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific; 15 min, RT) and permeabilised in 0.1% Triton X-100 (Thermo Fisher Scientific; 10 min, RT), then stained for intracellular targets in PBS with 0.05% Tween-20 and 3% FBS. Data were acquired on a CytoFLEX LX (Beckman Coulter) and analysed in FlowJo v10.8.1. All compounds and antibodies used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e5.1 γ-H2AX Assay for DNA Damage\u003c/h2\u003e\u003cp\u003eAfter surface staining, fixation, and permeabilization, cells were then incubated with anti-γ-H2AX-Alexa Fluor 488 (BD Biosciences, 1:20) for 1 hour, RT. It was followed by a washing step and then resuspended in FACS buffer. Gating strategy is outlined in Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e5.2. Ki-67 and PI Cell Cycle Assay\u003c/h2\u003e\u003cp\u003eAfter permeabilization, cells were incubated with Ki-67-FITC (Miltenyi Biotec, 1:50) for 20 minutes at room temperature in the dark. They were then counterstained with PI (propidium iodide, Miltenyi Biotec, 1:100) for 20 minutes at room temperature in the dark. Gating strategy is outlined in Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e. All compounds and antibodies used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e6. Real-Time Quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from co-cultured cells or RA-FLS only, at 24 hours or 5 days post-treatment using the NucleoSpin RNA isolation kit (Macherey-Nagel) following the manufacturer\u0026rsquo;s instructions. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed with SYBR Green Master Mix (Applied Biosystems) to quantify the expression of BCL-6, BAFF, AICDA, and other target genes. 18S rRNA was used as the reference gene, and relative gene expression was calculated using the ΔΔCt method. The primer sequences are given in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Note: RA-FLS are not antibody-producing cells; immunoglobulin and AICDA/GC-factor panels are included to document baseline/indirect transcriptional effects in the stromal compartment.\u003c/p\u003e\n\u003ch3\u003e7. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). For viability, DNA damage, cell cycle, cytokine/chemokine/immunoglobulin (day 8), and RT-qPCR analyses, we used repeated measures one-way ANOVA with Dunnett\u0026rsquo;s post hoc test to account for the paired and matched nature of samples derived from the same donors across multiple conditions. Significance thresholds: p\u0026thinsp;\u0026le;\u0026thinsp;0.05 (*), p\u0026thinsp;\u0026le;\u0026thinsp;0.01 (**), p\u0026thinsp;\u0026le;\u0026thinsp;0.001 (***), p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 (****). Analyses were performed in GraphPad Prism 8. All experiments used independent donor material. The language and grammar of this manuscript were checked using AI language editing assistance, with all content, data interpretation, and conclusions generated solely by the authors.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003eDifferential short and sustained loss of viability in RA-FLS/healthy PBMC co-cultures after a single γ-IR, 4-OOH IFA, or H₂O₂ challenge.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo characterize exposure response and temporal effects, we quantified viability at 24 hours and day 8 in RA-FLS/healthy PBMC co-cultures following a single graded exposure to γ-IR (Gy), 4-OOH IFA, or H₂O₂. γ-IR induced a monotonic decline in viability up to 2 Gy at 24 hours, with an estimated IC₅₀ of 0.4 Gy; viability stabilized at higher doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Despite inter-donor variability limiting post hoc significance, overall ANOVA confirmed a treatment effect. By day 8, both variance and viability further declined (IC₅₀ \u0026asymp; 0.9 Gy), but the concentration-response did not change beyond 2 Gy, suggesting stable cytotoxicity among surviving cells. RA-FLS monocultures remained fully viable as they were not irradiated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e4-OOH IFA exposure resulted in the most pronounced loss of viability. Statistically significant reductions began at 10 \u0026micro;M (IC₅₀, 14 \u0026micro;M), and were amplified by day 8 with IC₅₀ \u0026asymp; 7 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). High concentrations also impaired fibroblast viability (RA-FLS IC₅₀ = 7.7 \u0026micro;M), though FLS were generally more resistant than PBMCs. H₂O₂ yielded intermediate effects: a concentration-dependent viability loss was observed from 10 \u0026micro;M upward at 24 hours (IC₅₀, 23 \u0026micro;M), but viability partially recovered by day 8 (IC₅₀, 60 \u0026micro;M). RA-FLS monocultures were unaffected by H₂O₂ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Overall, γ-IR and H₂O₂ caused moderate, delayed cytotoxicity in PBMC/FLS co-cultures, whereas 4-OOH IFA induced rapid and sustained cell loss, including partial toxicity to FLS at high concentrations. These distinct profiles supported the selection of IC₂₀ and IC₅₀ concentrations for functional and mechanistic assays. Day 8 was prioritized for effector readouts to capture RA-FLS/PBMC crosstalk and stable mediator secretion, minimizing the influence of transient early effects.