Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multi-omics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. We present here a comprehensive multi-omics view of CTCL clonal evolution, incorporating exome, whole genome, epigenome, bulk, single cell (sc) TCR, and scRNA sequencing of 99 clinically annotated serial skin, peripheral blood, and lymph node samples from 34 CTCL patients. We leveraged this extensive dataset to define the molecular underpinnings of CTCL progression in individual patients at single cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, PI3K signaling, and PD-1 checkpoint pathways as evasion tactics deployed by malignant T-cells. We identified a gain of function mutation in STAT3 (D661Y) and demonstrated by CUT&RUN- and RNA-seq that it enhances binding to and transcription of genes in Rho GTPase pathways. With our previous work implicating this pathway in HDACi-resistant CTCL, these data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. Recurrent progression-associated mutations were common in the epigenetic modifier EZH2, suggesting that EZH2 inhibition may benefit patients with CTCL. Our findings support an approach in which genomic analysis is widely utilized for improved disease monitoring, biomarker-informed clinical trial design, and genome-guided therapeutic decision making. Moreover, these molecular changes present new opportunities for therapeutic targeting in this challenging and incurable cancer.

Despite extensive pretransplant serological screening and HLA matching, 10-15% of kidney allografts experience acute rejection within the first year. Currently, risk stratification for transplantation relies primarily on antibody reactivity to HLA molecules, with no assessment of the T cell compartment before or after transplantation. In our previously established longitudinal cohort of 54 patients, T cell receptor β (TCRβ) sequencing was performed on paired kidney biopsy and peripheral blood samples. Here, we further analyzed the data to construct a comprehensive set of sequence-similarity networks and quantify over 30 network metrics. After adjusting for repertoire size, graft status was the strongest signal for the underlying differences in network metrics. Individuals who rejected the kidney graft generally exhibited more fragmented and less connected networks at baseline, with fewer interconnect T cell clones and more isolated sequences. Notably, pre-transplant peripheral blood mononuclear cell (PBMC) network topology alone predicted non-stable outcomes with an area under the curve (AUC) of 0.81, sensitivity of 76%, and specificity of 76%. The performance of this prediction model was independent of HLA mismatch, while changes in network topology at three months post-transplantation further improved prediction to an AUC of 0.88 (permutation p = 0.009). Collectively, TCR sequencing and network analysis represent a potential novel, non-invasive approach for pre-transplant risk stratification and immune monitoring, capturing functional immunological risk that may not be accessible through HLA genotyping or serology.

Abstract Introduction T follicular helper (Tfh) cells are crucial in germinal center (GC) reactions, supporting B-cell maturation and long-term antibody production. While Tfh dynamics during primary and secondary immune responses are well-studied in mice, little is known about human Tfh behavior in draining lymph nodes after repeat vaccination. Recent advances, such as serial fine-needle aspiration (FNA) of lymph nodes, now make it possible to monitor these responses in humans. The present study aimed to track and characterize human spike-specific Tfh cells in the draining lymph node following SARS-CoV-2 mRNA booster vaccination, and to compare these memory responses to the primary vaccination phase. Methods We conducted serial lymph node FNAs in healthy immunologically naïve adults receiving primary and booster mRNA COVID-19 vaccines (1 year apart). We used single-cell RNA sequencing with paired T cell receptor (TCR) sequencing to analyze spike-specific Tfh clonotypes and transcriptional profiles. Results We show that the post-boost Tfh response was dominated by spike-specific TCR clonotypes already present after the primary vaccination, indicating robust memory recall. A minority of novel spike-specific Tfh clones emerged after boosting, suggesting limited recruitment of new Tfh populations during the secondary germinal center reaction. Secondary germinal center Tfh cells differed markedly from primary Tfh cells: 1) they exhibited a higher frequency of IL-10+ Tfh cells; 2) they had fewer classical germinal center Tfh (with high CXCR5 and PDCD1); and 3) boost-induced Tfh cells showed upregulation of transcription factors such as RUNX3, RUNX1, KLF2, and NFKB1, reflecting a memory-specific transcriptional signature. Conclusion Overall, our data shows that booster vaccination triggers a phenotypically and transcriptionally unique memory Tfh response in human lymph nodes, emphasizing that immune recall involves reactivation of existing memory clones to shape long-term humoral immunity. Funding Source R01AI173203 Topic Categories Immune Mechanisms of Human Disease (HUM)

