Summary Pluripotent stem cells must reconcile rapid replication with a highly dynamic transcriptional program, creating an inherent susceptibility to transcription–replication conflicts (TRCs). We demonstrate that embryonic stem cells (ESCs) operate in a “resilient” replication mode, tolerating high genomic traffic through the constitutive upregulation of R-loop and TRC resolution pathways. Through a targeted functional screen, we identify the RNA helicase Aquarius (AQR) as an essential safeguard of this state. AQR depletion downregulates these resolution factors, collapsing this stress-resistant program and driving ESCs into unstable, heterogenous states, marked by increased transcriptional entropy and cell-to-cell noise. Mechanistically, we show that key identity-defining genes are preferentially located within R-loop and TRC prone regions, making them uniquely vulnerable to AQR depletion. Our findings establish AQR as a critical governor of transcriptional fidelity, demonstrating that genomic resilience is fundamental to maintaining pluripotent cell identity.

Abstract The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role during S phase. It phosphorylates the replicative helicase (minichromosome maintenance, MCM complex), which leads to the initiation of replication. However, few other targets, besides the MCM complex, are known, leaving DDK an understudied kinase. Here, we determine the nuclear DDK-dependent phosphoproteome by a two-pronged mass spectrometry approach. Among ~ 400 DDK-dependent phosphorylation targets, we find the Arp8 subunit of the INO80 chromatin remodeling complex. Arp8 phosphorylation stabilises INO80’s complex integrity, finetunes its nucleosome spacing at replication origins, stimulates replication and improves the replication stress response. Taken together, we report the regulation of a chromatin remodeler with nucleosome-spacing activity by the cell-cycle machinery. DDK not only regulates the core replication machinery but also regulates a factor that generates replication-conducive chromatin architecture at replication origins.

BackgroundBlinatumomab (Blina), a CD19×CD3 bispecific T cell engager, is approved for the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), yet resistance remains a major challenge and the mechanisms driving treatment failure remain poorly understood.MethodsTo define the immunological determinants of resistance, we performed longitudinal profiling of peripheral blood T cells and the immune milieu of 34 patients receiving Blina using flow cytometry (n=19), single-cell CITE-seq (n=13), ex vivo Blina-induced cytotoxicity (n=26) and serum proteomics (n=17).ResultsAt baseline, Responders (R) were enriched for CD8+ effector memory T cells (TEM) expressing higher levels of cytotoxic genes and their transcriptional regulator ZNF683. Conversely, CD8+ TEM from Non-Responders (NR) displayed transcriptional features of activation without proportionate cytotoxic commitment. Over the course of the first treatment cycle, NR exhibited a progressive expansion of TIM3+CD8+ T cells that correlated with a rapid loss of ex vivo cytotoxic function. Linking baseline state to post-treatment T-cell exhaustion, the magnitude of TIM3+CD8+ expansion correlated inversely with baseline ZNF683 expression in CD8+TEM. Beyond T-cell-intrinsic features, NR harbored an immunosuppressive milieu characterized by higher circulating levels of M2-polarizing factors (CSF-1, HGF) and the TIM-3 ligand Galectin-9, which correlated positively with the magnitude of TIM3+CD8+ T-cell expansion.ConclusionsThese findings indicate that post-Blina CD8+ T-cell exhaustion is associated with resistance and it is shaped by both reduced ZNF683-dependent cytotoxic programming in CD8+ TEM and an immunosuppressive milieu. This provides a rationale for risk stratification based on baseline transcriptional profiling of CD8+ TEM and for combinatorial strategies targeting the suppressive microenvironment.

Maintaining a balanced immunity between pathogen defense and tolerance to environmental antigens in neonates is essential for survival and the establishment of life-long immune homeostasis. Instructed by environmental signals, type 1 conventional dendritic cells (cDC1) contribute to both processes but how the balance may be achieved is unclear. Here, we uncover an interferon (IFN)γ-driven regulatory circuit in early life that relays dietary cues to spleen cDC1. IFNγ-mediated STAT1-signaling induces an immunogenic maturation program in spleen cDC1 that enables them to shape the effector differentiation of antigen-experienced effector memory CD8⁺ T cells. This cDC1 program emerges during the transition from breastfeeding to solid food at weaning, occurs in germ-free mice, and remains operative to dietary intervention in adult mice. At weaning, this IFNγ signal enables spleen cDC1 to shape the effector phenotype of food-antigen-specific CD8+ T cells in a feedforward manner, thereby recalibrating the developing T cell pool. Our findings identify diet as a modifiable cue that can tune systemic cDC1-mediated immunity, opening new opportunities to steer immune responses during early life and beyond.

