Crohn’s disease is an inflammatory bowel disease (IBD) commonly treated through anti-TNF blockade. However, most patients still relapse and inevitably progress. Comprehensive single-cell RNA-sequencing (scRNA-seq) atlases have largely sampled patients with established treatment-refractory IBD, limiting our understanding of which cell types, subsets, and states at diagnosis anticipate disease severity and response to treatment. Here, through combining clinical, flow cytometry, histology, and scRNA-seq methods, we profile diagnostic human biopsies from the terminal ileum of treatment-naïve pediatric patients with Crohn’s disease (pediCD; n=14), matched repeat biopsies (pediCD-treated; n=8) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To resolve and annotate epithelial, stromal, and immune cell states among the 201,883 baseline single-cell transcriptomes, we develop a principled and unbiased tiered clustering approach, ARBOL. Through flow cytometry and scRNA-seq, we observe that treatment-naïve pediCD and FGID have similar broad cell type composition. However, through high-resolution scRNA-seq analysis and microscopy, we identify significant differences in cell subsets and states that arise during pediCD relative to FGID. By closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of T cell, innate lymphocyte, myeloid, and epithelial cell states in treatment-naïve pediCD (pediCD-TIME) samples which can distinguish patients along the trajectory of disease severity and anti-TNF response. By using ARBOL with integration, we position repeat on-treatment biopsies from our patients between treatment-naïve pediCD and on-treatment adult CD. We identify that anti-TNF treatment pushes the pediatric cellular ecosystem towards an adult, more treatment-refractory state. Our study jointly leverages a treatment-naïve cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict Crohn’s disease trajectory.

Tissue-specific T cell immune responses play a critical role in maintaining organ health but can also drive immune pathology during both autoimmunity and alloimmunity. The mechanisms controlling intratissue T cell programming remain unclear. Here, we leveraged a nonhuman primate model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation to probe the biological underpinnings of tissue-specific alloimmune disease using a comprehensive systems immunology approach including multiparameter flow cytometry, population-based transcriptional profiling, and multiplexed single-cell RNA sequencing and TCR sequencing. Transcriptional profiling revealed substantial biological differences between T cells infiltrating the lung and liver during aGVHD. These included enrichment for transcriptional pathways controlling extracellular matrix remodeling and chemotaxis in the lung and enrichment for transcriptional pathways linked to nucleic acid metabolism and proliferation in the liver. Single-cell RNA sequencing and TCR sequencing substantiated divergent organ-specific transcriptional programing of tissue-infiltrating T cells, which was linked to clonal expansion, with expanded clones progressively enriched for C-X3-C motif chemokine receptor 1 (CX3CR1)-expressing CD8 effector T cells in the lung and eomesodermin (EOMES)-expressing CD8 effector-memory T cells in the liver. This divergent evolution of T cells was maintained even for T cells sharing the same TCRs, indicating its independence from antigen specificity. Together, these results provide insights into the role that tissue microenvironment-derived signals play in local T cell transcriptional programming during alloimmune-mediated clonal expansion and suggest potential opportunities to develop tissue-specific therapeutics to curtail pathogenic immunity after transplant.

Following allogeneic hematopoietic cell transplantation (alloHCT) donor T cells infiltrate and damage multiple target organs, resulting in acute GVHD (aGVHD). However, the critical biological mechanisms underpinning the tissue-specificity of aGVHD remains only partially understood. Deciphering the organ-specific alloimmune mechanisms responsible for target organ damage offers new prospects for precisely targeted treatments for acute GVHD, minimizing impact on protective immunity and reducing overall toxicity.In the current study we investigated phenotypic and transcriptomic characteristics of T cells infiltrating liver and lungs following alloHCT in an established model of systemic aGVHD in Rhesus macaques. We found that donor-derived CD8 T cells with an effector-memory flow cytometrically-defined phenotype were the dominant population in liver and lung infiltrates. Despite their phenotypic similarity, T cells exhibited organ-specific transcriptomic characteristics with upregulated proliferation/cell cycle and cytokine signaling pathways in liver T cells and activated proinflammatory pathways associated with chemotaxis, the epithelial-mesenchymal transition, and extracellular matrix remodeling in lung T cells. This was also evident on a single-cell level with overrepresentation of a proliferating CD8 T cell cluster and a pro-inflammatory CX3CR1-expressing CD8 T effector cell cluster in liver and lung samples, respectively (Figure 1).To further elaborate on how the T cell-mediated alloimmune response evolves in different tissues, we used single cell transcriptomic profiling coupled with T cell receptor (TCR) sequencing. We discovered two distinct pathways of T cell pathogenic transformation during concurrent liver and lung aGVHD (Figure 2). One population of T cells differentiated into effectors prior to tissue infiltration and share both TCRs and gene expression signatures in both target organs. This was measured as a higher degree of cluster-distribution similarity of the shared expanded clones (Figure 2, left panel), and which was evident on the clonal level (Figure 3). In parallel, a distinct proportion of T cell clones, which likely expanded locally within each organ, exhibited divergent evolution and organ-specific adaptations, which was documented as a lower degree of cluster-distribution similarity of the expanded organ-specific clonotypes (Figure 2, right panel).Collectively, our data reveal a more complex mechanism of organ-specific GVHD than previously appreciated, and provide a rational for the development of both global and organ-specific anti-GVHD therapeutics.

Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a signi fi cant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CAR neg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CAR neg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8 + CAR neg T cells emerging during CAR T-cell expansion. These bystander CD8 + CAR neg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers ( KIR3DL2 , CD160 , and KLRD1 ), chemokines, and chemokine receptors ( CCL5, XCL1 , and CCR9 ), and downregulation of na & iuml;ve T-cell-associated genes ( SELL and CD28 ). A transcriptionally similar population was identi fi ed in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8 + T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype ef fi ciently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the fi rst comprehensive identi fi cation and pro fi ling of CAR neg bystander CD8 + T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www. ClinicalTrials.gov.

BackgroundRelapse following CD19 CAR-T cell (CAR-T) therapy has been linked to the upregulation of negative regulators of T cell function. Recent studies showed that inhibition of the negative T cell regulator PTPN2 using a small molecule inhibitor greatly improves T cell mediated tumor clearance in murine models. We now assessed the function, proliferation, persistence and toxicity of PTPN2 KO CAR-Ts in primary human CD19 CAR-Ts and in a clinical relevant model of Non-Human Primate (NHP) CD20 CAR-Ts.ResultsHuman PTPN2 CRISPR/Cas9 KO CD19 CAR-Ts showed enhanced in vitro effector function as evidenced by increased IFNg production (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001) and cytotoxicity against NALM6 B-ALL leukemia cells (59.6 + 9.8% vs 42.5 + 12.9% p = 0.03) when compared to control CD19 CAR-T.Furthermore, we assessed proliferation, persistence, and toxicity of PTPN2 KO CD20 CAR-Ts through a dose escalation study in 5 NHPs with Dose Level 1: 6x104, Dose Level 2: 6x105, Dose Level 3: 3x106, and Dose Level 4: 6x106 CAR-T/kg. Dose Level 1 and 2 showed no measurable expansion of CAR-Ts while all animals infused at dose level 3 (n=3, 3x106 CAR-Ts/kg) demonstrated CAR-T expansion (max expansion 18.6 + 6.8% CAR-Ts) and B cell aplasia, and all animals developed laboratory and clinical signs of CRS and ICANS (Fig 1). One animal at Dose Level 3 required treatment with Tocilizumab and Dexamethasone due to severe CRS and ICANS, which eventually resolved.A single NHP received the highest Dose Level (Level 4, 6x106 CAR-Ts/kg), exhibiting earlier and higher CAR-T expansion (Fig.1), severe CRS, and ICANS unresponsive to treatment with Tocilizumab and repeated doses of Dexamethasone, necessitating euthanasia. Importantly, previous animals receiving PTPN2-expressing (WT) CAR-Ts at higher doses (12x106) did not experience severe CRS or ICANS, suggesting that PTPN2 knockout CAR-Ts are associated with augmented effector function resulting in increased toxicity.Detailed analysis of the Dose Level 4 NHP revealed widespread cerebral edema and CAR-T cell infiltration in the central nervous system (CNS). Notably, there was an enrichment of PTPN2 knockout CAR-Ts in the blood (72%) and brain parenchyma (84%) when compared to the infused product (63%), indicating preferential expansion and CNS infiltration of PTPN2 KO CAR-Ts (Fig.2). Flow analysis conducted at the time of euthanasia revealed enrichment of CD8+ CAR-Ts (Fig.3) characterized by elevated expression levels of the proliferative and cytotoxic molecules Ki67 and GZMB. Extended CAR-T persistence was not seen in PTPN2 KO animals.ConclusionWe demonstrate that PTPN2 KO CAR-Ts possess increased effector function, proliferation, and CNS infiltration. Augmented effector function was associated with greater toxicities, highlighting the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models.