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSingle hit genotoxic stress uncouples lymphokine and immunoglobulin production from cell survival.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate functional consequences independent of early time points, we measured day-8 concentration\u0026ndash;response production of IL-10, IFN-γ, IL-2, sCD25/IL-2Rα, APRIL, and IgG/IgA/IgM across γ-IR, 4-OOH IFA, and H₂O₂ treatment. We then used Spearman correlation analyses to relate these effector outputs to corresponding viability measurements, clarifying the relationship between cell survival and functional changes.\u003c/p\u003e\u003cp\u003eFollowing γ-IR exposure, IL-10 and IFN-γ secretion were substantially reduced at concentrations that preserved cell viability, with both cytokines suppressed by approximately 70% at 2 Gy, where 8-day viability remained\u0026thinsp;\u0026ge;\u0026thinsp;80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, D). IgG and IgA levels were diminished to 40\u0026ndash;50%, while APRIL, sCD25, and IgM decreased to 50\u0026ndash;60% of CpG-stimulated controls at 2 Gy, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, D, G; Supplementary Fig.\u0026nbsp;5A, G). IL-2 was reduced by 60% at 1.5 Gy recovering at higher doses. (Supplementary Fig.\u0026nbsp;5D). Spearman correlation analysis revealed positive associations between cell viability and IL-10, IgG, IgM, and sCD25. In contrast, IgA, IL-2, and APRIL secretion did not correlate with viability, indicating partial decoupling of specific mediators from cell survival (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTreatment 4-OOH IFA elicited an abrupt shutdown of cytokine production, with IL-10 and IFN-γ reduced by over 50% at 5 \u0026micro;M, despite \u0026sim;70% viable cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, E). At 10 \u0026micro;M, IgG and IgA concentrations were reduced to 30% of control, accompanied by \u0026sim;60% viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, H). Reductions in APRIL and IgM approached 40%, CD25 declined to 70%, and IL-2 to 20% of control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;5B, E, H). All immune effectors determined under 4-OOH IFA treatment were positively correlated with cell viability (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eH₂O₂ induced a gradual suppression of function, with cell viability maintained above 90% up to 50 \u0026micro;M. IL-10 decreased by about 30% at 25 \u0026micro;M and transiently converged with the viability curve at 20 \u0026micro;M before separating at higher doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). IFN-γ remained consistently below the viability trajectory for the entire concentration range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). IgG and IgA were first inhibited at 25 \u0026micro;M without concurrent viability loss; parallel declines in both antibody secretion and viability emerged only at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;75 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, I). In contrast, IgM and CD25 each dropped to 40\u0026ndash;50% of control at 5 \u0026micro;M, APRIL declined by 60% at 40 \u0026micro;M, and IL-2 was suppressed by 20% at the lowest concentration tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Supplementary Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eC, F, I). Correlations between cell viability and IgG, IgA, and IgM weakened under H₂O₂, whereas CD25 and APRIL retained a positive association; IL-2 secretion was uncoupled from viability (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn summary, sublethal γ-IR, 4-OOH IFA, or H₂O₂ exposures consistently suppressed immunoglobulin output and accessory factor release most notably the release of IL-2, a cytokine mainly secreted by T cells that promotes their proliferation and survival. This pattern indicates a DNA damage checkpoint silencing immune effector programs at non-cytotoxic concentrations, prompting mechanistic investigation of γ-H2AX accrual in distinct lymphocyte subsets.