Multiciliated cells (MCCs) are specialized cells found in the brain, reproductive and respiratory tracts of mammals, and the epidermis of tadpole-stage Xenopus embryos. KMT5B and KMT5C are histone methyltransferases that deposit the dimethyl mark on histone 4 lysine 20 (H4K20). We previously showed that KMT5B/C double knockdown down-regulates H4K20me2 levels in bulk chromatin, as well as transcription of ciliary genes. MCCs of embryos lacking both enzymes, or only KMT5B, have depleted cilia. Here, we separate the function of KMT5B in multiciliogenesis and show that single knockdown of KMT5B, not KMT5C, leads to aberrant transcription and down-regulation of ciliary genes. This phenotype is rescued by catalytically active PHF8, an H4K20me1 demethylase, whereas hormone-inducible multicilin (MCI), master regulator of cilia, has no effect. Notably, the expression of key transcription factors of ciliogenesis is unaffected by KMT5B depletion, which dominates the transcriptional response to ectopic multicilin. Finally, ATAC-seq in animal caps shows KMT5B knockdown results in few differentially accessible peaks and does not compact chromatin at ciliary genes. This suggests KMT5B regulates MCCs via an alternative pathway to the canonical MCI-driven programme.

Abstract Balancing pathogen defence with maintaining tolerance to environmental antigens, such as food or commensals, in neonates is essential for survival and the establishment of life-long immune homeostasis. Instructed by environmental signals type 1 conventional dendritic cells (cDC1) drive either T cell tolerance or immunity. Here, we uncover an interferon (IFN)-γ-driven regulatory circuit in early life that relays dietary cues to spleen cDC1. Loss-of-function demonstrates that IFNγ-mediated STAT1-signaling induces an immunogenic maturation program in spleen cDC1 that instructs cDC1 to expand effector memory CD8⁺ T cells. This program emerges during weaning, when IFNγ production from lymphocytes rises, it occurs in germ-free mice and remains responsive to dietary intervention in adult mice. During the transition from breastfeeding to solid food at weaning, this circuit relays dietary information to spleen cDC1 to shape the effector phenotype of food-antigen specific CD8 + T cells in a feed-forward manner, allowing cDC1 to recalibrate the T cell pool at the moment of nutritional independence.

ABSTRACT:T-cell-based immunotherapies have revolutionized treatment paradigms in B-cell malignancies, yet their translation to acute myeloid leukemia (AML) has been hindered by a scarcity of tumor-restricted antigens and the risk of on-target off-leukemia toxicity. FLT3 has emerged as a promising therapeutic target with limited expression in healthy hematopoietic tissues. Here, we performed a head-to-head preclinical comparison of an FMS-like tyrosine kinase 3 (FLT3)-directed bispecific T-cell engager (BiTE) molecule and second-generation FLT3-specific chimeric antigen receptor (CAR) T cells. Both approaches induced potent cytotoxicity against AML cell lines and primary patient-derived cells but spared healthy hematopoietic stem and progenitor cells in vitro. Despite similar short-term efficacy, prolonged antigen exposure demonstrated progressive functional decline and metabolic exhaustion; however, CAR T cells maintained cytotoxic capacity and proliferative potential over time. In AML xenograft models, CAR T cells achieved superior tumor control, prolonged survival, and greater T-cell infiltration than BiTE molecule-treated counterparts. Transcriptomic profiling of T cells recovered from the bone marrow further revealed a distinct exhaustion-associated gene signature in samples from mice that had been treated with the FLT3 BiTE molecule. Importantly, provision of CD86-mediated costimulation enhanced antitumor activity of BiTE-redirected T cells in vitro and in vivo. These findings establish FLT3 as a viable and selective immunotherapeutic target in AML and underscore the functional and transcriptional differences between BiTE molecule-redirected T cells and CAR T cells. Moreover, they reveal a critical role for costimulatory signaling in sustaining the efficacy of T-cell-based therapies in vivo, offering a rationale for improving T cell-redirection strategies in myeloid malignancies.