Background: Despite the success of CD19 CAR-T cells (CAR-Ts) in inducing remission, relapse remains a major issue. Previous studies have shown that upregulation of negative T cell regulators can contribute to CAR-T failure, and that deletion of these regulators can enhance CAR-T efficacy. A potent negative regulator is PTPN2, which inhibits T cell function by modulating T cell receptor and cytokine signaling pathways. While murine models have shown that PTPN2-knockout T cells improve tumor control, these models fail to reliably predict clinical efficacy or toxicity. To improve this predictability, we investigated PTPN2 KO CAR-Ts in primary human cells and in a NHP model of B-cell directed CAR-T therapy. Methods: CRISPR/Cas9-mediated PTPN2 deleted (‘KO‘) human CD19 CAR-Ts were assessed for phenotype/function using cytokine secretion and cytotoxicity assays. PTPN2 KO NHP CD20 CAR-Ts were administered to lymphodepleted NHPs using our established model and a dose escalation with Level 1: 6x10 4, Level 2: 6x10 5, Level 3: 3x10 6, and Level 4: 6x10 6 CAR-T/kg, followed by assessment for CAR-T expansion, B cell aplasia, CRS and ICANS. Results: PTPN2 KO was successfully achieved in human CAR-Ts (83.8 + 5.2% deletion). IFNγ production was significantly higher in PTPN2 KO CD19 CAR-Ts vs control cells (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001). In cytotoxicity assays targeting the B-ALL cell line NALM6, PTPN2 KO CD19 CAR-Ts demonstrated significantly increased killing vs control cells (for example, at E:T ratio 1:1 59.6 + 9.8% vs 42.5 + 12.9% p = 0.03). We evaluated the proliferation, efficacy, and toxicity of PTPN2 KO CD20 CAR-Ts in 5 NHPs. The CAR-T PTPN2 KO rate ranged from 63-91%. During dose escalation, no expansion of CAR-Ts was observed at dose levels 1 and 2 (n=1 for each, Fig 1). However, all animals infused at dose level 3 (n=3, 3x10 6 CAR-Ts/kg) demonstrated CAR-T expansion (max expansion 18.6 + 6.8% CAR-Ts) and B cell aplasia, and all animals developed laboratory and clinical signs of CRS and ICANS ( Fig 1). One animal at dose level 3 experienced CRS on day +4 that was severe enough to require tocilizumab, with symptoms subsequently improving. On day +6, this animal also developed ICANS, including ataxia and muscle weakness, requiring 1 dose of Dexamethasone (1mg/kg), after which symptoms resolved. One animal received dose level 4 (6x10 6 CAR-Ts/kg). This recipient displayed earlier and higher expansion of CAR-Ts (day 3: 16.7%, day 7: 56.3% CAR-Ts, Fig.1), and developed severe CRS (with elevated CRP, LDH (not shown) and Ferritin, Fig.1) and ICANS that were unresponsive to treatment with 2 doses of tocilizumab and multiple doses of dexamethasone, with this recipient meeting humane euthanasia endpoints. Importantly, previous animals (n=5) receiving 6-12 x 10 6PTPN2 expressing (WT) CAR-Ts/kg, did not develop severe CRS nor ICANS (and did not require Toci or Dex). This suggests that PTPN2 KO CAR-Ts are associated with augmented toxicities. An extensive terminal analysis of the Dose Level 4 animal was performed; pathology demonstrated widespread cerebral edema and CAR-T CNS infiltration. Because the CAR-T infusion contained both PTPN2 KO and PTPN2 WT CAR-Ts, an assessment could be made for enrichment of PTPN2 KO CAR-Ts in the blood and brain parenchyma. Compared to the infused product (63% KO CAR-Ts, Fig.2), there was enrichment for KO CAR-Ts in the blood (72% KO CAR-Ts) and the brain parenchyma (84% KO CAR-Ts, Fig 2). This suggests increased expansion and CNS infiltration of PTPN2 KO versus PTPN2 WT CAR-Ts. Flow cytometry analysis of the PTPN2 KO CAR-Ts from the blood at maximum expansion revealed high expression of the proliferation and activation markers Ki67 and GZMB (84.6%, 74.5% of all CAR-Ts). Of note, at Dose Level 3, neither CAR-T persistence (15 + 2 days vs 20 + 5 days) nor duration of B cell aplasia (41+ 5 days vs 37 + 3 days) were increased with PTPN2 KO CAR-Ts vs historic controls infused with PTPN2 WT CAR-Ts. Conclusion: We demonstrate that PTPN2 KO CAR-T cells possess increased effector function, proliferation, and CNS infiltration versus PTPN2 WT CAR-Ts. However, this enhanced efficacy was associated with increased severity of CRS and ICANS, emphasizing that careful attention to the dose of PTPN2 KO CAR-Ts would be important. These results also highlight the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models, to thoroughly assess their efficacy and toxicity profiles.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell’s transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, ‘RM-scTCR-Seq’, which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR’s with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