\u003c/p\u003e\n\u003ch3\u003eKinetics of γ-H2AX accumulation reveal lineage-specific DNA damage responses at sublethal and half-lethal concentrations\u003c/h3\u003e\n\u003cp\u003eAfter establishing IC₂₀ and IC₅₀ concentrations from viability assays and observing durable suppression of cytokine and immunoglobulin secretion, we next investigated DNA damage responses in selected lymphocyte subsets by assessing γ-H2AX accumulation following exposure to γ-IR, 4-OOH IFA, or H₂O₂. At [IC₂₀] (sublethal), γ-IR produced a rapid increase in γ-H2AX across all investigated cell populations, peaking at 2 hours and remaining elevated above control at 24 hours, indicating incomplete repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-A). CD4⁺, CD8⁺ T cells, and memory B cells reached 6-7-fold above baseline, while na\u0026iuml;ve B cells increased to 10-fold at 24 hours. 4-OOH IFA produced a gradual increase in y-H2AX, reaching up to 6-fold by 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-B), and H₂O₂ elicited a modest early increase with a secondary peak at 8\u0026ndash;16 hours, stabilizing at ~\u0026thinsp;4-fold by 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-C). At [IC₅₀], similar cell specific patterns were observed but with higher amplitudes. γ-IR triggered 2-hour γ-H2AX peaks of 25-fold in CD4⁺, 15-fold in CD8⁺, 20-fold in memory B cells, and 9-10-fold in na\u0026iuml;ve B cells, decreasing by half at later timepoints except in na\u0026iuml;ve B cells, where the signal persisted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-D). 4-OOH IFA reached comparable levels at 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-E). H₂O₂ showed biphasic kinetics, y-H2AX peaking at 4-6-fold at 8 hours and declining to 2-3-fold by 16 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e-F). Adjusted p-values for all cell types/time points are reported in supplementary table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e. Basal γ-H2AX MFI levels were higher in na\u0026iuml;ve and memory B cells compared to CD4⁺ and CD8⁺ T cells (Supplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e), with significant fluctuations in B cell subsets over time even without treatment. All treated sample data were normalized to time-matched controls to enable accurate intra-lineage comparisons. Comparisons of γ-H2AX MFI across treatments at both [IC₂₀] and [IC₅₀] (Supplementary Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e) confirmed strongest early signals with γ-IR, especially at [IC₅₀]. These analyses demonstrate that DNA damage signalling is shaped by both lesion-specific chemistry and cell-intrinsic factors, influencing the dynamics and magnitude of repair responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDNA damage checkpoints translate into lineage-specific cell cycle blockade in B cells\u003c/h3\u003e\n\u003cp\u003eDistinct γ-H2AX signalling among lymphoid lineages suggests differential checkpoint engagement with functional consequences. To assess whether these lineage-specific signals affect cell-cycle progression, we analysed distributions in CD4⁺ and CD8⁺ T cells, as well as na\u0026iuml;ve and memory B cells, 24 and 48 hours following a single sublethal (IC₂₀) or IC\u003csub\u003e50\u003c/sub\u003e challenge with γ-IR, 4-OOH IFA, or H₂O₂. This links γ-H2AX burden to cell-cycle inhibition and proliferation arrest.\u003c/p\u003e\u003cp\u003eKi-67-FITC/PI staining showed that at 24 hours post-IC₂₀ γ-IR, memory B cells shifted out of quiescence into G₁, increasing from 18% \u0026plusmn; 2% to 64% \u0026plusmn; 6%; na\u0026iuml;ve B cells similarly accumulated in G₁ increasing from 4% \u0026plusmn; 1% to 21% \u0026plusmn; 4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), while IC₅₀ γ-IR produced no significant change. For 4-OOH IFA at [IC₅₀], memory B cells showed G₁ increase from 14% \u0026plusmn; 3% to 46% \u0026plusmn; 5% and S-G₂-M transition from 0% to 5% \u0026plusmn; 1%, with na\u0026iuml;ve B cells demonstrating modest G₁ and S-G₂-M enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). H₂O₂ at [IC₂₀] induced the strongest arrest: memory B cells redistributed into G₁ from 15% \u0026plusmn; 2% to 55% \u0026plusmn; 5% and S-G₂-M from 0% to 10% \u0026plusmn; 2%, while na\u0026iuml;ve B cells increased G₁ to 33% \u0026plusmn; 4% at [IC₂₀] and 23% \u0026plusmn; 3% at [IC₅₀] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 24 hours of CpG-ODN2006 stimulation (48 h total treatment), na\u0026iuml;ve B cells in all treatment groups reverted to quiescent status (G₀ \u0026gt;85%, G₁ \u0026lt; 10%). Memory B cells retained treatment-specific blocks: γ-IR IC₂₀ concentration G₁ stayed at 75% \u0026plusmn; 6% versus controls: 15% \u0026plusmn; 2%, S-G₂-M dropped from 3% \u0026plusmn; 1% to 0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). 