Peter Brazda

Immune reconstitution after allogeneic hematopoietic stem cell transplantation is influenced by graft-composition and viral reactivation, but the combined long-term impact on αβ and γδT cells remains unclear. We analyzed a cohort of 213 patients receiving either αβT cell-depleted grafts (n=146; graft engineering that removes donor αβT cells) or T cell-replete grafts (n=67; containing donor T cells). Longitudinal immune phenotyping was integrated with bulk and single-cell TCR repertoire and transcriptomic profiling. CMV reactivation was associated with expansion of CD8 ⁺ αβT cells across both transplant types and with numerical dominance of Vδ2 ⁻ γδT cells specifically in αβT cell-depleted recipients. Vδ2 ⁻ γδT cells underwent early polyclonal expansion followed by repertoire focusing, independent of CMV, whereas αβT cells remained clonally restricted. Reduced early Vδ2 ⁺ γδTCR diversity was associated with EBV reactivation. Single-cell and TCR tracking analyses revealed long-term persistence of donor-derived Vδ2 ⁺ γδTCRs, whereas Vδ1 ⁺ γδ and αβT cell repertoires were predominantly rebuilt de novo. Despite de novo rebuilding, αβTCR repertoire diversity diverged by platform at one year: αβT cell-depleted recipients exhibited marked (hyper)expansion of αβTCR clonotypes and lower diversity than T cell-replete recipients, indicating a durable imprint of graft engineering on αβTCR-clonality. Transcriptomic profiling showed that post-transplant T cells predominantly adopted effector programs, with platform-dependent polarization toward cytotoxic signatures in αβT cell–depleted recipients and toward AREG-associated tissue-repair signatures in T cell–replete recipients, consistent with wound-healing functions. In conclusion, transplantation platforms imprint durable clonal and transcriptional remodeling of αβ and γδT cells, while viral reactivation primarily amplifies expansion without fundamentally reshaping repertoire architecture. Abstract Figure:

Background: Osteoarthritis is a degenerative joint disease characterized by progressive loss of articular cartilage and limited capacity for intrinsic repair. A major barrier to developing effective regenerative strategies is the incomplete understanding of the molecular mechanisms regulating chondrogenesis and cartilage maintenance. Cell surface proteins are key mediators of extracellular communication, adhesion, and signaling, yet the chondrogenic surfaceome remains incompletely mapped, with prior studies focusing primarily on mature or cytokine-activated chondrocytes. The aim of this study was to provide a temporal profile of the surfaceome during in vitro chondrogenic differentiation and to identify novel membrane proteins with potential roles in cartilage biology. Methods: We applied a sialoglycoprotein-targeted glycocapture strategy to selectively enrich plasma membrane proteins from chick embryonic limb bud-derived micromass cultures undergoing chondrogenesis. Enriched samples underwent high-resolution shotgun proteomic analysis, and differentially expressed candidates were validated by western blotting, immunocytochemistry, and transient gene silencing. Functional effects on extracellular matrix gene regulation were assessed by quantitative RT-PCR and matrix histochemistry. Results: This approach generated the temporal surfaceome map of chondrogenic progenitors. Among identified candidates, two proteins not previously linked to chondrogenesis, podocalyxin (PODXL) and ciliary neurotrophic factor receptor (CNTFR), were detected at the plasma membrane and confirmed at the protein and transcript levels. Both proteins exhibited time-dependent downregulation during differentiation. Targeted knockdown revealed differential regulation of the fibrocartilage marker COL1A1 expression, indicating non-redundant roles in cell-matrix signaling and survival pathways. Single-cell transcriptomic meta-analysis confirmed expression of both proteins in discrete human articular chondrocyte subpopulations. Conclusions: This study expands the molecular framework of chondrogenesis, identifying PODXL and CNTFR as novel, temporally regulated surfaceome components with distinct roles in extracellular matrix signaling. These findings complement prior proteomic analyses of cytokine-activated mature articular chondrocytes and suggest new candidates for developmental cartilage biomarkers and therapeutic targets for osteoarthritis. Our results provide a resource for future cross-species surfaceome studies and highlight key pathways for further investigation into cartilage lineage specification and matrix adaptation.

Colorectal cancer (CRC) arises in the colorectal tissue driven by genetic disorder or the accumulation of somatic mutations, leading to abnormal epithelial cell growth. In this study, we employed single‐nucleus multi‐omics analysis, including single‐nucleus RNA‐seq and single‐nucleus ATAC‐seq, on over 100,000 high‐quality nuclei to investigate the molecular landscape of both primary tissue and patient‐derived organoids (PDOs). Our analysis showed that normal PDOs (N‐PDOs) derived from tissue adjacent to tumors replicate the cellular composition and differentiation trajectory of colorectal crypts. In contrast, tumor PDOs (T‐PDOs) showed patient‐specific transcriptomic and epigenomic heterogeneity yet consistently maintained a stem cell‐like state. T‐PDOs retained the somatic mutation profile of the primary tumor while also exhibiting de novo mutations not detected in either the primary tumor or N‐PDOs. Notably, inferred cell–cell interaction analysis highlighted the activin signaling pathway as a potential unique feature of fibroblast‐epithelial interactions within the tumor microenvironment. This study provides a comprehensive view of the transition from normal to malignant colorectal epithelium and underscores the utility of PDOs as a faithful model for capturing both conserved and patient‐specific features of colorectal cancer.