Background: Approximately 50% of patients receiving CAR-T cells will relapse, with half of these relapses due to CD19neg antigenic escape. To address this, we have explored an alternative pathway for targeting antigen-negative leukemic cells through activated endogenous CARneg bystander T cells, which could provide additional leukemic control through CAR-independent mechanisms. Here we leverage data from a NHP CAR-T cell model, and from clinical studies, to identify mechanisms of CARneg T cell bystander activation and their potential antileukemic role. Methods: NHP: Anti-CD20 CAR-T cells were transferred into lymphodepleted NHP as previously described (PMID: 29563103). Expansion of these cells resulted in B cell aplasia, and induced clinical Cytokine Release Syndrome (CRS). The transferred CAR-T cells persisted for ~4 weeks, at which point loss of CAR-Ts was followed by B cell recovery. Flow cytometry, scRNA- and scTCR Seq were performed on 5 NHP CAR-T cell recipients. Clinical Samples: Flow cytometry, scRNA- and scTCR-Seq were also performed on T cells from 6 pediatric B-ALL patients receiving Tisangenlecleucel. ScRNA-Seq and scTCR-Seq libraries were aligned with cellranger, preprocessed and analyzed with the Python packages scanpy and scVI. Results: NHP recipients of CD20-CAR T cells demonstrated CAR-T cell maximum expansion on Days 7-11 post-infusion. Coincident with CAR-T cell expansion, we also observed a 5-fold expansion of activated, CD8+ CARneg T cells. To rigorously profile the expanded CARneg T cells, we performed scRNASeq on sorted CARneg T cells from the infused product, at the time of maximum CAR-T proliferation (Day 11), and during the CAR-T contraction phase (Day 20). Longitudinal clone tracking and associated gene expression analysis demonstrated that the CD8 CARneg T cell clones formed a unique cluster that emerged in parallel with expansion of CARpos T cells. This CD8 CARneg population made up <1% of T cells in the blood prior to CAR-T cell infusion or in the infused CAR-T cell product, suggesting evolution and expansion of these cells in the post-CAR-T cell infusion/CRS milieu. These 'bystander' CD8 CARneg T cells demonstrated high expression of the cytokine receptors for IL15 and IL2, and of the cytokines IL-18, IL2 and IFNg, along with expression of canonical cytotoxicity molecules, including Perforin and Granzyme M. In addition, the CARneg CD8 T cells expressed high levels of KLRK1 (NKG2D) and FASL, receptors that have been associated with direct tumor lysis. T cell repertoire analysis demonstrated high TCR diversity, suggesting that these cells were bone fide T cells, and not NK-T or MAIT cells. To better understand this population, we subsetted all clusters enriched for CARneg CD8 T cells at the time of maximum CAR-T cell proliferation and reclustered this data, which enabled the further identification of these CARneg CD8 T cells to express multiple NK markers, including KLRK1 (NGG2D), KLRD1 (CD94), CD160, CCL5, NKG7 (Panel 1), a transcriptional signature that is similar to CD8 T cells recently found to emerge as cytotoxic bystander cells in viral infections (PMID: 31827070), and in the inflammatory tumor microenvironment (PMID: 33468558), where they provide additional anti-viral/tumor control. We next performed scRNA-Seq on samples from 6 patients receiving Tisagenlecleucel CAR-T cells. At the time of maximal CAR-T cell expansion, we identified a CD8 CARneg T cell population in all patients, which mirrored the transcriptional signature originally observed in NHP (Panel 2). To determine whether cytokines released by CRS could induce the transformation of human CD8 CARneg T cells into these highly activated cells, we stimulated primary human T cells with an array of CRS-associated cytokines, and found that the gamma cytokines IL-2 and IL-15 induced a CD8+ T cell population that upregulated a similar group of phenotypic markers (CD160, NKG2D and CCL5) observed in the bystander cells obtained from CAR-T cell patients. Conclusions: These data demonstrate for the first time that a CAR-T cell induced bystander effect is predominantly elicited in CD8 CARneg T cells, in both NHP and patients. These CD8 CARneg T cells express several canonical activation and NK markers in response to CRS-associated cytokines, and also express both NKG2D and