4-OOH IFA [IC₅₀] memory G₁ persisted at 65% \u0026plusmn; 5%, S\u0026ndash;G₂\u0026ndash;M at 7% \u0026plusmn; 1%. H₂O₂ [IC₂₀] memory G₁ was 62% \u0026plusmn; 4%, S-G₂-M at 9% \u0026plusmn; 2%, and [IC₅₀] memory G₁ at 34% \u0026plusmn; 3%, despite maintained viability. CD4⁺ and CD8⁺ T cells showed only transient G₁ increases (γ-IR IC₂₀) or S-phase spikes (H₂O₂ [IC₅₀]) at 24 hours, both resolving by 48 hours (Supplementary Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e). These findings demonstrate that DNA damage-induced checkpoints establish durable, lineage-specific cell cycle arrest, especially in memory B cells, likely contributing to selective impairment in immune cell proliferation and function.\u003c/p\u003e\n\u003ch3\u003eTranscriptional Responses to DNA Damage in PBMC/RA-FLS Co-cultures and RA-FLS Monocultures\u003c/h3\u003e\n\u003cp\u003eTo associate γ-H2AX and cell-cycle checkpoint data with the functional readouts, we profiled 28 transcripts in PBMC/RA-FLS co-cultures and RA-FLS monocultures (supplementary figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e) by RT-qPCR, collecting RNA at 24 hours after [IC₂₀] or [IC₅₀] genotoxic challenge, and again on day 5 for differentiation markers. At [IC₂₀], all stressors triggered a robust sensor burst in co-culture: ATM, APEX1, XRCC6, and XRCC5 increased by 20-30-fold after γ-IR and 4-OOH IFA, and 15-20-fold after H₂O₂ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). At [IC₅₀], for γ-IR, the four sensors returned to baseline, whereas 4-OOH IFA and H₂O₂ maintained strong sensor upregulation (10-25-fold). RAD51 but not RAD50 was upregulated 4-6-fold at [IC₂₀] and [IC₅₀] for 4-OOH IFA and at IC₂₀ for γ-IR and H₂O₂ (Supplementary Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). RA-FLS monocultures showed limited transcriptional changes: H₂O₂ selectively increased XRCC5 at 24 hours, and 4-OOH IFA elevated XRCC5, RAD50, and RAD51 levels (Supplementary Figure \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). TP53 transcript was only slightly reduced at 4-OOH IFA [IC₂₀] and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [IC\u003csub\u003e50\u003c/sub\u003e], while CDKN1A/P21 did not change. BAX increased 3-fold at γ-IR [IC₅₀], 2-fold at 4-OOH IFA [IC₅₀], but decreased at H₂O₂ [IC₅₀] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). FAS did not change, while FASLG showed specific upregulation with each agent. BCL-2 was enhanced 2-fold at γ-IR IC₅₀ and H₂O₂ IC₂₀ doses but declined at H₂O₂ [IC₅₀]. BRCA1, BRCA2, and PDCD1 were unchanged; BCL-6 increased 600-700-fold under all treatments at IC\u003csub\u003e20\u003c/sub\u003e and IC\u003csub\u003e50\u003c/sub\u003e doses/concentrations. BAFF was upregulated by 10-fold at both doses of γ-IR and at 4-OOH IFA by 4-fold at [IC\u003csub\u003e20\u003c/sub\u003e] and [IC\u003csub\u003e50\u003c/sub\u003e]with a more limited increase for H₂O₂ at [IC₂₀] (Supplementary Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). In RA-FLS, H₂O₂ [IC₂₀] significantly induced BCL-6, BAX, and TP53; 4-OOH IFA [IC₅₀] elevated FAS, FASLG, and PDCD1 levels, while BCL-6 and BAX were upregulated at [IC₂₀]. BRCA2 and TP53 increased after [IC₂₀] (Supplementary Figure \u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003e). These data reveal treatment-specific transcriptomic responses in DNA damage sensors, repair genes, and apoptotic regulators, underlying the observed functional impairments under genotoxic stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEarly blockade and late persistence of the B cell differentiation program\u003c/h3\u003e\n\u003cp\u003eTo relate the functional loss of Ig secretion to transcriptional checkpoints, we quantified canonical differentiation and plasma cell genes in PBMC/RA-FLS co-cultures at 24 hours and day 5 post-IC₂₀ or IC₅₀ pulse, focusing on B cell genes due to the clear impact on Ig secretion, and did not profile T cell differentiation genes in this study.\u003c/p\u003e\u003cp\u003eTwenty-four hours after exposure, all three stressors triggered distinct but overlapping bursts in B cell program genes. γ-IR induced\u0026thinsp;\u0026gt;\u0026thinsp;200-fold rises in XBP1 and IRF4 (both at [IC₂₀] and [IC₅₀]), and BACH2, PAX5 (at [IC₂₀]), with AICDA strongly repressed under both concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). 