The presence of B cells within high-grade serous ovarian cancer (HGSOC) tumors associates with favorable prognoses. We examined the contribution of tumor-draining lymph nodes (TDLNs) to the anti-tumor B cell response. Patient-derived TDLNs were largely devoid of active germinal center (GC) structures, plasma cells (PCs), and T follicular helper cells and were instead dominated by quiescent memory B cells (MBCs) that expressed mutated, class-switched antibodies reactive to ovarian tumor cells. GC B cells and PCs largely resided within the tumor, whereas classical class-switched MBCs were present both in the tumor and in matched TDLN samples. MBCs located in the TDLNs were clonally related to MBCs and PCs within the tumor. Increased frequencies of DC-SIGN⁺ macrophages in TDLNs were correlated with reduced presence of GC B cells, suggesting a regulatory role. Thus, in HGSOC, TDLNs fail to sustain active GC responses, serving instead as reservoirs of tumor-reactive memory B cells that contribute to the intra-tumoral B cell response.

Abstract Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer, characterized by high genomic instability. Because of higher mutational load and genetic heterogeneity in BLBC, cancer cells tend to upregulate DNA repair pathways. Therefore, DNA repair-based therapies are considered to have significant potential for BLBC patients. PARP (Poly (ADP-ribose) polymerase) inhibitors are approved by FDA to treat a subset of BLBC patients with BRCA1/2 mutations. However, most BLBC patients have wildtype BRCA1/2 and lack good therapeutic targets. We analyzed all available DNA repair genes and proteins using TCGA RNA-sequencing and RPPA (Reverse Protein Phase Array) data. Our investigation identified that mismatch repair (MMR) proteins MSH2 and MSH6 (referred to as MutSα) are highly elevated in BLBC and their higher expressions are correlated to poor survivals of BLBC patients. Conversely, MLH1 and PMS2 (referred to as MutLα), the second major component of the MMR machinery, are downregulated at the mRNA level and cannot predict patient survival in BLBC. In contrast to the known tumor suppressor functions of MMR proteins, our data indicates that MSH2 promotes BLBC metastasis; MLH1, on the other hand, is associated with decreased tumor progression and metastasis. The contrasting functions of MSH2 and MLH1 have never been reported. At the mechanistic level, our data strongly indicate that MSH2, in contrast to MLH1, regulates the expression of chemokines and tumor infiltrating immune cells. Further investigation at the genomic level suggests that MSH2 regulates the expression of interferon alpha/beta receptor 1 (IFNAR1), which plays various roles in the tumor microenvironment (TME) for potential antitumor effects. Deletion of MSH2 initiates a chain of immune reactions via the upregulation of IFNAR1 expression which explains a highly immune active TME in tumors with MSH2-deficiency. Our study supports the contrasting functions of MSH2 and MLH1 in BLBC progression are due to their distinct transcriptional regulation of immune related genes, not related to their canonical mismatch repair activity. These findings challenge the universal paradigm that all MMR proteins have similar effects on tumor progression or suppression. Citation Format: Tanzia Islam Tithi, Jiao Mo, Nicholas Borcherding, Sung Jo, Heather R Kates, Chandra Maharjan, Seyedehalaleh Anvar, Richard L. Bennett, Jixiu Shan, Rohan A. Desai, Kailey E Cash, Masayoshi Honda, Lei Wang, Kawther K. Ahmed, Kalyanee Shirlekar, Li Chen, Katherine N. Gibson-Corley, Ronald Weigel, Jonathan D. Licht, Maria Spies, Ryan Kolb, Weizhou Zhang. Contrasting roles of MSH2 and MLH1 in basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5719.