Background: Osteoarthritis is a degenerative joint disease characterized by progressive loss of articular cartilage and limited capacity for intrinsic repair. A major barrier to developing effective regenerative strategies is the incomplete understanding of the molecular mechanisms regulating chondrogenesis and cartilage maintenance. Cell surface proteins are key mediators of extracellular communication, adhesion, and signaling, yet the chondrogenic surfaceome remains incompletely mapped, with prior studies focusing primarily on mature or cytokine-activated chondrocytes. The aim of this study was to provide a temporal profile of the surfaceome during in vitro chondrogenic differentiation and to identify novel membrane proteins with potential roles in cartilage biology. Methods: We applied a sialoglycoprotein-targeted glycocapture strategy to selectively enrich plasma membrane proteins from chick embryonic limb bud-derived micromass cultures undergoing chondrogenesis. Enriched samples underwent high-resolution shotgun proteomic analysis, and differentially expressed candidates were validated by western blotting, immunocytochemistry, and transient gene silencing. Functional effects on extracellular matrix gene regulation were assessed by quantitative RT-PCR and matrix histochemistry. Results: This approach generated the first temporal surfaceome map of chondrogenic progenitors. Among identified candidates, two proteins not previously linked to chondrogenesis, podocalyxin (PODXL) and ciliary neurotrophic factor receptor (CNTFR), were detected at the plasma membrane and confirmed at the protein and transcript levels. Both proteins exhibited time-dependent downregulation during differentiation. Targeted knockdown revealed differential regulation of the fibrocartilage marker COL1A1 expression, indicating non-redundant roles in cell-matrix signaling and survival pathways. Single-cell transcriptomic meta-analysis confirmed expression of both proteins in discrete human articular chondrocyte subpopulations. Conclusions: This study expands the molecular framework of chondrogenesis, identifying PODXL and CNTFR as novel, temporally regulated surfaceome components with distinct roles in extracellular matrix signaling. These findings complement prior proteomic analyses of cytokine-activated mature articular chondrocytes and suggest new candidates for developmental cartilage biomarkers and therapeutic targets for osteoarthritis. Our results provide a resource for future cross-species surfaceome studies and highlight key pathways for further investigation into cartilage lineage specification and matrix adaptation. Plain English Summary: Osteoarthritis is the most common joint disease worldwide, causing pain, stiffness, and reduced mobility as the smooth cartilage that covers the ends of bones gradually breaks down. Cartilage has a very limited ability to heal itself, and there are currently no treatments that can stop or reverse the disease. Scientists are working on new ways to repair or replace damaged cartilage, but to do this effectively, they need to understand the molecular “language” that cartilage-forming cells use to develop and maintain healthy tissue. In this study, we examined the set of proteins that are present on the surface of cartilage-forming cells, known as the “surfaceome.” These surface proteins play important roles in how cells connect to their surroundings, receive signals, and work together to build the cartilage matrix. Using a method to selectively capture and analyze these proteins, we discovered two that had never been linked to cartilage development: podocalyxin and ciliary neurotrophic factor receptor. We found that these two proteins may have opposite effects on cartilage quality. Because they can be detected on the outside of cells, they could be developed into markers for early osteoarthritis detection or as targets for new therapies. Our findings open the door to new strategies for diagnosing and treating joint disease by focusing on cell-surface communication.

The rise of cellular immunotherapy for cancer treatment has led to the utilization of immune oncology cocultures to simulate T cell interactions with cancer cells for assessing their antitumor response. Previously, we developed BEHAV3D, a three-dimensional live imaging platform of patient-derived tumor organoid (PDO) and engineered T cell cocultures, that analyzes T cells’ dynamics to gain crucial insights into their behavior during tumor targeting. However, live imaging alone cannot determine the molecular drivers behind these behaviors. Conversely, single-cell RNA sequencing (scRNA-seq) allows researchers to analyze the transcriptional profiles of individual cells but lacks spatio-temporal resolution. Here we present an extension to the BEHAV3D protocol, called Behavior-Guided Transcriptomics (BGT), for integration of T cell live imaging data with single-cell transcriptomics, enabling analysis of gene programs linked to dynamic T cell behaviors. BGT uses live imaging data processed by BEHAV3D to guide the experimental setup for cell separation based on their PDO engagement levels subsequently followed by fluorescence-activated cell sorting and scRNA-seq. It then integrates in silico simulations of these experiments to computationally infer T cell behavior on scRNA-seq data, uncovering new biomarkers for both highly functional and ineffective T cells, that could be exploited to enhance therapeutic efficacy. The protocol, designed for users with fundamental cell culture, imaging and programming skills, is readily adaptable to diverse coculture settings and takes one month to perform.

Vγ9Vδ2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of Vγ9Vδ2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable Vγ9Vδ2 TCR binding to tumor cells. However, full activation of T cells through a Vγ9Vδ2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed Vγ9Vδ2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a Vγ9Vδ2TCR.

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αβ T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28_WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BB_CD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BB_CD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8⁺ TEGs, the NKG2D-4-1BB_CD28TM chimera mainly skewed CD4⁺ TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8⁺ subset toward a proliferative state.

Celiac disease is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine leading to destruction of the lining epithelium. Current treatment focusses on lifelong adherence to a gluten-free diet. Gluten-specific CD4⁺ T cells and cytotoxic intraepithelial CD8⁺ T cells have been proposed to be central in disease pathogenesis. Here we use unbiased single-cell RNA-sequencing and explore the heterogeneity of CD45⁺ immune cells in the human small intestine. We show altered myeloid cell transcriptomes present in active celiac lesions. CD4⁺ and CD8⁺ T cells transcriptomes show extensive changes and we define a natural intraepithelial lymphocyte population that is reduced in celiac disease. We show that the immune landscape in Celiac patients on a gluten-free diet is only partially restored compared to control samples. Altogether, we provide a single cell transcriptomic resource that can inform the immune landscape of the small intestine during Celiac disease.