4-OOH IFA drove all five factors above 200-fold at [IC₂₀] and maintained high levels except PRDM1 at [IC₅₀]; AICDA was unchanged. H₂O₂ largely mirrored γ-IR for XBP1, IRF4, and PAX5, with BACH2 steeply elevated only at [IC₂₀]. PRDM1 was repressed at [IC₅₀], and AICDA was only reduced by γ-IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFive days after the single stressor exposure, expression profiles diverged. After γ-IR, XBP1, IRF4, and PAX5 remained\u0026thinsp;\u0026ge;\u0026thinsp;200-fold [(IC₂₀]) and ~\u0026thinsp;300-fold ([IC₅₀]); PRDM1 persisted at [IC₂₀]; BACH2 returned to baseline, AICDA remained high to \u0026gt;\u0026thinsp;200-fold. With 4-OOH IFA, only PRDM1 and AICDA stayed elevated; others were reduced to almost baseline levels. For H₂O₂, PRDM1 remained high at [IC₂₀], IRF4 showed modest increase ([IC₅₀]), AICDA surpassed 500-fold, others stayed near baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F). In RA-FLS alone (24h), B-cell differentiation genes showed modest, limited changes (Supplementary Fig. \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGene panels five days after challenge linked germinal center survival signals and class switch recombination with heavy chain transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-I). γ-IR (at IC₂₀/IC₅₀ doses) increased BCL-6, BAFF, and \u0026micro; heavy chain\u0026thinsp;~\u0026thinsp;200\u0026ndash;300-fold. IgG and IgA rose sharply only at IC₂₀, not at IC₅₀. For 4-OOH IFA, BAFF transcripts increased at both concentrations, IgM dropped at [IC₂₀] but rebounded at [IC₅₀], and IgG and IgA increased five-fold. H₂O₂ enhanced transcription of BAFF and all heavy chains (4-200-fold), with BCL-6 unchanged. This suggests differentiation blockade and lingering transcriptional shifts, rather than full germinal centre output. These late timepoint data reveal that different genotoxins and y-IR uniquely reactivate B cell effector gene expression, suggesting varied impacts on functional recovery after damage.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study delineates how a single genotoxic stressor from DNA damage to checkpoint activation impacts immune cell function, especially memory B cells, as shown by y-H2AX and cell cycle analysis in RA-FLS/healthy PBMC co-cultures. By integrating viability, γ-H2AX kinetics, cell cycle, and transcriptomic analyses, we show that γ-IR, 4-OOH IFA, and H₂O₂ exert distinct effects across immune lineages, especially memory B cells, which seem uniquely sensitive to sustained DNA damage signalling and functional shutdown. While classical DNA damage signalling in lymphocytes usually involves ATM and p53 activation, our co-culture system induced ATM but not p53, suggesting established DNA damage models may not fully apply in mixed cultures. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Early versus sustained cytotoxicity: how stressor type sets the tone\u003c/h2\u003e\u003cp\u003eEach genotoxic stressor induced a distinct viability trajectory in the RA-FLS/healthy PBMC co-culture. γ-IR produced only modest early death, yet induces a delayed attrition. In contrast, 4-OOH IFA caused the steepest and most sustained loss of PBMC viability. H₂O₂ had an intermediate effect between these extremes. Early viability dropped, but partial recovery by day 8, which might suggest the selection or adaptation of ROS-tolerant cells, consistent with the transcriptional upregulation of antioxidant programs seen in other models (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The stromal compartment behaved differently. In our design, RA-FLS were not irradiated, so no inference about radioresistance can be drawn. Under 4-OOH IFA, RA-FLS monocultures retained substantially higher viability than PBMC-rich co-cultures at matched concentrations but did show impairment at the higher concentrations, indicating relative but not absolute resilience. Using H₂O₂, RA-FLS were largely unaffected across the tested concentrations. Therefore, PBMCs are the primary cytotoxic target of all three stressors, whereas FLS are comparatively spared by H₂O₂ and only partly affected by high-concentration 4-OOH IFA, consistent with previous studies demonstrating the resilience of fibroblast-like synoviocytes to oxidative damage (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Functional silencing precedes cell death: A transcriptional checkpoint\u003c/h2\u003e\u003cp\u003eAcross all three stressors, functional silencing of IL-10, IFN-γ, IgG, and IgA routinely occurred before overt cytotoxicity, suggesting regulation at a transcriptional checkpoint while cells remained viable. This pattern fits an established DNA damage response hierarchy, in which ATM sensor activation triggers chromatin tightening and transient repression of differentiation programs (AICDA-PRDM1-XBP1 axis), prioritizing repair over specialized immune functions (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Our findings support this mechanism, as robust induction of ATM and associated sensors occurred rapidly with only modest changes in apoptotic genes and cell cycle effectors (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). However, positive correlations between immune effector production and viability also indicate a relationship with cell death, and thus cannot, on their own, exclude a model in which protein loss simply results from cytotoxicity. Indeed, the inverse correlation between cell survival and effector loss could reflect either checkpoint-regulated suppression or progressive cell death. The most rigorous demonstration of independent checkpoint regulation would require finding a factor positively associated with cell death or specifically upregulated in surviving cells post-stress a criterion that DNA repair factors could fulfill if de novo synthesis coincides with increased cell death. Our transcriptomic data show that ATM, APEX1, XRCC6, and XRCC5 are rapidly upregulated after stress, matching this expectation (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAgent-specific chemistries further shape the depth and breadth of functional silencing. γ-IR activates ATM signalling, leading to moderate and sustained suppression of gene expression, while 4-OOH IFA likely induces robust ATR-CHK1 response and uniform transcriptional slowdown, and H₂O₂ evokes mixed base damage mechanisms. Notably, APRIL and IL-2 demonstrate regulation independent of viability, implicating additional pathways (e.g., NF-κB, AP-1, STATs) beyond ATM-driven checkpoints (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Antibody isotypes also showed distinct regulation. IgG and IgA were more sensitive to early shutdown than IgM, consistent with the metabolic demands of class-switch recombination (CSR), dependent on AID and ER/UPR programs tightly monitored by the DNA damage response. Basal IgM production, being less energetically demanding, persisted under stress, while CSR and high-level antibody output were selectively suppressed. These patterns corroborate prior findings and reinforce a model of stress-dependent transcriptional throttling (\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Given that these class-switched isotypes predominantly arise from memory B cells, and our γ-H2AX and cell cycle data substantiate vulnerability in this subset, we infer that the observed isotype suppression under genotoxic stress might be driven primarily by memory B cell checkpoint engagement. However, as na\u0026iuml;ve B cells can also become activated and undergo class-switch recombination under CpG stimulation, we cannot fully exclude their contribution to the observed effects. Quantifying the ratio of reactivated memory versus newly activated na\u0026iuml;ve B cells would be required to substantiate this inference, which therefore remains interpretive but central to our model (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, robust sensor induction and selective effector suppression at sublethal exposures, in the absence of strong apoptotic signals, argue most convincingly for early checkpoint regulation as the principal mechanism, rather than cell death per se. This aligns with increasing evidence that DDR pathways can directly downregulate immune effector gene expression via ATM/ATR signalling before apoptosis is initiated (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). While our study cannot disambiguate single-cell checkpoint effects from population-level coordination, the observed association between repair factor induction and cell death strengthens the case for differential regulation in this coculture setting, mimicking the situation in inflamed RA joints.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Lesion kinetics and cell cycle checkpoint responses\u003c/h2\u003e\u003cp\u003eOur kinetic analyses revealed stressor-specific γ-H2AX and cell cycle checkpoint engagement, with prolonged and synchronous activation specifically demonstrated in memory B cells. γ-IR drove a rapid and synchronous γ-H2AX peak across lymphoid lineages, with T cells displaying the highest early activation and swift resolution, whereas na\u0026iuml;ve B cells exhibited sustained γ-H2AX at late timepoints, suggesting slower repair capacity. In contrast, 4-OOH IFA produced a delayed, monotonic γ-H2AX increase, consistent with interstrand crosslink damage and prolonged checkpoint activation. H₂O₂ triggered a biphasic γ-H2AX response, resulting in mixed and transient cell cycle checkpoint patterns (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Notably, 4-OOH IFA's prolonged