Lung tissue-resident CD8+ T cells facilitate viral clearance and protective immunity to influenza viruses in animal models. Their role during acute human infection is not clear. Here we use bronchoalveolar lavage samples collected from human subjects naturally infected with influenza B virus to show that influenza-specific CD8+ T cells are expanded in the lower airways during acute infection and target only a few epitopes from phylogenetically conserved internal influenza virus proteins. The lower airway influenza-specific CD8+ T-cell immunodominance hierarchy is different from the hierarchy observed in matched blood samples. Transcriptional and protein-level analyses using HLA class I tetramers reveal a tissue-resident profile and less expression of cytotoxic effector molecules in lower airway influenza-specific CD8+ T cells. Collectively, our data show that high-frequency influenza-specific CD8+ T cells with a tissue-resident phenotype are found in the lower airways during viral clearance. These cells recognize a handful of conserved viral epitopes and exhibit a functional phenotype different from cells found in blood. These cells may play a role in controlling human influenza infection.

[This corrects the article DOI: 10.3389/fimmu.2025.1680437.].

Human Leukocyte Antigen (HLA) class-II genes, particularly HLA-DR2 and HLA-DR3, and the gut microbiota are intricately linked to the pathobiology of multiple sclerosis (MS) through their ability to regulate host immunity, a critical factor in disease pathogenesis. An imbalance between anti-inflammatory CD4+ Tregs and pro-inflammatory IL-17A-secreting CD4+ Th17 cells is thought to drive disease. However, a key unresolved question is whether HLA-class II-restricted CD4+IL-17A cells can influence Treg populations and the extent to which gut microbiota regulate this IL-17A-Treg axis. Therefore, we utilized humanized transgenic mice expressing the HLA class-II gene and deficient in mouse class-II molecules, where all CD4+ T cells are selected on the human HLA class-II molecule, closely mimicking human immune responses. Utilizing IL-17A-deficient (DR3.IL-17A−/−) mice expressing HLA-DR3 (HLA-DRβ1*0301), we show that IL-17A deficiency enriches beneficial gut bacteria, including Prevotella species, enhances peroxisome proliferator–activated receptor (PPAR) signaling, and increases FoxP3+ regulatory T (Treg) cells and IL-10 production. The importance of gut microbiota in promoting Tregs and anti-inflammatory responses was confirmed by administering Prevotella copri, a common commensal in human gut, which mirrored the effects observed in IL-17A-deficient mice by inducing PPAR signaling and Treg population. Moreover, DR3.IL-17A−/− mice exhibited a marked reduction in EAE severity compared to IL-17A-sufficient (DR3) mice, underscoring the enhanced functional capacity of the Treg population in mitigating disease progression. Cohousing experiments validated the role of gut microbiota in immune regulation including Treg induction, as demonstrated by the transfer of Prevotella species from IL-17A-deficient mice to IL-17A-sufficient mice, increased Treg populations and attenuated EAE severity in recipient DR3 mice. This study redefines IL-17A's role in immune regulation, emphasizing its ability to directly influence gut microbiota composition and the abundance of Treg-promoting bacteria. Thus, gut microbiota-targeted therapies, particularly those promoting Treg-inducing bacteria like Prevotella species, hold promise for treating autoimmune diseases by modulating host immune responses.