Bispecific T-cell engagers (BiTE® molecules) have transformed the treatment of B-cell malignancies, yet clinical activity in AML has been modest. Resistance is driven in part by the genetic heterogeneity of AML, most notably TP53 mutations, present in 10-15% of de novo and up to 25% of therapy-related AML. Thus, we hypothesized that TP53 aberrations in AML contribute to cell-intrinsic and extrinsic resistance against T-cell-based immunotherapy. Cytotoxicity against TP53-deleted (DEL) primary AML cells and TP53-knockdown (KD) AML cell lines was reduced in co-cultures with T cells stimulated with the BiTE molecule AMG 330 (CD3×CD33). In addition, T-cell proliferation and proinflammatory cytokine secretion was impaired in co-cultures with TP53 KD cells. Transwell assays identified the secretome of TP53 KD AML cells as a key contributor to the immunosuppressive effects. Proteomic analysis revealed TGF-β1 in TP53 KD co-cultures as a mediator of T-cell suppression. RNA sequencing of T cells co-cultured with TP53 KD cells uncovered a transcriptional shift toward a senescent cell cycle profile. Our data collectively identify the immunosuppressive secretome of TP53-deficient AML as a key barrier to T-cell-engaging immunotherapies, underscoring an unmet clinical need for strategies able to restore T-cell function in TP53 KD AML.

Circadian clocks underlie daily rhythms in physiology by coordinating temporal patterns of gene expression and protein function throughout the body. At the core of this system in mammals is CLOCK/BMAL1, a ubiquitously expressed heterodimeric transcription factor complex that orchestrates tissue-specific circadian gene expression. The basis for this specificity remains unclear, but tissue-specific interactions at chromatin could provide one. Here we used chromatin immunoprecipitation coupled to mass spectrometry to map CLOCK/BMAL1-associated protein complexes on chromatin in mouse liver, kidney and lung. We detected 1,510 associated proteins, most of which were tissue-specific and not explained by protein abundance. Among these, we identified the homeodomain transcription factors PROX1, HNF1B and HOXA5 as tissue-enriched interactors that bind BMAL1, co-occupy most BMAL1 genomic sites and establish organ-restricted circadian transcription. Our findings demonstrate that tissue-specific transcription factors confer cellular identity on the core clock, thereby contributing to organ-specific patterns of rhythmic gene expression.

T cell-based immunotherapy has revolutionized the treatment of B-cell malignancies, yet applying it to acute myeloid leukemia (AML) is challenging due to difficulties in identifying suitable target antigens without on-target off-leukemia toxicity. Prior studies identified FLT3 as a promising target antigen with restricted expression in the healthy hematopoietic compartment1. Here, we evaluate a FLT3-directed BiTE® molecule and 2nd generation FLT3-specific CAR T cells in a preclinical AML model. We hypothesize that positive costimulatory molecules on AML cells enhance BiTE® molecule effectiveness, while CAR T cells may be less dependent due to their built-in costimulatory domain. Cytotoxicity was assessed in cocultures over time using MPFC. The impact of positive costimulation was tested using our established Ba/F3 model system lacking any human costimulatory molecules. On-target off-leukemia toxicity was evaluated in cytotoxicity and CFU assays of pAML cells and healthy bone marrow (hBM). T-cell exhaustion was examined in a longterm culture system with continuous antigen exposure. Lastly, xenograft AML models and RNA sequencing explored differences between the two platforms in vivo. BiTE® molecule and CAR effectively mediated cytotoxicity against various AML cell lines and pAML cells. Overexpression of the costimulatory CD86 on the target cells significantly increased BiTE® molecule-mediated cytotoxicity cells, while CAR-mediated lysis was unaltered. In both scenarios, low E:T-dependent toxicity was observed against hBM. Mixing experiments showed efficient pAML cell lysis with minimal impact on hBM. CFU assays indicated no effect on hematopoiesis of healthy CD34+ stem cells. 28-day cocultures led to T-cell exhaustion in both settings, marked by reduced proliferation, cytokine secretion, metabolic fitness, and cytotoxicity. CAR T cells demonstrated superior antileukemic activity, with enhanced proliferation and splenic homing in an AML xenograft model. RNA sequencing revealed more exhaustion-related markers in BiTE® molecule-redirected T cells, while CAR T cells showed upregulated glycolysis and fatty acid metabolism. Repeating our in vivo xenograft experiment with CD86-overexpressing AML cells supported our hypothesis that costimulation might be a key factor providing a head start to CAR T cells by increasing T-cell proliferation and persistence in vivo. Our in vitro data show similar cytotoxicity and specificity, while xenograft mouse models suggest CAR T cells offer a significant survival advantage over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR constructs boosts T-cell activation, homing, and efficacy, thereby delaying T-cell exhaustion in vivo. Future studies are needed to further dissect differences between BiTE® molecule vs CAR T cell-based immunotherapy and identify suitable patient groups. 1Brauchle et al 2020 Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Adrian Gottschlich, Gordon Hoffmann, Nora Philipp, Gerulf Hänel, Martin Kirmaier, Anetta Marcinek, Maryam Kazerani, Alica-Joana Emhardt, Giulia Magno, Rebecca L. Goldstein, Sebastian Theurich, Tobias Straub, Sebastian Kobold, Tara Arvedson, Veit L. Bücklein, Marion Subklewe. BiTE® molecule vs CAR-T targeting FLT3 in AML: How positive co-stimulation tips the scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3174.

