Abstract In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.

Multiple Myeloma (MM) is a hematological malignancy associated with poor prognosis and relapses. MM has a strong inherited component, and while the mutational and transcriptomic landscapes of MM have been characterized extensively, the molecular consequences of germline genetic variation in MM have not been previously explored. Here, we have leveraged matched genotype and expression data from 607 patients to map the germline and somatic regulatory variation in MM. We find that non-coding variation shapes the transcriptional landscape of MM, contributing to overall survival. These regulatory variants alter the binding of transcription factors with known importance in MM, such as IRF4, providing insights into the underlying mechanisms. These data contribute to the increasing understanding of the genetic, molecular, and cellular forces shaping MM risk and outcomes. ### Competing Interest Statement The authors have declared no competing interest.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis. Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA sequencing of lung tissue from 66 individuals with pulmonary fibrosis and 48 unaffected donors. Using a pseudobulk approach, we mapped expression quantitative trait loci (eQTLs) across 38 cell types, observing both shared and cell-type-specific regulatory effects. Furthermore, we identified disease interaction eQTLs and demonstrated that this class of associations is more likely to be cell-type-specific and linked to cellular dysregulation in pulmonary fibrosis. Finally, we connected lung disease risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression and implicates context-specific eQTLs as key regulators of lung homeostasis and disease. Single-cell transcriptomics and expression quantitative trait locus mapping in 114 lung tissue samples, including 66 with interstitial lung disease, highlight the cell-type-specific functions of risk variants contributing to disease pathobiology.

ABSTRACTOrgan- and body-scale cell atlases have the potential to transform our understanding of human biology. To capture the variability present in the population, these atlases must include diverse demographics such as age and ethnicity from both healthy and diseased individuals. The growth in both size and number of single-cell datasets, combined with recent advances in computational techniques, for the first time makes it possible to generate such comprehensive large-scale atlases through integration of multiple datasets. Here, we present the integrated Human Lung Cell Atlas (HLCA) combining 46 datasets of the human respiratory system into a single atlas spanning over 2.2 million cells from 444 individuals across health and disease. The HLCA contains a consensus re-annotation of published and newly generated datasets, resolving under- or misannotation of 59% of cells in the original datasets. The HLCA enables recovery of rare cell types, provides consensus marker genes for each cell type, and uncovers gene modules associated with demographic covariates and anatomical location within the respiratory system. To facilitate the use of the HLCA as a reference for single-cell lung research and allow rapid analysis of new data, we provide an interactive web portal to project datasets onto the HLCA. Finally, we demonstrate the value of the HLCA reference for interpreting disease-associated changes. Thus, the HLCA outlines a roadmap for the development and use of organ-scale cell atlases within the Human Cell Atlas.

Outcomes for pediatric brain tumor patients remain poor, and there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. Here, we present interim results from the first six pediatric patients treated on an ongoing phase I clinical trial (NCT04510051) of IL13BBζ-CAR T cells delivered weekly into the lateral cerebral ventricles, identifying clonal expansion of endogenous CAR-negative CD8 + T cells in the cerebrospinal fluid (CSF) over time. Additionally, of the five patients evaluable for disease response, three experienced transient radiographic and/or clinical benefit not meeting protocol criteria for response. The first three patients received CAR T cells alone; later patients received lymphodepletion before the first infusion. There were no dose limiting toxicities (DLTs). Aside from expected cytopenias in patients receiving lymphodepletion, serious adverse events possibly attributed to CAR T cell infusion were limited to one episode of headache and one of liver enzyme elevation. One patient withdrew from treatment during the DLT period due to a Grade 3 catheter-related infection and was not evaluable for disease response, although this was not attributed to CAR T cell infusion. Importantly, scRNA- and scTCR-sequence analyses provided insights into CAR T cell interaction with the endogenous immune system. In particular, clonally expanded endogenous CAR − T cells were recovered from the CSF, but not the peripheral blood, of patients who received intraventricular IL13BBζ-CAR T cell therapy. Additionally, although immune infiltrates in CSF and post-therapy tumor did not generally correlate, a fraction of expanded T cell receptors (TCRs) was seen to overlap between CSF and tumor. This has important implications for what samples are collected on these trials and how they are analyzed. These initial findings provide support for continued investigation into locoregionally-delivered IL13BBζ-CAR T cells for children with brain tumors.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

Recent advances in immunotherapy, particularly chimeric antigen receptor (CAR)-engineered T cell therapy, have shown promise for the treatment of many tumor types including progressive recurrent glioblastoma (GBM). While early phase clinical trials have illuminated the potential for CAR T cell therapy to effectively treat GBM, they have also highlighted the unique challenges regarding the efficacy and safety of immunotherapy for brain tumors, and many patients continue to progress during therapy. We seek to overcome these challenges and ultimately extend the time of survival for patients diagnosed with GBM by investigating the immune- and tumor-mediated mechanisms driving variation in response to CAR T cell therapy. We generated the first multi-omics time-series dataset of CAR T cells, endogenous immune cells, and tumor cells from 59 GBM patients treated with CAR T cell therapy. Using single cell RNA-sequencing and simultaneous quantification of nearly 200 cell surface proteins, we comprehensively profiled the cellular phenotypes and signaling pathways within tumor and circulating immune cells that are associated with treatment response. The combination of mRNA and protein expression allowed us to resolve cell states beyond what either modality was capable of alone. Additionally, we found differentially expressed genes and proteins between tumor biopsies collected before and after CAR T cell therapy as well as differential expression between pre-infusion CAR T cells and those identified within the tumor following infusion. By evaluating the CAR T cell phenotypes prior to and during treatment we sought to address the outstanding question of how intrinsic variability impacts the activity and persistence of CAR T cells and to determine the phenotypes that confer the greatest therapeutic benefit for patients with GBM. Our results have direct implications for precision medicine and future clinical trials investigating the use of CAR T cell therapy for GBM as well as other solid tumors.