Abstract Background and aims Atherosclerotic plaque rupture is a major cause of myocardial infarction and stroke. However, the precise drivers of plaque destabilisation remain elusive. We hypothesised that antigen-driven, autoimmune-like T cell responses are central to the destabilisation and rupture of atherosclerotic plaques. Methods To dissect T cell responses specifically in unstable compared to stable plaques, we leveraged near-infrared autofluorescence (NIRAF) imaging–guided dissection of human carotid plaques. We also used our tandem stenosis model reflecting plaque instability as seen in patients to differentiate between unstable and stable plaques in mice. To explore T cell involvement, we studied T cell differentiation states and T cell receptor (TCR) repertoires by single-cell multi-omics. Then, testing if antigen-driven CD8 + T cell responses drive plaque instability in mice, we applied a combination of AAV8-PCSK9-induced atherosclerosis, tandem stenosis and TCR transgenic mice. Finally, we leveraged data from the AtheroExpress Biobank Study to link T cell immunity to histology-defined instability and cardiovascular outcomes. Results T cell responses in unstable versus stable atherosclerosis were distinct. Unstable human plaques contained highly expanded, autoimmune-like CD8⁺ T cells with markedly increased cytotoxic signatures, reduced exhaustion and distinct clonal repertoires compared to stable regions. Most plaque CD8⁺ T cells exhibited a pronounced tissue-resident transcriptional program. Moreover, the transcriptional signature of these plaque resident T cells was distinct from multiple other human tissues. Autoimmune-like cytotoxic and tissue-resident CD8 + T cell responses were also evident in murine atherosclerosis, where restricting the activation of antigen-driven CD8 + T cells prevented plaque destabilisation. Importantly, analysis of carotid endarterectomy samples from >1000 patients identified that intraplaque cytotoxic CD8⁺ T cell gene signatures strongly correlated with histological instability and predicted future strokes. Conclusions Integrated human, murine and clinical analyses demonstrate that autoimmune-like, cytotoxic CD8⁺ T cell responses are central drivers of plaque instability and major cardiovascular events. Targeting pathogenic CD8⁺ T cell responses may thus offer a compelling immunomodulatory strategy to stabilise plaques and reduce the risks of stroke and myocardial infarction. Graphical Abstract Key Question Rupture of unstable atherosclerotic plaques is a typical cause of myocardial infarction and stroke. To understand the underlying cause and to prevent plaque rupture, we addressed the central hypothesis that autoimmune-like T cell responses drive plaque destabilisation and rupture. Key Findings CD8 + T cells are clonally expanded with increased cytotoxic signatures in unstable versus stable plaques (mice and humans) and require antigen recognition to drive plaque instability. Cytotoxic CD8 + T cell signatures in excised plaques correlate with increased future cardiovascular events. Take Home Message Autoimmune-like adaptive immune reactions, dominated by CD8 + T cells, are a major driver of plaque instability/rupture. Therefore, targeting pathogenic CD8⁺ T cell responses offers a compelling immunomodulatory strategy to stabilise plaques and reduce the risk of myocardial infarction and stroke.

Abstract Tumor necrosis factor (TNF) receptor–associated factor (TRAF)–interacting protein with forkhead-associated domain B (TIFAB), an inhibitor of NF-κB signaling, plays critical roles in hematopoiesis, myelodysplastic neoplasms, and leukemia. We previously demonstrated that Tifab enhances KMT2A::MLLT3–driven acute myeloid leukemia (AML) by either upregulating Hoxa9 or through ubiquitin-specific peptidase 15–mediated downregulation of p53 signaling. In this study, we show that Tifab deletion in KMT2A::MLLT3–induced AML impairs leukemia stem/progenitor cell (LSPC) engraftment, glucose uptake, and mitochondrial function. Gene set enrichment analysis reveals that Tifab deletion downregulates MYC, HOXA9/MEIS1, mTORC1 signaling, and genes involved in glycolysis and oxidative phosphorylation. By comparing genes upregulated in TIFAB-overexpressing LSPCs with those downregulated upon Tifab deletion, we identify hepatocyte nuclear factor 4 alpha (Hnf4a) as a key TIFAB target, regulated through the inhibition of NF-κB component RelB, which suppresses Hnf4a in leukemia cells. HNF4A, a nuclear receptor involved in organ development, metabolism, and tumorigenesis, rescues the metabolic defects caused by Tifab deletion and enhances leukemia cell engraftment. Conversely, Hnf4a knockdown attenuates TIFAB-mediated enhancement of LSPC function. These findings highlight the critical role of the TIFAB-HNF4A axis in KMT2A::MLLT3–induced AML and uncover a novel regulator in leukemia biology.