In vertebrate embryos, gene expression is first initiated at zygotic genome activation (ZGA). Maternally expressed transcription factors are essential for this process. However, it is unknown whether active chromatin modifications established in gametes are present in early embryos and contribute to ZGA and embryonic development. Here, we show that in Xenopus laevis, H3K4me3 occurs at common genomic loci in gametes, in transcriptionally quiescent pre-ZGA embryos, and in transcriptionally active ZGA embryos. These loci exhibit high H3K4me3 intensities and breadth, DNA hypomethylation, and elevated CpG content. We show that H3K4 methylation pre-marking is required for successful ZGA and development, including expression of the key ZGA transcription factor Pou5f3.2. We demonstrate that the H3K4 methyltransferase Cxxc1 ensures establishment of H3K4me3 and proper ZGA. These findings reveal a role for H3K4 methylation in defining active chromatin states in Xenopus laevis embryos and highlight its importance for accurate ZGA and embryonic development.

Background Challenges to developing immunotherapies for acute myeloid leukemia (AML) include the identification of suitable target antigens due to on-target-off-leukemia toxicity. CD70, expressed on AML bulk and leukemic stem cells with limited expression on healthy cells, has emerged as a promising target.Methods This study evaluated CD70 as a target for NK-cell-based immunotherapy using a sugar-engineered antibody (PF-08046040, SEA-CD70). CD70 surface expression was assessed in primary AML samples by multiparameter flow cytometry. The cytotoxic capacity of SEA-CD70 was analyzed through antibody-dependent cellular cytotoxicity (ADCC) assays using AML cell lines, primary AML samples, and a severe combined immunodeficiency (SCID) mouse xenograft model. The effects of cytokines on CD70 expression and ADCC were investigated by exposing AML cells to conditioned medium (CM) derived from activated T cells or recombinant cytokines.Results Flow cytometry revealed CD70 expression ranging from 0.2% to 89.6% (median=7.0%, n=86) in primary AML cells across genetic subgroups; this expression remained unchanged at relapse (median=3.9%, n=14). SEA-CD70 showed potent, dose-dependent cytotoxicity against AML cell lines, primary cells, and in an SCID mouse model, which correlated with CD70 expression levels. Notably, AML cells exposed to CM from activated T cells upregulated CD70. TNF-α was identified as the driver of CD70 upregulation, translating into enhanced ADCC against AML cells (cytotoxicity w/o TNF-α = 17.9% vs with TNF-α = 34.3%, n=13–15). Conversely, IFN-γ exposure led to reduced ADCC (cytotoxicity w/o IFN-γ = 17.9% vs with IFN-γ = 9.2%, n=15), which is attributed to increased expression of NK inhibitory receptor ligands (HLA-ABC, HLA-E). Blocking of the corresponding inhibitory NK receptors (KIR/CD158b and NKG2A) partially reversed this effect. Similar findings were observed with a CD33-directed antibody, indicating a universal resistance mechanism against ADCC-based immunotherapy in AML.Conclusions CD70 is a promising target for NK cell-based immunotherapy in AML. However, IFN-γ-dependent upregulation of HLA molecules on AML cells contributes to resistance to ADCC. These findings underscore the need for rationale combination strategies in clinical trials to overcome this inducible immune escape mechanism.