checkpoint engagement, reflected by delayed RAD51 induction and persistent γ-H2AX in memory B cells, coincided with selective loss of antibody outputs and differentiation markers (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). H₂O₂, by contrast, allowed functional recovery following checkpoint resolution. These findings highlight a nuanced picture: memory B cells are uniquely vulnerable to prolonged checkpoint activation and functional suppression under genotoxic conditions, a central finding of our study. Although an initial DNA damage checkpoint attenuates antibody secretion, oxidative lesions appear to be repaired or tolerated sufficiently allowing CpG stimulation to activate the secretory program, including class-switch output at later time points (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). This transient, stress-adapted phenotype aligns with prior studies showing that efficient repair of oxidative DNA damage enables rapid B-cell recovery and renewed antibody production (Mori et al., 2009). Similar observations have also been reported by Valverde et al. (2018), who demonstrated that cells exposed to sublethal oxidative stress upregulate DNA repair pathways and undergo adaptive responses enabling genomic protection and functional recovery (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSome limitations need to be discussed. Direct measurement of DNA lesion spectra, repair kinetics, and protein-level validation in disease-relevant, single-cell assays would strengthen mechanistic conclusions. Our approach, leveraging population-level analyses in pathophysiologically relevant co-cultures, nonetheless demonstrates coordinated DNA damage checkpoint engagement and immune suppression. The inclusion of PBMC co-cultures introduces the potential for paracrine effects but more closely reflects in vivo immune-stromal interactions, an essential consideration for RA pathogenesis.\u003c/p\u003e\u003cp\u003eIn summary, our findings show that sublethal, type-dependent genotoxic stress can selectively engage DNA damage checkpoints in memory B cells while largely preserving overall cell viability in a physiologically relevant PBMC co-culture. By characterizing these coordinated responses, this study contributes to a clearer understanding of how genotoxic stress affects immune function, and establishes a foundation for future investigations in disease contexts and more refined cell populations.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, we delineate a coherent sequence linking DNA damage response dynamics, measured by \u0026gamma;-H2AX kinetics and associated sensor gene expression, to a cell cycle checkpoint engagement and subsequent transcriptional alterations that explain how DNA damage can selectively recalibrate B cell function. These findings might provide a rationale for precision, low-concentration genotoxic modulation of target cell in autoimmune diseases, suggesting that a brief exposure to an alkylator or radiotherapeutic agent might silence pathogenic B cells while leaving stromal fibroblasts and most T cells intact. Mapping this cascade provides a rational basis for coupling genotoxic agents with targeted immune modulation in rheumatoid arthritis and related B-cell-driven disorders.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRA- Rheumatoid Arthritis\u003c/p\u003e\n\u003cp\u003eRA-FLS — rheumatoid-arthritis fibroblast-like synoviocytes\u003c/p\u003e\n\u003cp\u003ePBMC(s) — peripheral blood mononuclear cell(s)\u003c/p\u003e\n\u003cp\u003eγ-IR — gamma irradiation\u003c/p\u003e\n\u003cp\u003e4-OOH IFA — 4-hydroperoxyifosfamide\u003c/p\u003e\n\u003cp\u003eH₂O₂ — hydrogen peroxide\u003c/p\u003e\n\u003cp\u003eIC₂₀ / IC₅₀ — concentration causing 20%/50% reduction in viability or function\u003c/p\u003e\n\u003cp\u003eγ-H2AX — phosphorylated histone H2AX (Ser139)\u003c/p\u003e\n\u003cp\u003eMFI — median fluorescence intensity\u003c/p\u003e\n\u003cp\u003eCpG-ODN2006 — CpG oligodeoxynucleotide 2006\u003c/p\u003e\n\u003cp\u003eNHEJ / HR / BER — non-homologous end joining / homologous recombination / base-excision repair\u003c/p\u003e\n\u003cp\u003eSer139-Serine 139\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies involving animal subjects\u003c/p\u003e\n\u003cp\u003eNo animal studies are presented in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudies involving human subjects:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe studies involving humans were approved by the local ethics committee and conducted according to the ethical principles outlined in the Declaration of Helsinki. Written informed consent has been obtained from the patients to publish this paper. The studies were conducted in accordance with local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions :\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, D.B., G.P., T.L., and G.F., methodology, D.B. G.P., T.L., and G.F, validation, D.B., investigation, data curation, D.B., writing original draft preparation, D.B., G.P., T.L., and G.F., . All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—417677437/GRK2578.