Interleukin-2 (IL-2) is a pleiotropic cytokine playing important roles in both lymphoid and non-lymphoid cells to maintain tissue-immune homeostasis. Until now, active T cells are known to be the primary cellular sources of IL-2 although natural killer (NK) cells, dendritic cells, and intestinal innate lymphoid cells have also been found to produce IL-2 in different contexts. Datamining our previously published mouse mammary gland single-cell RNA-seq dataset revealed significant Il2 expression in mammary epithelial cells, which we validated by detecting the IL-2 protein in a flow cytometric analysis. We found that prolactin induces Il2 expression in mammary epithelial cells by activating the STAT5 signaling pathway. To specifically evaluate the in vivo significance of mammary epithelial IL-2, we conditionally deleted IL2 within these cells by crossing MMTV-Cre and IL2fl/fl mice. We found that mammary epithelial IL-2 is required for the maintenance of NK cell number and function in the mouse mammary gland. Loss of NK cells in the epithelial IL-2-deficient mammary tissue was consistent with compromised tumor immunosurveillance evidenced by expansion of luminal mammary epithelial cells in young epithelial IL-2 KO mice culminating in mammary tumor development as they grew old. Our results establish mammary epithelial cells as novel producers of IL-2, critical for NK cell-mediated cancer immunosurveillance in the mammary tissue. NIH grants CA200673, CA203834, and CA260239 Cytokines and Chemokines and Their Receptors (CCR)

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive and chemo-resistant sarcomas with poor survival rates. Loss of CDKN2A or P53 following NF1 disruption is a key event in MPNST development. Here, we used CRISPR-Cas9 somatic tumorigenesis in mice to identify transcriptomic and metabolomic features distinguishing CDKN2A- versus P53-deleted MPNSTs. Convergent, multiomic analyses revealed that CDKN2A-deleted MPNSTs are especially dependent on the pentose phosphate pathway (PPP) and NADPH metabolism for growth and viability. Disruption of glucose-6-phosphate dehydrogenase (G6PD), the PPP rate-limiting enzyme, slowed CDKN2A-deleted MPNST growth and sensitized MPNSTs to standard-of-care chemotherapy. Knockdown of the redox-regulated transcription factor NRF2 slowed MPNST growth and decreased G6PD transcription. Analysis of patient MPNSTs identified a NRF2 gene signature correlating with tumor transformation. Furthermore, G6PD and NRF2 expression in PanCancer TCGA samples correlates with patient survival. This work identifies NRF2-PPP dependency as a targetable vulnerability in these difficult-to-treat MPNSTs, particularly in the NF1/CDKN2A-deleted majority.

Integration of single-cell RNA-sequencing (scRNA-seq) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) is extremely powerful in studying lymphocyte development. A python-based package, Dandelion, introduced the VDJ-feature space method, which addresses the challenge of integrating single-cell AIR data with gene expression data and enhances trajectory analysis results. However, no R-based equivalent or similar methods currently exist. To fill this gap, we present dandelionR, an R implementation of Dandelion’s trajectory analysis workflow, bringing the VDJ feature space construction and trajectory analysis using diffusion maps and absorbing Markov chains to R, offering a new option for scRNA-seq and scVDJ-seq analysis to R users.

The mismatch repair (MMR) pathway is known as a tumor suppressive pathway and genes involved in MMR are commonly mutated in hereditary colorectal or other cancer types. However, the function of MMR genes/proteins in breast cancer progression and metastasis are largely unknown. We found that MSH2, but not MLH1, is highly enriched in basal-like breast cancer (BLBC) and that its protein expression is inversely correlated with overall survival time (OS). MSH2 expression is frequently elevated due to genomic amplification or gain-of-expression in BLBC, which results in increased MSH2 protein to pair with MSH6 (collectively referred to as MutSα). Genetic deletion of MSH2 or MLH1 results in a contrasting phenotype in metastasis, with MSH2-deletion leading to reduced metastasis and MLH1-deletion to enhanced liver or lung metastasis. Mechanistically, MSH2-deletion induces the expression of a panel of chemokines in BLBC via epigenetic and/or transcriptional regulation, which leads to an immune reactive tumor microenvironment (TME) and elevated immune cell infiltrations. MLH1 is not correlated with chemokine expression and/or immune cell infiltration in BLBC, but its deletion results in strong accumulation of neutrophils that are known for metastasis promotion. Our study supports the differential functions of MSH2 and MLH1 in BLBC progression and metastasis, which challenges the paradigm of the MMR pathway as a universal tumor suppressive mechanism.

IntroductionSingle-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of immune heterogeneity. Although previous studies have mapped the single-cell transcriptomic atlases of peripheral leukocytes in healthy dogs, the identification and functional characterization of distinct immune subsets remain incomplete. MethodsWe constructed a single-cell atlas of peripheral leukocytes from six healthy small-breed dogs using the 10x Genomics platform and the updated canFam4 genome. Results and discussionAnalysis of 30,040 high-quality transcriptomes revealed 51 distinct immune subsets, including CD14+CD33+ monocytes, XCR1+CD1D+ dendritic cells, CEACAM1+CD24+ neutrophils, and IL32+BATF+ regulatory T cells, which were underrepresented in canFam3.1-based studies. Interferon-enriched CD14+ monocytes and CD4+ T subsets associated with myxomatous mitral valve disease were also identified. Functional enrichment analyses suggested that PDCD1 is associated with attenuated TCR signaling, whereas LAG3 was associated with malate metabolism pathways in CD4+ T cells and reduced TBX21 expression in CD8+ T cells linked to antiviral responses. CD274, which encodes PD-L1 was linked to IL-10 production in neutrophils, and CTLA4 represented an initial activation of double-negative T subsets. T cell exhaustion scores and proliferative fractions varied across cohorts, reflecting differences in environmental antigenic exposures. ConclusionTo our knowledge, this study represents the first comprehensive, gene-resolved single-cell analysis to reveal immunoregulatory checkpoint mechanisms underlying immune homeostasis in healthy dogs. Our dataset will serve as a valuable resource for future comparative and translational immunology research in dogs.