Multiciliated cells are a specialized cell type found in the brain, reproductive tract and respiratory tract of mammals. Multiciliated cells resembling those of the mammalian lung can also be found on the surface of the epidermis of tadpole stage Xenopus embryos, making the frog an ideal model organism to study this cell type. KMT5B/suv4-20h1 is a histone methyltransferase that writes the dimethyl mark on histone 4, lysine 20 (H4K20). We have previously shown that the multiciliated cells of embryos lacking KMT5B have depleted cilia and a reduced actin cap, and that knockdown of both KMT5B and KMT5C/suv4-20h2 leads to downregulation of ciliogenic genes. Here we further tease out the independent function of KMT5B in multiciliogenesis, and show that single knockdown of KMT5B, but not KMT5C leads to aberrant transcription and the downregulation of cilia genes. This phenotype is ameliorated by overexpression of catalytically active PHF8, an H4K20me1 demethylase, while a hormone inducible variant of multicilin (MCI), the master regulator of cilia tuft formation, has no effect. Notably, the expression of key transcription factors of ciliogenesis is unaffected by KMT5B depletion, and the transcriptional effect of KMT5B depletion dominates the response to ectopic multicilin. Finally, ATAC seq analysis in animal caps reveals that knockdown of KMT5B results globally in very few peaks with differential activity and does not compact chromatin at ciliary genes. Taken together, this suggests that KMT5B regulates multiciliated cells through an alternative pathway to the canonical MCI-driven multiciliogenic program. ### Competing Interest Statement The authors have declared no competing interest.

The centrosome is a microtubule orchestrator, nucleating and anchoring microtubules that grow radially and exert forces on cargos. At the same time, mechanical stresses from the microenvironment and cellular shape changes compress and bend microtubules. Yet, centrosomes are membraneless organelles, raising the question of how centrosomes withstand mechanical forces. Here, we discover that centrosomes can deform and even fracture. We reveal that centrosomes experience deformations during navigational pathfinding within motile cells. Coherence of the centrosome is maintained by Dyrk3 and cNAP1, preventing fracturing by forces. While cells can compensate for the depletion of centriolar-based centrosomes, the fracturing of centrosomes impedes cellular function by generating coexisting microtubule organizing centers that compete during path navigation and thereby cause cellular entanglement in the microenvironment. Our findings show that cells actively maintain the integrity of the centrosome to withstand mechanical forces. These results suggest that centrosome stability preservation is fundamental, given that almost all cells in multicellular organisms experience forces.

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14, and 28 d after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on days 3 and 7 after vaccination as well as cell type-specific responses and response kinetics. Single-cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines, and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses.

The physiological significance of thymic positive selection and its reliance on a single stromal cell type, cortical thymic epithelial cells, remain incompletely understood. The lysosomal cysteine protease cathepsin L (CTSL) has been implicated in generating major histocompatibility complex class II-bound peptides in cortical thymic epithelial cells for efficient CD4+ T cell differentiation. Here, we addressed the extent and nature of the CD4+ T cell repertoire changes associated with CTSL deficiency. In the absence of CTSL, a highly selective loss of T cell receptors resulted in a markedly reduced repertoire diversity. However, a similarly large proportion of nominally 'CTSL-independent' T cell receptors were retained. Clones representative of the second category experienced weaker positive selection signals in the absence of CTSL, which were sufficient for further maturation yet imprinted aberrant responsiveness to agonist stimulation and impaired homeostatic behavior. Together, these findings demonstrate that CTSL is crucial for both shaping full repertoire diversity and optimizing CD4+ T cell functionality.

BACKGROUND:Patients with eosinophilic esophagitis (EoE) require long-lasting resolution of inflammation to prevent fibrostenosis and dysphagia. However, the dissociation between symptoms and histologic improvement suggests persistent molecular drivers despite histologic remission. OBJECTIVE:We characterized persisting molecular alterations in pediatric patients with EoE using tissue transcriptomics and proteomics. METHODS:Esophageal biopsy samples (n = 247) collected prospectively during 189 endoscopies from pediatric patients with EoE (n = 36, up to 11 follow-up endoscopies) and pediatric controls (n = 44, single endoscopies) were subjected to bulk transcriptomics (n = 96) and proteomics (n = 151). Intercellular junctions (desmoglein-1/3, desmoplakin, E-cadherin) and epithelial-to-mesenchymal transition (vimentin:E-cadherin ratio) were assessed by immunofluorescence staining. RESULTS:Active EoE (≥15 eosinophils per high-power field [eos/hpf]), inactive EoE (<15 eos/hpf), and deep-remission EoE (0 eos/hpf) were diagnosed in 107 of 185, 78 of 185, and 41 of 185 biopsy samples, respectively. Among the dysregulated genes (up-/downregulated 310/112) and proteins (up-/downregulated 68/16) between active EoE and controls, 17 genes, and 6 proteins remained dysregulated in inactive EoE. Using persistently upregulated genes (n = 9) and proteins (n = 3) only, such as ALOX15, CXCL1, CXCL6, CTSG, CDH26, PRRX1, CLC, EPX, and periostin (POSTN), was sufficient to separate inactive EoE and deep-remission biopsy samples from control tissue. While 32 differentially expressed genes persisted in deep-remission EoE compared to controls, the proteome normalized except for persistently upregulated POSTN. Epithelial-to-mesenchymal transition normalized in inactive EoE, whereas desmosome recovery remained impaired as a result of desmoglein-1 downregulation. CONCLUSION:The analysis of molecular changes shows persistent EoE-associated esophageal dysregulation despite histologic remission. These data expand our understanding of inflammatory processes and possible mechanisms that underlie tissue remodeling in EoE.

Variable responses to platinum chemotherapy and the emergence of resistant disease drive high mortality in high-grade serous ovarian cancer (HGSOC). To study resistance mechanisms, we developed the organoid drug resistance assay (ODR-test) with patient-derived organoids from our ovarian cancer biobank and identified sustained phenotypic reprogramming and cellular plasticity of organoids under carboplatin pressure as a conserved mechanism irrespective of the basal resistance level. Transcriptional and proteomic analyses revealed changes in cell adhesion and differentiation as adaptive responses that lead to an increase in resistance. We identified Keratin 17 (KRT17) as a mediator of platinum resistance and validated its function by CRISPR-Cas9 and overexpression. Additionally, we found that KRT17 expression status (K-score) is a significant negative prognostic histopathological biomarker in a large cohort (N = 384) of patients with advanced HGSOC. In organoids, increased KRT17 levels enhanced sensitivity to PI3K/Akt inhibitors alpelisib and afuresertib, highlighting the potential of KRT17 as a stratification biomarker for targeted therapies. Video abstract

Objective: Retinitis pigmentosa (RP) is a hereditary retinal disease characterized by progressive photoreceptor degeneration, leading to vision loss. The best hope for a cure for RP lies in gene therapy. However, given that RP patients are most often diagnosed in the midst of ongoing photoreceptor degeneration, it is unknown how the retinal proteome changes as RP disease progresses, and which changes can be prevented, halted, or reversed by gene therapy. Methods: Here, we used a Pde6b-deficient RP gene therapy mouse model and performed untargeted proteomic analysis to identify changes in protein expression during degeneration and after treatment. Results: We demonstrated that Pde6b gene restoration led to a novel form of homeostatic plasticity in rod phototransduction which functionally compensates for the decreased number of rods. By profiling protein levels of metabolic genes and measuring metabolites, we observed an upregulation of proteins associated with oxidative phosphorylation in mutant and treated photoreceptors. Conclusion: In conclusion, the metabolic demands of the retina differ in our Pde6b-deficient RP mouse model and are not rescued by gene therapy treatment. These findings provide novel insights into features of both RP disease progression and long-term rescue with gene therapy.

Introduction: Emerging evidence suggests that immune dysregulation drives resistance to cellular therapies. In particular, low CAR T-cell expansion and inferior patient outcomes have been associated with an inflamed immune milieu. Here, we investigated whether myeloid cells and serum proteomics are linked to T-cell dysfunction and treatment failure. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel, brexu-cel, or tisa-cel in the third- or later-line setting were retrospectively included (n=74). Aliquots of EDTA-anticoagulated peripheral blood and serum were collected. Immune checkpoint (IC) expression (PD-1, TIM-3, and LAG-3) was assessed through flow cytometry before CAR T-cell infusion (time of leukapheresis, day -5 and day 0) as a surrogate for T-cell exhaustion. More specifically, the abundance of different co-expression profiles of ICs was compared and defined as non-exhausted (PD-1-TIM-3-LAG-3-), progenitor-exhausted (PD-1+TIM-3+LAG-3-), and terminally-exhausted (PD-1+TIM-3+LAG-3+). Using the Olink® Immuno-Oncology panel, 92 immune-related proteins were measured at day 0. A next-generation single-cell proteogenomics approach (BD RhapsodyTM) was applied to analyze the transcriptome of myeloid cells before and after infusion (day -5 and day 7). Responding patients (R, complete or partial remission) were compared to non-responding patients (NR, stable or progressive disease) according to 3-month (PET-) CT scans. Results: At baseline, NR showed lower frequencies of non-exhausted (apheresis, p = 0.025; day -5, p = 0.0236; day 0, p = 0.0496) and higher frequencies of terminally-exhausted T-cells (-5, p = 0.0053) compared to R. Interestingly, these surrogates for pre-existing T-cell dysfunction in NR were linked to an inflammatory state. Patients with low levels of non-exhausted T-cells also had higher levels of CRP (p = 0.0059), Ferritin (p = 0.0333), and LDH (p = 0.0268) compared to patients with high levels of non-exhausted T-cells before infusion. Network analysis correlating the abundance of immune-modulatory proteins with baseline IC expression on T cells revealed numerous connections for NR but merely associations for R, suggesting an impact of the serum protein milieu on IC expression in NR. We dissected these networks in terms of the degree centrality differences for the different proteins (i.e., the variations in the number of connections among the nodes between NR and R). While we identified a higher degree centrality for co-stimulatory proteins such as IL-18, CD8A and ICOSLG in R, we noted higher degree centrality for immune-inhibitory and tumor-related proteins such as CXC3L1, TNFRSF21, VEGFA and IL-6 in NR. Accordingly, we linked a higher abundance of CD8A and a lower abundance of TNFRSF21 and IL-6 to the frequency of non-exhausted T-cells at baseline using a linear regression model. Finally, we asked whether the myeloid compartment at baseline drives these differences in the serum protein milieu observed between NR and R. We identified four phenotypic and transcriptomic different monocytic and dendritic cell populations. Interestingly, two classical monocyte-like populations (CD14+CD16+HLA-DRlo) showed divergent population dynamics in NR but not R when assessing pre- and post-infusion time points. Notably, gene set enrichment analysis revealed an upregulation of immunosuppressive pathways, such as genes regulated by NF-KB in response to TNF, in several myeloid populations in NR but not R (classical monocyte-like 2, p < 0.0001; non-classical monocyte-like, p < 0.0001; DC-like, p = 0.0024). Surprisingly, monocytic populations in R revealed an upregulation of genes with immune stimulatory functions linked to responses to interferon-alpha or -gamma proteins (classical monocyte-like 1 and 2, p = 0.0058 and p = 0.0003; non-classical monocyte-like, p = 0.0003). Conclusion: These data suggest that CAR-T non-responders exhibit pre-existing T-cell dysfunction resulting from a systemic inflamed proteomic and cellular environment. Notably, the profound alterations within the myeloid compartment highlight diverging immune-modulating functions in non-responding and responding B-NHL patients. These findings set the further scope for scientific investigations not only to mitigate immune dysregulation but also to target the innate immune compartment for enhanced CAR T-cell responses.