DIISCO: A Bayesian framework for inferring dynamic intercellular interactions from time-series single-cell data.

Characterizing cell-cell communication and tracking its variability over time is essential for understanding the coordination of biological processes mediating normal development, progression of disease, or responses to perturbations such as therapies. Existing tools lack the ability to capture time-dependent intercellular interactions, such as those influenced by therapy, and primarily rely on existing databases compiled from limited contexts. We present DIISCO, a Bayesian framework for characterizing the temporal dynamics of cellular interactions using single-cell RNA-sequencing data from multiple time points. Our method uses structured Gaussian process regression to unveil time-resolved interactions among diverse cell types according to their co-evolution and incorporates prior knowledge of receptor-ligand complexes. We show the interpretability of DIISCO in simulated data and new data collected from CAR-T cells co-cultured with lymphoma cells, demonstrating its potential to uncover dynamic cell-cell crosstalk.

ZNF683 marks a CD8+ T cell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.

Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia.

In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists.

Combined quadriceps medial patellofemoral ligament reconstruction and osteochondral fixation offers good patient-reported outcomes and low rates of recurrent instability for osteochondral defects secondary to acute patella dislocation.

PURPOSE: Chondral injuries secondary to traumatic patella dislocation are common, and a subgroup of these are significant defects with fragments amenable to fixation. There is a paucity of published evidence assessing patients managed with combined acute patellofemoral stabilisation and osteochondral fixation. The purpose of this study is to report the outcomes of patients with osteochondral injuries secondary to acute traumatic patella dislocation treated with combined early fragment fixation and MPFL reconstruction using a quadriceps tendon turndown technique which has distinct advantages for this cohort, including preventing chondral overloading and non-violation of the patella bone. METHODS: Patients who underwent combined quadriceps tendon MPFL reconstruction and osteochondral fixation were included. Patient demographics, defect characteristics, complications and reoperations were evaluated. Patients were assessed with Lysholm, Kujala, KOOS-PF scores and satisfaction scale at follow up. Pre-operative MRI was assessed for presence of radiological risk factors for patella dislocation and post-operative MRI was used to assess cartilage quality with MOCART 2.0 score. RESULTS: A total of 19 patients (63.2% female) were included. The mean age was 17.4 ± 4.8 years and patients were followed up at a mean 15.8 ± 5.1 months post-surgery. The mean defect size was 2.4 cm2 ± 1.3 cm2, with the most common defect location being the patella (13/19; 68.4%) followed by the lateral femoral condyle (5/19; 26.3%). At final follow up, the overall mean Lysholm, Kujala, and KOOS-PF scores were 84.9 ± 11.1, 89.7 ± 5.8 and 80.6 ± 13.6, respectively. Seventeen patients (89.5%) were satisfied with their outcome. The mean MOCART 2.0 score at final follow-up was 72.5. One patient required medial capsular plication with removal of a loose chondral body and microfracture and 3 knees required minor reoperations. CONCLUSION: Combined acute osteochondral fragment fixation and MPFL reconstruction using a quadriceps tendon graft offers good radiological and patient-reported outcomes with high satisfaction and low rates of recurrent patella dislocation. To our knowledge, this is currently the largest series of its kind in the literature and the results of this study provide a rationale for a combined approach using a quadriceps tendon graft for this cohort. LEVEL OF EVIDENCE: Level IV.

The immune microenvironment shapes transcriptional and genetic heterogeneity in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions, and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared with PB and in immunoglobulin heavy-chain variable (IGHV) region unmutated compared with mutated cases. Single-cell RNA sequencing (scRNA-seq) distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival (TFS) and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing (WES) of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives the clinical outcome. Clonal trajectories are shaped by the LN milieu, where T-cell immunity may contribute to suppressing clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

cyCombine allows for robust integration of single-cell cytometry datasets within and across technologies.

Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.

Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment.

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.

Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy.

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.

Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.

Chimeric antigen receptor (CAR) T-cells have emerged as an efficacious modality in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP), a state in which mutations in hematopoietic cells give rise to a clonal population of cells, is more common in patients exposed to cytotoxic therapies, has been shown to influence inflammatory immune programs, and is associated with an adverse prognosis in patients with NHL and MM receiving autologous transplantation. We therefore hypothesized that CHIP could influence clinical outcomes in patients receiving CAR T-cell therapy. In a cohort of 154 patients with NHL or MM receiving CAR T-cells, we found that CHIP was present in 48% of patients and associated with increased rates of complete response and cytokine release syndrome severity, but only in patients younger than age 60 years. Despite these differences, CHIP was not associated with a difference in progression-free or overall survival, regardless of age. Our data suggest that CHIP can influence CAR T-cell biology and clinical outcomes, but, in contrast to autologous transplantation, CHIP was not associated with worse survival and should not be a reason to exclude individuals from receiving this potentially life-prolonging treatment.

Coevolving JAK2V617F+relapsed AML and donor T cells with PD-1 blockade after stem cell transplantation: an index case.

Relapse of myeloproliferative neoplasms (MPNs) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes, as therapeutic approaches to reinstate effective graft-versus-leukemia (GVL) responses remain suboptimal. Immune escape through overexpression of PD-L1 in JAK2V617F-mutated MPN provides a rationale for therapeutic PD-1 blockade, and indeed, clinical activity of nivolumab in relapsed MPN post-HSCT has been observed. Elucidation of the features of response following PD-1 blockade in such patients could inform novel therapeutic concepts that enhance GVL. Here, we report an integrated high-dimensional analysis using single-cell RNA sequencing, T-cell receptor sequencing, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and assay for transposase-accessible chromatin using sequencing (scATAC-seq), together with mass cytometry, in peripheral blood mononuclear cells collected at 6 timepoints before, during, and after transient response to PD-1 blockade from an index case of relapsed MPN following HSCT. Before nivolumab infusion, acute myeloid leukemia (AML) blasts demonstrated high expression of chemokines, and T cells were characterized by expression of interferon-response genes. This baseline inflammatory signature disappeared after nivolumab infusion. Clinical response was characterized by transient expansion of a polyclonal CD4+ T-cell population and contraction of an AML subpopulation that exhibited megakaryocytic features and elevated PD-L1 expression. At relapse, the proportion of the AML subpopulation with progenitor-like features progressively increased, suggesting coevolution of AML blasts and donor-derived T cells. We thus demonstrate how single-cell technologies can provide complementary insight into cellular mechanisms underlying response to PD-1 blockade, motivating future longitudinal high-dimensional single-cell studies of GVL responses in relapsed myeloid disease.

Longitudinal Single-Cell Dynamics of Chromatin Accessibility and Mitochondrial Mutations in Chronic Lymphocytic Leukemia Mirror Disease History.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics. SIGNIFICANCE: Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution. See related commentary by Hilton and Scott, p. 2965. This article is highlighted in the In This Issue feature, p. 2945.