Introduction: Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells. One possible consequence of mitochondria transfer is energetic support of neighboring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism, as shown in cultured ρ0 cells or Ndufs4-/- peritoneal macrophages. Mitochondria transplantation has been explored in multiple settings, including ex vivo exposure of hematopoietic stem cells prior to autologous transplantation for patients with large-scale mitochondrial DNA mutations, as well as mitigation of ischemic injury to the heart, brain, and limbs. However, the therapeutic potential of mitochondria transfer-based therapies for inherited mitochondrial disorders remains unclear. Leigh syndrome (LS) is a fatal pediatric mitochondrial disease characterized by progressive neurodegeneration. No effective treatments exist for this devastating condition. Ndufs4-/- mice, which lack a subunit of complex I in the electron transport chain, serve as a robust LS model. We previously demonstrated that in vivo administration of wild-type mitochondria can restore aerobic respiration in Ndufs4-/- peritoneal macrophages. Immune cells are known to donate mitochondria to metabolically compromised cells in several tissues, raising the possibility that leveraging mitochondria transfer could ameliorate systemic mitochondrial disease. Methods: We evaluated mitochondria transfer-based strategies to improve morbidity and mortality in Ndufs4-/- mice. Two approaches were tested: (1) intraperitoneal injection of purified mitochondria from wild-type (WT) or Ndufs4-/- mice, either as a single administration or repeated weekly injections; and (2) bone marrow transplantation (BMT) from WT or Ndufs4-/- donors, including experiments using the mtDendra2Flox/Flox (PhAM Flox; referred to as mtD2F/F) mitochondrial reporter system. Metabolic function of peritoneal macrophages was assessed using Seahorse-based oxygen consumption rate (OCR) analysis. Whole-body metabolism was measured in metabolic cages, neurological performance was evaluated using the rotarod assay, and survival was monitored. Circulating and tissue-associated donor mitochondria were tracked to assess transfer to recipient immune and non-immune cells. Finally, cross-species experiments were performed using human mitochondria administered to Ndufs4-/- mice. Results: Single intraperitoneal injection of WT mitochondria into Ndufs4-/- mice increased both basal and maximal OCR in peritoneal macrophages, indicating improved mitochondrial respiration. Repeated weekly injections further enhanced whole-body energy expenditure and neuromotor performance, ultimately extending survival. In contrast, mitochondria derived from Ndufs4-/- mice failed to improve neurological function (median 84 vs 68 days, p=0.0052). BMT from WT donors led to the release of extracellular mitochondria into circulation and subsequent transfer to host immune (B cells, neutrophils) and non-immune (epithelial, endothelial, stromal) cells in blood, spleen, and liver. This transfer was associated with increased energy expenditure, improved rotarod performance, and significantly prolonged survival compared to Ndufs4-/- donor BMT (median 74 vs 40 days, p<0.0001). Importantly, cross-species administration of purified human mitochondria to Ndufs4-/- mice also resulted in enhanced neurological function and extended lifespan (median 80.5 vs 67.5 days, p=0.0079), supporting the translational potential of this approach. Conclusion: Our findings demonstrate that mitochondria transfer-based interventions, including systemic delivery of isolated WT mitochondria and BMT from WT donors, ameliorate morbidity and extend lifespan in the Ndufs4-/- model of Leigh syndrome. These benefits are linked to enhanced macrophage respiration, systemic metabolic improvements, and donor mitochondria transfer to recipient tissues. Moreover, successful cross-species rescue with human mitochondria highlights the potential of mitochondria transfer as a therapeutic strategy for primary mitochondrial diseases such as LS.

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) and single-cell RNA sequencing (scRNA-seq) provide a transformative approach to profiling immune responses at unprecedented resolution across diverse pathophysiologic contexts. This work presents scRepertoire 2, a substantial update to our R package for analyzing and visualizing single-cell immune receptor data. This new version introduces an array of features designed to enhance both the depth and breadth of immune receptor analysis, including improved workflows for clonotype tracking, repertoire diversity metrics, and novel visualization modules that facilitate longitudinal and comparative studies. Additionally, scRepertoire 2 offers seamless integration with contemporary single-cell analysis frameworks like Seurat and SingleCellExperiment, allowing users to conduct end-to-end single-cell immune profiling with transcriptomic data. Performance optimizations in scRepertoire 2 resulted in a 85.1% increase in speed and a 91.9% reduction in memory usage from the first version over the range repertoire size tested in benchmarking, addressing the demands of the ever-increasing size and scale of single-cell studies. This release marks an advancement in single cell immunogenomics, equipping researchers with a robust toolset to uncover immune dynamics in health and disease.

Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multi-omics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. We present here a comprehensive multi-omics view of CTCL clonal evolution, incorporating exome, whole genome, epigenome, bulk-, single cell (sc) VDJ-, and scRNA-sequencing of 114 clinically annotated serial skin, peripheral blood, and lymph node samples from 35 CTCL patients. We leveraged this extensive dataset to define the molecular underpinnings of CTCL progression in individual patients at single cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified a large number of recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, PI3K signaling, and PD-1 checkpoint pathways as evasion tactics deployed by malignant T cells. We also identified a gain of function mutation in STAT3 (D661Y) and demonstrated by CUT&RUN-seq that it enhances binding to transcription start sites of genes in Rho GTPase pathways, which we previously reported to have activated chromatin and increased expression in HDACi-resistant CTCL. These data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. A striking number of progression-associated mutations occurred in chromatin methylation modifiers, including EZH2, suggesting that EZH1/2 inhibition may also benefit patients with CTCL. Knowledge of these molecular changes should be leveraged for improved disease monitoring, biomarker-informed clinical trial design, and new therapeutic strategies in this challenging and incurable cancer.