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to extend our sincere gratitude to the Department of Rheumatology and Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, for their instrumental role in facilitating the execution of this study within their esteemed research facility.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang Z, Gao X, Liu S, Wang Q, Wang Y, Hou S, et al. 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DNA Repair (Amst). 2018;65:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7912272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7912272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRheumatoid arthritis (RA) features lymphocyte-driven inflammation in which B cells, alongside T cells, play key effector roles (autoantibody production, antigen presentation, cytokines, or chemokines). Within B cells, during normal diversification, activation-induced cytidine deaminase (AID) introduces targeted DNA lesions in immunoglobulin loci (class-switch recombination/somatic hypermutation), creating a potential vulnerability to sublethal genotoxic stress. T cells also contribute to RA pathogenesis through cytokine production and cell-mediated responses, and are exposed to similar genotoxic stressors in the inflamed joint environment. Given this, we asked whether a single, sublethal insult can modulate lymphocyte effector function without overt cytotoxicity.\u003c/p\u003e\u003cp\u003ePeripheral blood mononuclear cells from healthy donors were co-cultured with RA fibroblast-like synoviocytes and exposed once to an IC₂₀ or IC₅₀ concentration of γ-irradiation (γ-IR), hydrogen peroxide (H₂O₂), or the oxazaphosphorine metabolite 4-hydroperoxyifosfamide (4-OOH IFA). Viability, γ-H2AX kinetics, cell cycle status, cytokine and immunoglobulin secretion, and a 28-gene damage response/differentiation panel were quantified at either 24 hours or 5 days.\u003c/p\u003e\u003cp\u003eTogether, the data indicate that a single, carefully titrated low-concentration genotoxic hit can selectively suppress lymphocyte effector programs, with B cells being more durably affected than T cells. At 2 Gy, overall cell viability remained above 80%, whereas IL-10 expression declined by approximately 70%, demonstrating functional silencing in the absence of substantial cytotoxicity. Targeting this vulnerability may selectively dampen pathogenic B cell activity in RA while sparing overall immune viability and T-cell competence.\u003c/p\u003e","manuscriptTitle":"Sublethal DNA Damage Switches Off B-Cell Effector Programs in an RA-FLS- PBMC Co-culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 17:08:47","doi":"10.21203/rs.3.rs-7912272/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-12-02T10:45:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-29T10:37:50+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-23T22:04:10+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-10T02:07:36+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-07T09:21:34+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-11-05T23:43:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-27T14:43:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T18:00:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2025-10-24T18:00:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b20933a0-5dbc-4574-8621-f62563e917ee","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57514947,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":57514948,"name":"Biological sciences/Drug discovery/Toxicology"},{"id":57514949,"name":"Health sciences/Diseases/Rheumatic diseases/Rheumatoid arthritis"}],"tags":[],"updatedAt":"2026-04-01T07:11:10+00:00","versionOfRecord":{"articleIdentity":"rs-7912272","link":"https://doi.org/10.1038/s41420-026-03021-1","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2026-03-21 04:00:00","publishedOnDateReadable":"March 21st, 2026"},"versionCreatedAt":"2025-11-17 17:08:47","video":"","vorDoi":"10.1038/s41420-026-03021-1","vorDoiUrl":"https://doi.org/10.1038/s41420-026-03021-1","workflowStages":[]},"version":"v1","identity":"rs-7912272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7912272","identity":"rs-7912272","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}