FASL, suggesting that they could contribute to anti-leukemic effects through CAR-independent mechanisms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Chemorefractory primary and secondary central nervous system lymphomas (CNSL) have a dismal prognosis, and represent a major unmet need in oncology. To address this, we conducted a pilot trial of axi-cel in patients with relapsed/refractory CNSL. All patients had Ommaya reservoirs, such that after CAR-T infusion, paired CSF and blood could be collected and analyzed daily, allowing the interrogation of single-cell CAR-T transcriptional profiles at an unprecedented level of detail. This analysis enabled us to address key mechanistic questions relevant to CAR-T therapy for CNS lymphoma, including determining the impact of tumor site (brain vs lymphoid tissue) on CAR-T efficacy and toxicity, and discovering the drivers of CAR-T cell transcriptional evolution as these cells move between their sites of action. Methods: Adult patients with either primary or secondary relapsed/refractory CNSL were enrolled on the 'Axi-cel In CNS Lymphoma' Trial (Clinicaltrials.gov# NCT04608487). Patients received fludarabine/cyclophosphamide lymphodepletion followed by axi-cel infusion. Blood and CSF were sampled daily between D0-14 after infusion, with data from CAR-T maximum expansion (Days 5-9) reported here. 5' 10x Single-cell RNA-Seq (scRNA-Seq) and TCR-Seq were performed on peripheral blood mononuclear cells (PBMC), enriched T cells, and CSF-derived cells. scRNA-Seq data was analyzed with the Python packages 'scanpy' and 'scVI' to identify and characterize high-quality CD4+ and CD8+ T cells, and to conduct differential expression tests. Gene set enrichment analysis (GSEA) was used to compare CSF and blood CD4+ and CD8+ CAR-T cells using the 'clusterProfiler' package in R. Results: Detailed clinical results from this trial are reported in an accompanying abstract. In brief, data from the first 7 patients on-study demonstrated no treatment-limiting toxicities and an ORR of 86%, with 6 of 6 responding patients achieving a CR by 3 months. 2 of the responding patients have progressed, 1 at 6 months and 1 at 15 months. To interrogate the mechanisms driving axi-cel efficacy in inducing CNS responses, we analyzed 129,088 CAR+ and non-CAR T cells from the peripheral blood (64,337 cells), the CSF (37,070 cells), and the axi-cel product (27,681 cells) from 5 enrolled patients, with blood/CSF collected around the time of maximum CAR-T expansion (Days 5-9 post-infusion). Enumeration of CAR-T cells in the blood vs CSF revealed a mean 13% CAR-Ts of total blood T cells (range 7-24%), and a mean 18% (range 13-28%) in the CSF. CD4+ CAR-T cells predominated in the infused product (mean 79% CAR-T cells were CD4+, range 68-95%), with the CD4:CD8 balance maintained at peak expansion (with 77% (range 60-83%) CAR-T cells in the blood being CD4+, and 95% (range 91-97%) CAR-T cells in the CSF being CD4+). Sc-TCR sequencing enabled an evaluation of the T cell repertoire, revealing a similarly broad CAR-T repertoire in both the blood (mean 1,375 clones (range 313-2,563) and CSF (mean 1,090 clones, range 268-1,965). scRNA-Seq gene expression and clustering analysis revealed a striking distinction between blood and CSF CAR-Ts: While blood CAR-Ts exhibited a prominent proliferation gene expression signature (analyzed by single cell GSEA), CSF CAR-Ts, obtained on the same days as those from the blood, exhibited strong enrichment for interferon-pathway associated genes (Panel 1). Although the majority of CAR-T clones identified were unique to either the blood or CSF, we were able to identify 54 shared clones between these compartments. Analysis of these clones further substantiated the transcriptional evolution of CAR-Ts between the blood and CSF. Thus, individual shared clones tracked in blood and CSF from multiple patients exhibited an evolution from a more diverse proliferation/IFN pathway profile in the blood, to predominantly IFN pathways enriched in CSF CAR-Ts (Panel 2). Conclusions: Daily examination of the blood and CSF by scRNA-Seq and scTCR-Seq has identified a major distinction between blood and CSF CAR-Ts at the level of individual CAR-T clones, revealing the evolution of a prominent interferon pathway transcriptomic signature of CAR-Ts in the CSF. These results suggest that IFN pathway enrichment may play a major role in CAR-T efficacy for the eradication of CNS disease in patients with CNSL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal