Immunology & Cell Biology

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

The cohesin ring complex organizes and mediates functional activity of the genome through chromatin loop extrusion1,2. Cohesin comprises a core SMC1-SMC3-RAD21 ring associated with multiple regulatory proteins, including SA1 and SA2 paralogs that bind mutually-exclusively to a RAD21 site3,4. Functional studies of cohesin complexes in vivo often employ inactivation of particular components2,3. However, inactivation of SA1 or SA2 in mice is limited by mouse embryonic lethality5-7 and, for dual SA1/2-inactivation by cycling cell lethality8-10. For these reasons, it remains unknown whether the requirement for SA1/2 in loop extrusion in vitro11,12 applies in vivo1,2, and whether these factors have overlapping or distinct functions in regulating this process3. Here, we employ G0/G1-arrested v-Abl pro-B lines, which are viable in the absence of cohesin-complex factors13,14, to dissect SA1 and SA2 functions in V(D)J recombination, a strictly G0/G1-specific process that harnesses loop extrusion to effect distinct Igh and Ig{kappa} locus recombination mechanisms15,16. Robust long-range Igh or Ig{kappa} V(D)J recombination continued unabated in the absence of SA1 or SA2. However, inactivation of both abrogated most positioned chromatin loops genome-wide and also abolished long-range Igh V(D)J recombination with a phenotype similar to RAD21 (cohesin) inactivation13. Yet, robust Ig{kappa} V(D)J recombination continued upon dual SA1/2-inactivation, which along with Hi-C findings, demonstrates ongoing loop-extrusion in the absence of SA1/2. Correspondingly, replacement of weak Igh V(D)J recombination signal sequences (RSSs) that target V(D)J recombination activity, with strong Ig{kappa} counterparts15 restored long-range Igh V(D)J recombination in SA1/2 inactivated cells. We conclude that SA1/2 together maintain loop extrusion impediments and genome-wide architecture in G0/G1-arrested cells; and, in their absence, loop extrusion proceeds unimpeded. Remarkably, strong RSSs allow Ig{kappa} to function normally in the absence of internal positioned loop structure across the 3.1-mega-base Ig{kappa} locus. We discuss implications of our findings for long-range gene regulation in G0/G1-arrested cells more broadly.

Hematopoiesis sustains lifelong blood production and immunity and is strongly influenced by biological sex. Males and females differ in their hematopoietic composition and susceptibility to infection and hematologic disease. Because current models cannot readily disentangle the independent contributions of sex chromosomes versus hormones, the mechanisms underlying sexual dimorphism remain poorly understood. Zebrafish have proven to be a powerful, translatable system for studying hematopoiesis, and offer a unique opportunity to isolate hormonal contributions due to their absence of sex chromosomes. However, whether sex is regulated in the zebrafish hematopoietic system remained unknown. Here, we discovered pronounced sexual dimorphism in the adult zebrafish marrow, with compositional differences peaking at reproductive age. Transcriptome analysis of marrows revealed conservation of sex-specific programs between zebrafish and human datasets. Upon immune challenge with lipopolysaccharide, all major blood lineages exhibited sex-biased changes in their composition and transcriptomes with the largest responses in erythroid and myeloid cell types. Among these sex differences was an unexpected immunoregulation of the erythroid lineage across maturation states. These findings establish biological sex as a major regulator of zebrafish hematopoiesis and immunity, paralleling humans, and lay the groundwork necessary for future mechanistic studies on sexual dimorphism in hematologic health and disease.

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

The human host defence peptide LL-37 mediates pleiotropic immunomodulatory functions that can be both pro- and anti-inflammatory. Under inflammatory conditions in the lungs, LL-37 is susceptible to the post-translational modification (PTM) citrullination. The impact of this PTM on LL-37's immunomodulatory functions is not fully understood. Therefore, we characterized the transcriptome and cytokine profile in response to LL-37 and citrullinated-LL-37 (citLL-37), in human bronchial epithelial cells (HBEC). Cells were stimulated with either LL-37 or citLL-37 (0.5 M), or a scrambled peptide (sLL-37). RNA after 4 hours (h) was analyzed with NanoString nCounter Host Response Panel, and 96 cytokines were examined (after 24 h) in tissue culture supernatants (Luminex platform). Genes and cytokines with a Log2 fold-change [&ge;]0.5 with p<0.05 compared to unstimulated cells were considered differentially expressed (DE). These studies revealed overlapping and distinct biosignatures. There were 118 DE genes with LL-37 and 71 with citLL-37, of which magnitude of change was significantly different for 32 genes. 16 out of 17 DE genes altered by both peptides were directly related to inflammation, and these were significantly less (by ~45%) with citLL-37 compared to LL-37. Similarly, inflammatory cytokines enhanced in response to LL-37 were significantly less (by 37-94%) with citLL-37. Overall, our findings indicate that citrullination of LL-37 does not abrogate cellular response in HBEC, instead dampens the inflammatory biosignature induced by LL-37.

Somatic mutations in the de novo DNA methyltransferase DNMT3A are frequent in clonal hematopoiesis, but how they alter the function of differentiated myeloid cells remains unclear. Here, we used isogenic human embryonic stem cell-derived macrophage models to define the consequences of DNMT3A dysfunction during myeloid differentiation. DNMT3A-mutant macrophages selectively upregulated bivalent Polycomb target genes, accompanied by DNA hypomethylation, reduced H3K27me3 and increased promoter accessibility. Despite broad epigenetic changes across bivalent promoters, transcriptional derepression occurred at a subset of loci characterized by lower baseline DNA methylation and higher H3K27me3, identifying pre-existing chromatin state as a determinant of transcriptional response. This Polycomb-associated program was conserved in murine macrophages and enriched for genes involved in cell migration and wound repair. DNMT3A-mutant macrophages exhibited enhanced migration and preferential early recruitment to injured tissue. These findings link clonal hematopoiesis-associated epigenetic alterations to selective transcriptional reprogramming and altered function of differentiated myeloid cells.

Asthma severity and immune responses are regulated by circadian rhythms, yet the role of circadian timing in fungal allergen-induced airway inflammation remains poorly defined. Alternaria alternata, a clinically relevant fungal allergen, is strongly associated with severe asthma exacerbations and persistent airway dysfunction. Using a preclinical mouse model, we investigated whether pulmonary responses to Alternaria exposure are influenced by time of day and sex. We demonstrate that Alternaria induces allergic airway inflammation, airway hyperresponsiveness (AHR), and epithelial barrier disruption in a circadian- and sex-dependent manner. Female mice exhibited enhanced eosinophilic, neutrophilic, interstitial macrophage, and dendritic cell infiltration predominantly at Zeitgeber Time 0 (ZT0), whereas males showed stronger inflammatory responses at ZT12. Alternaria exposure increased IL-33 release and promoted ILC2 and Th2 cell accumulation, with distinct temporal patterns between sexes. Cytokine and chemokine profiling revealed robust Th2, proinflammatory, and Th17 responses that varied with exposure time, with higher responses at ZT12 in females and ZT0 in males. Markers of tissue injury, including lactate dehydrogenase and uric acid, followed similar circadian-dependent trends. Functionally, Alternaria-exposed mice displayed greater AHR during the resting phase, particularly in males. Additionally, Alternaria disrupted epithelial junctional proteins (E-cadherin and ZO-1) and altered expression of core circadian clock proteins CLOCK, BMAL1, and REV-ERB. Mechanistically, peak inflammation coincided with enhanced STAT1, STAT3, and STAT6 activation. Collectively, these findings identify circadian timing and sex as key determinants of fungal allergen-induced airway inflammation and suggest chronotherapeutic approaches may improve asthma outcomes.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

Clinical translation of mesenchymal stromal cell (MSC) therapies derived from primary tissue sources is limited by manufacturing inconsistencies driven by donor-to-donor variability, which reduces predictability of clinical response. Induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) offer a promising donor-independent, clonal alternative strategy that is also more amenable to engineering, but whether engineered iMSCs reproduce the potency attributes of primary MSCs remains unclear. Here, we benchmarked a doxycycline-inducible, hTERT-immortalized iMSC line engineered to overexpress TGFB1 (TGFB1-iMSCs), a key regulator of macrophage polarization, against multiple adipose tissue-derived MSC (MSC(AT)) donors across predefined immunomodulatory and angiogenic potency attributes. TGFB1-iMSCs were smaller and more circular with comparable or higher proliferative rates than primary MSC(AT) donors. Interestingly, they had a distinct angiogenic signature (EDIL3, EDN1, PDGFA), and nine differentially expressed microRNAs were predicted to target secretory trafficking and growth factor receptor signalling. Functionally, however, TGFB1-iMSCs secreted less VEGF and showed intermediate HUVEC tube formation, but matched or exceeded all primary MSC(AT) donors in a monocyte-macrophage transwell immunomodulatory assay. Notably, in a murine destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis, a single intra-articular injection of TGFB1-iMSCs, but not MSC(AT), reduced total synovial macrophage numbers and lowered the MHCII/CD206 ratio at eight weeks, although it did not reduce cartilage degeneration, synovitis, or fibrosis. Together, these findings show that TGFB1-engineered iMSCs have a macrophage-directed potency profile distinct from that of primary MSC(AT). Immortalized iMSCs therefore provide a reproducible platform for mechanistically dissecting engineered MSC potency and its effects on the immune environment.

Hematopoietic stem and progenitor cells (HSPC) depend on interactions with nearby stromal cells to support their homeostatic functions and mount responses to perturbations. The murine bone marrow (BM) niche can be profiled using single-cell RNA sequencing (scRNA-seq), but there is considerable inconsistency in cell type identification, and uncertainty over how populations can be isolated prospectively by flow cytometry. Here, we produced new scRNA-seq reference datasets using purified populations of murine BM stromal cells, which we used to create a bespoke classifier for stromal cells. Our NicheScribe approach is consistent, accurate, and generalizable to query datasets irrespective of methods used for cell isolation. Moreover, consistent annotation permits comparative analyses of the same stromal cells across different bone types and perturbations like inflammation, neoplasia, and aging. Our work provides new tools for consistent cell type annotation in the murine BM niche and new insights for combined molecular and functional analyses.

Tuberculosis (TB) disease consistently causes the highest number of annual deaths from infectious disease and is responsible for over 100 million disability-adjusted-life-years annually. Despite the dramatic impact on human health, TB treatments are intensive and moderately effective, while Bacille Calmette-Guerin (BCG), the primary TB vaccine strategy and the oldest vaccine in circulation, needs updating. One gap in knowledge is that the vaccine primed lung immune environment has not been systematically profiled following TB challenge, making it challenging to interpret how various vaccine strategies provide protection. Here we profiled the male and female mouse lung immune environment using Mass Cytometry by Time of Flight (CyTOF) and bacterial burden (colony forming units, CFU), to systematically identify immune cells contributing to Mycobacterium tuberculosis (Mtb) infection or vaccine-induced protection. We investigated the mouse immune system after various infection outcomes including primary Mtb infection, Mtb reinfection, Mtb challenge after BCG vaccination and Mtb challenge after vaccination with a live attenuated Mtb vaccine. Across all conditions we identified Thy1.2 expression on NK cells as a new candidate for a sex-specific correlate of protection. Our results further replicate and support known immune responses for vaccine-induced protection, such as T-cell activation and infiltration. These findings highlight the pan-immune response to vaccine-induced protection, showcase the importance of sex in rodent models of TB, and identify novel host immune candidates to protect the lung against TB disease.

Herpes zoster (HZ), caused by varicella zoster virus (VZV) reactivation, is common in older adults with declining cell-mediated immune protection. A modestly efficacious live attenuated (ZVL) and a highly efficacious recombinant adjuvanted (RZV) zoster vaccine protect against HZ. To uncover the mechanism(s) associated with the superior efficacy of RZV, we developed a controlled human infection model consisting of intradermal administration of attenuated vOka VZV to older adults previously vaccinated with RZV or ZVL. Infiltration of VZV-specific CD4+ and CD8+ T cell clonotypes at the infection site was quicker and higher in RZV than ZVL recipients [&ge;]5 years post-immunization due to the persistence of RZV-induced gE-reactive CD4+ and CD8+ T cell clonotypes in blood. High CD4+ cytotoxic T lymphocyte frequencies in blood on Day 1 post-inoculation predicted control of VZV replication. Persistence of circulating RZV-induced CD4+ and CD8+ CTL and rapid migration to sites of VZV replication are potential mechanisms underlying durable protection against HZ.

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR+) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR+ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1Cre; Scf fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance.

B-cell activation requires extensive metabolic reprogramming, yet how B cells acquire and utilize extracellular fatty acids and adapt to changes in lipid availability remains poorly understood. Here, we show that B-cell activation and the extracellular lipid environment dynamically regulate B cells. B-cell receptor (BCR) and Toll-like receptor 4 (TLR4) stimulation induced distinct mitochondrial and lipid-handling programs, differentially regulating fatty-acid uptake, cholesterol acquisition, lipid storage and mitochondrial respiration. Disrupting long-chain fatty-acid uptake, acyl-CoA activation, or mitochondrial utilisation impaired B-cell proliferation, demonstrating that multiple steps of fatty-acid metabolism are required for B-cell expansion. However, increasing extracellular fatty-acid availability alone did not enhance proliferation, indicating that fatty-acid acquisition and metabolic utilisation are functionally distinct. B cells also acquired fatty acids directly from adipocytes, identifying the local microenvironment as a potential source of metabolic substrates. In vivo, dietary fatty-acid chain length differentially shaped the germinal-centre response. A long-chain fatty-acid-rich diet increased germinal-centre B cells and T follicular helper cells and induced mitochondrial and transcriptional changes that partially converged with those observed following obesogenic feeding. Circulating lipidomics further revealed shared remodelling of lipid species between long-chain fatty-acid-rich and obesogenic diets, including altered partitioning of stearate-containing lipids. Exogenous fatty acids also modified membrane order in activated B cells, suggesting that lipid availability can influence both metabolic and biophysical properties. Together, these findings establish fatty-acid uptake, activation, and utilization as integral components of B-cell metabolic adaptation and demonstrate that dietary fatty-acid composition can remodel the lipid environment and germinal-centre response.

Pulmonary lymphocytosis is a common feature of chronic lung disease, but the mechanisms governing lymphocyte accumulation within the lung remain poorly understood. Here, we identify granulocyte-macrophage colony-stimulating factor (GM-CSF) as a central regulator of pulmonary lymphocyte homeostasis. Using genetic models, single-nucleus and spatial transcriptomics, cell trafficking analyses, and pulmonary macrophage replacement studies, we show that loss of GM-CSF signaling causes profound pulmonary lymphocytosis characterized by lymphoid follicle formation, local immunoglobulin production, and expansion of multiple lymphocyte lineages. Mechanistically, GM-CSF signaling deficiency blocks terminal alveolar macrophage differentiation and reprograms alveolar macrophages into inflammatory states marked by reduced PU.1 and PPAR{gamma} expression, enhanced NF-{kappa}B signaling, and expression of lymphocyte-recruiting chemokines. These changes promote lymphocyte recruitment, local proliferation, and lymphoid lineage commitment. Restoration of GM-CSF-responsive alveolar macrophages re-established macrophage identity, suppressed inflammatory chemokine programs, and reversed pulmonary lymphocytosis. Together, these findings identify alveolar macrophages as gatekeepers of adaptive immune homeostasis in the lung.

Background Vitiligo is an acquired depigmenting disorder characterised by melanocyte loss. While immune responses against melanocytes are implicated in its pathogenesis, melanocyte detachment, melanocyte senescence and aberrant epidermal differentiation are increasingly characterised as additional pathological features. Granzyme B (GzmB), classically recognised as an intracellular effector of perforin-dependent cytotoxicity, is increased in vitiligo lesions; however, its extracellular contribution to vitiligo pathology remains unclear. Objectives To determine whether extracellular GzmB contributes to depigmentation and epidermal pathological features relevant to vitiligo, and to define its cellular source and underlying mechanisms. Methods Human vitiligo skin samples, murine depigmentation models and cultured human epidermal cells were analysed. The functional effects of extracellular GzmB were examined by subcutaneous administration of recombinant GzmB in mice. A rhododendrol (RD)-induced leukoderma model was used to evaluate the disease relevance of GzmB and the therapeutic effect of topical GzmB inhibition with VTI-1002. Results GzmB-positive cells were markedly increased in active vitiligo lesions and were predominantly associated with tryptase-positive cells, with limited co-distribution with CD8 or perforin. Vitiligo lesions also showed melanocyte detachment, increased p16INK4A-positive melanocytes and aberrant keratinocyte differentiation. These pathological features were recapitulated in vivo by extracellular GzmB administration, accompanied by focal depigmentation. In cultured melanocytes, GzmB exerted no cytotoxic effects but reduced attachment strength and induced a senescence-associated phenotype characterised by decreased extracellular matrix- and adhesion-related gene expression, increased p16INK4A level, elevated senescence-associated {beta}-galactosidase activity and activation of transforming growth factor-{beta}/SMAD signalling. In keratinocytes, extracellular GzmB promoted aberrant differentiation associated with activation of p53 signalling. Topical inhibition of GzmB in RD-induced leukoderma attenuated depigmentation progression, melanocyte detachment, melanocyte senescence and abnormal epidermal differentiation. Conclusions Extracellular GzmB promotes depigmentation associated with inducing melanocyte detachment, melanocyte senescence and aberrant keratinocyte differentiation. These findings identify extracellular GzmB as a previously underrecognised pathogenic mediator and potential therapeutic target in vitiligo-associated depigmentation.

Abstract Background: Experimental models that retain endogenous tumor immune complexity are needed to investigate heterogeneous responses to immune checkpoint inhibitors (ICIs) in melanoma. We established patient-derived melanoma organoids (PDMOs) to examine retention of parental tumor cellular components and investigate patient-specific and context-dependent responses to checkpoint blockade. Methods: Fresh melanoma specimens (n=50), including primary tumors, neoadjuvant-treated tumors and tumor-infiltrating lymphocyte (TIL) associated samples were processed for Matrigel-embedded culture of patient-derived melanoma organoids (PDMOs). Characterization and functional studies were performed in subsets of established cultures. Immunofluorescence assessed tumor, stromal, immune, and checkpoint-marker expression in PDMOs and matched parental tissues. NGFR expression was evaluated in relation to organoid establishment and growth. Responses to anti PD1 alone or combined with anti-LAG3 or anti-CTLA4 therapies were evaluated using viability and morphological analyses, with selected models undergoing immune phenotyping and multiplex cytokine profiling. Available clinical outcomes were used for exploratory comparison. PDMOs and matched two-dimensional cultures were evaluated under 21% and 5% oxygen conditions. Results: PDMOs were established from 30 of 50 specimens (60%) and retained melanoma, stromal, lymphoid, and myeloid components identified in matched parental tissues during early culture, together with immune checkpoint expression. Higher NGFR (CD271) expression was associated with greater organoid-forming capacity in an exploratory subset. Responses to checkpoint blockade varied across patient-derived models and regimens, with combination blockade not uniformly producing lower viability than PD1 monotherapy. Ex vivo responses showed parallels with available clinical outcomes, with discordant cases also observed. In three selected PDMOs, divergent PD-1 responses were accompanied by differences in T-cell and myeloid representation, MHCII and AXL expression, and inflammatory versus immunoregulatory cytokine profiles. Under normoxic conditions, PDMOs and matched two-dimensional cultures exhibited divergent treatment sensitivities, including clinically responding cases in which sensitivity was retained in PDMOs but attenuated in monolayers. Changing oxygen tension further altered checkpoint sensitivity within individual PDMOs, with the direction and magnitude of the observed shifts varying by model and regimen. Conclusions: Early passage PDMOs retain key endogenous tumor, immune, and stromal components and support functional investigation of patient-derived melanoma biology. Their heterogenous responses across checkpoint regimens and oxygen conditions provide a platform for investigating how tumor-immune composition and environmental context influence treatment sensitivity.

The non-canonical NF-kappa B pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c-terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2p.Tyr867*). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5+ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4+ and CD8+ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4+ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM+IgD- transitional cells and a marked reduction of IgD+ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

Adult stem cells maintain tissue homeostasis and regenerative capacity, but mechanisms preserving their integrity under stress remain incompletely understood. Here, we identify BRN2 (POU3F2), a POU-domain transcription factor, as a regulator of melanocyte lineage homeostasis and response to genotoxic stress. Single-cell transcriptomic analyses identified Brn2 among transcription factors preferentially active in melanocyte stem cells (McSCs), with BRN2 activity declining with age in human hair follicle melanocytic cells. Melanocyte-specific Brn2 deletion increased melanocyte differentiation and accumulation in the hair bulb, while downregulating DNA-repair and chromosome-maintenance programs. Despite these changes, Brn2 loss caused neither spontaneous hair greying nor detectable McSC depletion under steady-state conditions. Following ionizing radiation, however, Brn2-deficient cells showed altered DNA-damage responses, reduced mitotic signatures, increased p53 and p21, and enhanced melanocytic differentiation. Irradiated Brn2-deficient mice developed rapid, dose-dependent, permanent hair depigmentation associated with McSC and melanocyte depletion. Thus, BRN2 links POU-dependent transcriptional control to stem-cell resilience under genotoxic stress, preserving melanocyte regenerative capacity and protecting against irradiation-induced hair greying.