Cellular Indexing Of Transcriptomes Research Articles

Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients. CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells. To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.

Free from Drug Therapy in Multiple Myeloma (FREEDMM): MRD-Based Cessation of Maintenance

Background. Treatment of newly diagnosed multiple myeloma (NDMM) consists of induction therapy followed by autologous stem cell transplant (ASCT) and indefinite maintenance. Continuous lenalidomide carries both significant clinical and financial toxicity. Minimal residual disease (MRD) has proven to be a powerful surrogate for PFS and OS but is not yet used to guide treatment. We hypothesize that MRD could be used to predict a subset of patients who can safely stop maintenance therapy and enter into a disease surveillance phase. Study Design and Methods. This prospective phase 2 study (NCT 05192122) is designed to stop post-ASCT maintenance therapy once sustained MRD negativity is achieved. MM patients are eligible if they have completed ≥2 years of maintenance therapy post-ASCT, and meet IMWG criteria for ≥ PR. Bone marrow (BM) MRD testing is performed twice, one year apart, using NGS. Sustained MRD-negativity is defined at a threshold of 10 -6, with negative MRD sustained between the Year 1 and Year 2 interval. Patients with sustained MRD negativity and absence of new bony lesions on PET then undergo maintenance therapy cessation. MRD-positive patients are removed from study. In patients who discontinue maintenance, BM testing to re-assess for MRD is repeated yearly for 3 years. At the time of MRD testing, peripheral blood (PB) samples are collected for MALDI-TOF mass spectrometry testing and circulating multiple myeloma cell (CMMC) enumeration. These are done in parallel with MRD assessment. Health-related quality of life (HRQoL) using the EORTC QLQ-MY20 questionnaire is measured before and after maintenance cessation. This study plans to enroll 50 patients to meet the primary objective of assessing sustained MRD-negativity 1 year after maintenance cessation. For all patients, 5-mL PB and BM samples are collected, processed, and kept frozen until analysis. Cellular indexing of transcriptomes and epitopes (CITE-seq) to simultaneously quantify cell surface protein and transcriptome data is used to characterize differences in the immune microenvironment between MRD-negative, MRD-positive, as well as between patients with biochemical and/or clinical progression and patients with MRD-resurgence but biochemically and clinically stable. Preliminary Results. FREEDMM enrollment began December 2021, and 31 patients have been enrolled, 29 of whom have undergone Year 1 BM for MRD assessment. Median follow up of our cohort is 414 (0-577) days. The median age is 69.5 (40-78) years, and the majority are either Black (n=9; 29%) or Hispanic (n=11; 35%). 18 (58%) patients had R-ISS 1 or 2, while 2 patients had high risk disease with presence of 17p deletion. The median number of years of maintenance therapy completed prior to Year 1 BM is 3.4 (2-7.9) years. 28 (96.6%) were in CR at time of Year 1 BM. Year 1 MRD results are pending in 13 patients. At Year 1, 11 (68.8 %) of 16 patients were found to be MRD-negative, while 5 (31.3%) patients were found to be MRD-positive with a median of 206 (1-9,748) cells per 10 6 nucleated cells detected. 24 (85%) of 28 patients had a negative MALDI-TOF result at the time of the Year 1 BM. MALDI-TOF results were false negative in 3 MRD-positive patients. PB CMMC samples were concordant with MRD results in all 5 (100%) patients who were tested. There was concordance between MALDI-TOF and CMMC results in 9 (75%) of 12 patients. At year 2, 4 patients continued to be MRD negative, while 1 patient who was previously MRD-negative became MRD positive. At most recent follow-up, 3 (42.9%) of 7 MRD positive patients at Year 1 experienced clinical progression and were removed from the study. 2 (50%) of 4 patients with sustained MRD-negativity have undergone maintenance cessation, with the remaining 2 awaiting confirmation of MRD-negative results. 59 paired PB (n=29) and BM (n=30) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 10,000-15,000 cells with 33,000-56,000 reads/ cell on one MRD-positive BM and paired PB sample. CITE-seq results for remaining patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to maintenance therapy cessation in MM patients and represents one of the first studies introducing transcriptome analysis of the tumor and immune microenvironment in the maintenance setting. Ongoing results will be reported at the conference.

Immune Dynamics Associated with Patient Outcomes Identified By Multimodal Single-Cell Analysis in Multiple Myeloma Patients Receiving Talquetamab Monotherapy

Background: Talquetamab (Tal) is a CD3xGPRC5D bispecific antibody (BiAb) that redirects endogenous T cells to mediate killing of GPRC5D-expressing multiple myeloma (MM) cells. In the recent phase I/II MonumenTAL-1 trial (NCT03399799), Tal showed ≥ 70% response rate when given at the recommended phase 2 doses (RP2D) in heavily pretreated MM patients (Chari A et al., NEJM 2022). However, there is still a need to identify predictive biomarkers and better understand the immune dynamics associated with treatment response and durability with this novel agent. Methods:We identified 30 patients who received Tal monotherapy and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and PB collected at cycle 3 day 1 (C3D1). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed to understand transcriptional changes and cell surface proteomics. Total cells were isolated by Ficoll separation from PB and BM samples and were then analyzed separately and only samples of good quality were included in the analysis. Samples were filtered for good quality based on total cell count: at least 1000 cells with more than 500 unique molecular identifiers (UMIs) or genes and less than 4-5 standard deviations above median for UMI, genes and mitochondrial transcripts. A total of 60,851 BM cells (15 pts at baseline) and 299,996 PB cells (23 pts at baseline and 17 pts on C3D1) were analyzed. Downstream analysis was performed using the Seurat R package. All patients included in this study had signed consent for an IRB-approved institutional sub-study (GCO # 18-00456). Results: The 30 patients had a median age of 66.2 years and 57% were female. They received a median of 6 prior lines of therapy, with 83% being triple-class refractory and 50% penta-drug refractory. Only 2 patients were previously treated with anti-BCMA CAR-T cell therapy and none were treated with other BiAbs. 43% of patients had high-risk cytogenetics (including t(4;14), t(14;16) and 17p deletion) and 13% had extramedullary disease at time of initiating therapy. Ten patients (30%) received the recommended phase 2 dose (RP2D) of Tal (0.4 mg/kg weekly or 0.8 mg/kg every other week). Patients were followed for a median of 32.5 months and had a median progression-free survival (PFS) of 6.7 months. 22/30 patients (73%) achieved a partial response (PR) or better, and 14 patients had a short PFS of <150 days. There was no association between the prevalence of different immune cell subsets and depth of response, but rather with PFS. In the baseline BM, a longer PFS was associated with a higher percentage of CD8+ cytotoxic T cells (central memory p=0.00031, effector memory p=0.0022, EMRA p=0.014) and a trend towards higher CD4+ cytotoxic T cells (p=0.072). Of note, we did not find the same associations in the baseline PB samples, which may indicate that the PB may not always reflect the tumor microenvironment in the BM. When we next looked at immune changes in the PB of patients with longer PFS, we found that they exhibited higher percentage of NK cells (p=0.0018 for CD56 low, p=0.04 for CD56 high) and Gamma Delta T cells (p=0.04) at C3D1. Gamma Delta T cells are novel players that warrant further investigation, as they only constitute 1-5% of the T cell repertoire yet are involved in early immune response and do not require major histocompatibility complex (MHC) antigen presentation to function. Profiling of activation and exhaustion markers of these immune subsets is underway. Conclusion: Our findings highlight that the baseline BM immune repertoire and the PB immune changes during Tal treatment may predict long term outcomes. Further in vitro mechanistic studies of T cell function and fitness and validation of these results in independent cohorts will be critical to develop these into predictive biomarkers of long-term response to Tal.

Single-Cell Transcriptomic Profiling of T Cells from Blinatumomab-Treated Patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Reveals Circulating CD8 T Cell Subsets Associated with Treatment Response

Background Blinatumomab (Blina), a bispecific CD3xCD19 T-cell engager (BiTE®), is approved for patients with relapsed/refractory (R/R) and measurable residual disease (MRD) positive B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL). However, a significant percentage of patients do not respond to Blina, and the determinants of resistance are currently unknown. Here, we used Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to identify pre-treatment T cell features associated with response. Methods PBMCs of 13 patients with BCP-ALL who received Blina at the LMU Munich University Hospital from 2016 to 2021 were collected before treatment start. CITE-seq(BD Rhapsody Platform) was performed on a total of 34.907 cells isolated via magnetic-activated cell sorting based on the expression of CD3. An AbSeq panel containing 7 surface markers (CD4, CD8, CCR7, CD45RA, CX3CR1, CD127 and PD1) and a customized T-cell targeted RNA sequencing panel (477 genes) were employed. A Monte-Carlo permutation test was used to compare the proportions of cells in different clusters between Responders (R) and Non-Responders (NR). Significant differences were assumed with a fold change threshold of at least 1.5 (log2FC (LFC) ≥ 0.58) and a false discovery rate (FDR) cutoff of < 0.05. Results We used CITE-seq to characterize T cells in peripheral blood (PB) samples obtained from R/R (n=3) and MRD+ (n=10) BCP-ALL patients. Ten patients (9 MRD+, 1 R/R) responded to the Blina treatment. An integrated strategy based on Reciprocal Principal Component Analysis(RPCA) was implemented and bioinformatic analysis of gene expression mapped in two dimensions via uniform manifold approximation and projection (UMAP) identified 12 CD3 pos distinct clusters. Within CD8 pos T cells of R, we found an increased proportion of CCR7 low Granzyme K(GZMK) high Effector Memory-like cells (GZMK high TEM-like, LFC 0.78, 95CI: 0.66 to 0.92; FDR=0.0012) and a reduced percentage of Naïve cells(CCR7 high LEF1 high CD62L high), when compared to NR (LFC -0.59, 95CI: -0.54 to -0.64; FDR =0.0012, Figure1A). Atrend towards an increased proportion of CCR7 lowCX3CR1 highGZMB high terminal effector-like cells (GZMB high TE-like, LFC 0.57, 95CI: 0.45-0.70; FDR=0.0013) was also noted in CD8 pos T cells of R vs NR. Peripheral CD4 pos T cells of R exhibited a tendency towards an increased proportion of GZMK high cytotoxic T cells (LFC 0.43, 95CI: 0.26 to 0.58; FDR=0.0019) and a reduced percentage of FOXP3 high T regulatory cells (LFC -0.19, 95CI: -0.03 to -0.34; FDR=0.025), compared to NR. Furthermore, we investigated gene expression differences between R and NR cells in each cell cluster. Naïve T cells (CD8 and CD4) of NR showed a significantly increased (LFC>1, FDR<0.05, Figure1B) PRDM1 (Blimp-1) expression, a key promoter of T-cell terminal differentiation that is known to be involved in CD19 CAR-T dysfunction. CD8GZMB high TE-likecells of NR showed upregulated expression of genes involved in chemokine production and cytokine signaling (CCL3, IFNGR1) and downregulated LAIR2, a decoy receptor protecting CD8 T cells from collagen-induced exhaustion. Additionally, TOX, a regulator of T cell exhaustion, was moderately upregulated in CD8GZMB high TE-like cells of NR (LFC 0.41, FDR=0.007). Conclusions Taken together, we were able to identify CD8 T cell subsets that are associated with a response to Blina treatment, as well as gene sets involved in BiTE-mediated T-cell function. Interestingly, the PRDM1 overexpression we found in both CD4 and CD8 naive T cells of Non-Responders has already been linked to T-cell dysfunction in the context of CD19 CAR-T cell therapy. We plan to confirm the results of our analysis in an independent patient cohort. Concomitantly, we will enrich the dataset with results from multi-parameter flow cytometry and in vitro T-cell functional assays conducted using longitudinally collected PB samples from Blina-treated patients. Figure1 A)Log Fold Change(log FC) of the proportion of Responders vs Non Responders CD8 pos cells in each cluster. The dots represent the observed logFC, the horizontal line the bootstrapped 95%Confidence Intervals; PTGDR2= prostaglandin D2 receptor 2; ns= not significant B) Differentially expressed genes in Non Responders vs Responders CD8 pos naïve T cells. FDR=False Discovery Rate; CCL5= Chemokine (C-C motif) ligand 5 ; PRDM1= PR/SET Domain 1;SGK1= serum/glucocorticoid regulated kinase 1.

IMMU-34. SYSTEMIC IMMUNE SUPPRESSION IN GLIOBLASTOMA IS TARGETED BY CONCURRENT VEGF AND PD-1 INHIBITION IN A DOSE-DEPENDENT MANNER: TRANSCRIPTOMIC FINDINGS THROUGH CITE-SEQ FROM A RANDOMIZED CONTROLLED TRIAL

Abstract INTRODUCTION Anti-PD1 blockade has had poor efficacy in recurrent glioblastoma (rGBM) across three RCTs. VEGF, a proangiogenic factor that is upregulated in rGBM, contributes to tumor-associated immunosuppression and is widely targeted through bevacizumab. Preclinical data indicate a potential dose-dependent effect of anti-VEGF therapy on immunomodulation. Hence, a combination of anti-PD-1 and anti-VEGF agents is a promising approach for rGBM, for which an RCT was conducted. This work sought to utilize a multimodal approach capturing single-cell gene and protein expression by ‘cellular indexing of transcriptomes and epitopes by sequencing’ (CITE-Seq) to interrogate the systemic immunological changes in response to concurrent VEG and PD-1 inhibition. METHODS Patients with rGBM were administered nivolumab (240mg IV q2weeks) plus either standard-dose bevacizumab (10mg/kg) or low-dose bevacizumab (3mg/kg) IV q2weeks, with 1:1 randomization. Stratification factors were age, performance status, extent of resection, and MGMT methylation status. Primary endpoint was overall survival (OS) at 12 months. CITE-Seq was performed on 16 patients across trial arms, both pre- and on-therapy, yielding total 32 immunological profiles. For control, CITE-seq was done on three patients receiving anti-VEGF therapy alone. RESULTS 90 patients were enrolled with median age of 60.6 years and 45 patients per arm. Both arms had similar OS at 12 months (P=0.14). Significantly higher OS was found in posthoc analysis for patients aged ≥60 years with standard dose. Distinct immune cell populations were identified, and differential changes in myeloid-derived suppressor cells (MDSCs) found across trial arms on-therapy. Network medicine approach identified VEGF as target to reduce MDSCs, with VEGF primarily expressed by responders’ MDSCs. Differential gene expression analysis identified increased pro-inflammatory signatures in standard-dose arm. CONCLUSIONS Standard dose bevacizumab was associated with increased inflammatory response and reduction of immunosuppressive MDSC. These mechanistic insights potentially explain survival benefit seen with concurrent VEGF and PD-1 inhibition in elderly rGBM patients.

A single-cell atlas of immune checkpoint molecules in atherosclerosis in ApoE-/- mice

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

RAMP up: Risk Adapted Post-Autologous Transplant Maintenance Therapy in MRD Positive Myeloma Patients

Background. Post-autologous stem cell transplant (ASCT) maintenance therapy for patients with newly diagnosed multiple myeloma (MM) continues to evolve. Currently, single agent lenalidomide maintenance remains standard of care but outcomes for patients with high-risk cytogenetics are inferior to those with standard risk disease; optimal maintenance strategies for these patients need to be developed. Minimal residual disease (MRD) has proven to be a powerful prognostic factor for PFS and OS but is not yet used to guide treatment. Isatuximab is a cytolytic CD38-directed, immunoglobulin G1 (IgG1) chimeric monoclonal antibody FDA-approved for the treatment of relapsed or refractory MM that is overall well tolerated and efficacious when used in combination regimens. Our upcoming trial, RAMP UP (NCT05344833), investigates the safety and efficacy of isatuximab combined with lenalidomide as a maintenance regimen in patients who are MRD-positive after autologous transplant. Study Design and Methods. This prospective, multicenter single-arm phase 2 (Figure 1) trial will enroll patients that are MRD-positive (10 -5) by Adaptive next generation sequencing within 90 days (+/- 30 days) post-autologous transplant. We plan to enroll: R-ISS stage 1, 2 or 3; <2 lines of induction therapy prior to transplant; > PR per IMWG criteria post-transplant; and ECOG Performance Status <2. Patients will be treated with isatuximab (10 mg/kg weekly for cycle 1 and every other week thereafter) and lenalidomide (10 mg daily on days 1-21) on 28-day cycles for a total of 3 years. Patients will be monitored for disease response every 3 months and for marrow MRD every 12 months. The primary endpoint is the 1-year rate of CR/MRD negativity (10 -5); secondary endpoints include 1-year rate of CR/MRD negativity (10 -6), IMWG-defined response rates, 3-year overall CR/MRD-negativity, PFS, OS, treatment-related adverse events, and health related quality of life (HRQoL) measured by EORTC-QLQ-C30, EQ-5D-5L, EORTC QLQ-MY20. We propose enrollment of 50 patients to allow for approximately 10% dropout rate in order to have 43 patients analyzed for the primary endpoint; for 50 patients to be treated, we estimate that 75-100 patients may have to be enrolled. Correlative objectives include correlation of isatuximab pharmacokinetics and receptor occupancy with MRD-negative response and time to progression, characterization of differences in the BM and peripheral blood T, B, and myeloid cell compartments using cellular indexing of transcriptomes and epitopes (CITE-Seq) between responders and nonresponders, and concurrent assessment of other disease biomarkers, including peripheral blood circulating multiple myeloma cells (CELLSEARCH), with MRD testing. Discussion. Our study started June 2023. One patient has been enrolled, and two have signed informed consent for enrollment. Ultimately, our study aims to inform MRD risk-adapted approaches for maintenance therapy in myeloma.

Biomarker Correlates of Response to Ciltacabtagene Autoleucel in Patients with Relapsed or Refractory Multiple Myeloma from CARTITUDE-1, a Phase 1b/2 Open-Label Study, at the ~3 Year Follow-up

Ciltacabtagene autoleucel (cilta-cel) consists of autologous T cells genetically modified to express a two-binding domain chimeric antigen receptor (CAR). The target antigen of the CAR is B-cell maturation antigen (BCMA), which is highly expressed on malignant plasma cells (PC) from multiple myeloma subjects. In the Phase 1b/2 CARTITUDE-1 study, cilta-cel led to early, deep, and durable responses and was approved for the treatment of patients with relapsed or refractory multiple myeloma (RRMM) after four or more prior lines of therapy by FDA. Despite cilta-cel's outstanding overall response rate (97.9%) and duration of response (DOR) in RRMM patients, relapse still occurs. Biomarkers associated with this durable response and acquired resistance remain to be elucidated. We present updated correlative data from the CARTITUDE-1 study. At the ~3 year follow-up (October 2022 data cutoff), 97 patients had received a single infusion of cilta-cel (median 0.71 x10 6 cells/kg [range 0.52 x10 6-0.94 x10 6]); the median follow up was 33.4 months (range 1.5-45.2), median DOR was 33.9 months (95% CI, 25.5-not estimable [NE]) and median PFS was 34.9 months (95% CI, 25.2-NE) (Lin Y et al, ASCO 2023). Drug product (DP), baseline and on-treatment whole blood and bone marrow samples from enrolled patients were analyzed by methods including flow cytometry, MSD immunoassays, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and TCR sequencing. In CARTITUDE-1, the DP contained a mixture of transduced and non-transduced T cells; the median transduction efficiency was 16% (range 5-32%), with a balanced distribution of CAR+CD4+ and CAR+CD8+ cells, median frequency 12% (range 2-28%) and 6% (range 2-20%), respectively. Further, the DP T cell subset composition was variable but balanced between central (Tcm) and effector memory (Tem) phenotypes. CAR+ T cells expanded reaching a median peak concentration (C max of 730 cells/µL; range 3-13805 cells/µL) in blood between days 12-14 post-infusion and persisted in circulation for a median of 100 days (range 20-912 days). High efficacy (best response) and DOR were achieved despite variable CAR-T cell expansion and lack of detectable CAR-T cell persistence over time. At C max, the CD4:CD8 ratio (based on % of CAR+ cells) was 0.29, demonstrating a CAR+CD8+ T cell preferential expansion, and both CAR+CD4+ and CAR+CD8+ T cells were predominantly of a Tcm phenotype, median 95% (range 62-99.5%) and 96% (range 33-99.7%), respectively. Higher frequency of early-memory T cell phenotypes has been associated with clinical responses to CAR-T therapies in MM (Dancy et al., Blood 2018). CITE-seq analyses showed that DP characteristics such as a high CD8+ stem-like phenotype and a low CD4+ Treg-like phenotype in the CAR+ T cell compartment associated with longer DOR. Some of the most abundant clones in the DP single cell TCR data were CAR+CD8+ T cells expressing granzyme B, suggesting high cytotoxic potential. Additional bulk TCR sequencing data suggest both diversity and individual clonal expansion longitudinally after cilta-cel infusion. We also evaluated tumor and patient intrinsic characteristics at baseline. BCMA expression on bone marrow PC was prevalent and highly variable among RRMM patients; however, expression did not associate with best response or DOR. Further, high ORR was observed in patients with high-risk cytogenetics, high tumor burden (>= 60% bone marrow PC), or baseline plasmacytomas, although with a non-statistically significant shorter PFS. Sustained MRD negativity strongly associated with longer DOR. Conversely, patient-intrinsic characteristics associated with inflammation (CRP, ferritin, neutrophils, pro-inflammatory cytokines) correlated with shorter DOR. Treatment benefit of the magnitude seen with cilta-cel had not been reported with any other approved therapy in this setting. Correlative analyses suggest an emerging profile to help understand such outstanding clinical activity: a balanced CD4/CD8 ratio and Tcm/Tem phenotype of the manufactured DP and preferential expansion of CAR+CD8+ Tcm cells. Further, certain baseline patient-intrinsic and tumor characteristics may explain the longer DOR in the RRMM setting. These investigations help identify markers of response to cilta-cel and may lead to CAR-T cell design or manufacturing strategies that enhance drug product characteristics and thus clinical efficacy.

Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11–13 years) and older adults (aged 55–65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

MZB1-expressing cells are essential for local immunoglobulin production in chronic rhinosinusitis with nasal polyps

BackgroundThe expression of MZB1 genes is significantly elevated in patients who have chronic rhinosinusitis with nasal polyp (CRSwNP) disease compared with healthy controls. ObjectiveTo characterize MZB1-positive B cells in CRSwNP and to estimate the contribution of distinct subsets of B cells to the local overproduction of immunoglobulins. MethodsSingle-cell RNA-sequencing with Cellular Indexing of Transcriptomes and Epitopes by Sequencing technology, Switching Mechanism At the 5′ end of RNA Template sequencing, flow cytometry, immunohistochemistry and immunofluorescence staining, Western blot, QuantiGene Plex assay, B-cell ImmunoSpot assay, Luminex assay, and enzyme-linked immunosorbent assay were performed. ResultsSignificantly higher mRNA expression of MZB1 and HSP90B1 was found in type 2 CRSwNP compared with controls. In CRSwNP, MZB1 expression correlated with the local production of IgE. MZB1 could be colocalized with plasma and mature B cells, especially marginal zone (MZ) B cells. Single-cell transcriptome and epitope studies revealed prominent populations of B cells in type 2 CRSwNP with unexpectedly high MZB1 gene expression. The MZ B-cell population was significantly increased in CRSwNP compared with healthy controls in both peripheral blood mononuclear cells and nasal tissue single-cell suspensions. When those single cells were cultured overnight, the MZ B-cell numbers were positively correlated with local IgE production but negatively correlated with local IgM production. In vitro, MZB1 stimulation up-regulated the mRNA expression of IgE. ConclusionMZB1 was primarily expressed by plasma and mature B cells in nasal mucosa. MZB1 expression level was increased in CRSwNP compared with controls. MZB1 contributed to the local IgE production in type 2 CRSwNP.

Systematic identification of gene combinations to target in innate immune cells to enhance T cell activation

Genetic engineering of immune cells has opened new avenues for improving their functionality but it remains a challenge to pinpoint which genes or combination of genes are the most beneficial to target. Here, we conduct High Multiplicity of Perturbations and Cellular Indexing of Transcriptomes and Epitopes (HMPCITE-seq) to find combinations of genes whose joint targeting improves antigen-presenting cell activity and enhances their ability to activate T cells. Specifically, we perform two genome-wide CRISPR screens in bone marrow dendritic cells and identify negative regulators of CD86, that participate in the co-stimulation programs, including Chd4, Stat5b, Egr2, Med12, and positive regulators of PD-L1, that participate in the co-inhibitory programs, including Sptlc2, Nckap1l, and Pi4kb. To identify the genetic interactions between top-ranked genes and find superior combinations to target, we perform high-order Perturb-Seq experiments and we show that targeting both Cebpb and Med12 results in a better phenotype compared to the single perturbations or other combinations of perturbations.

Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy-to-use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye-oligo conjugates by flow cytometry. Using dye-oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye-oligo conjugates using a flow cytometry cell sorter and processed for downstream single-cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye-oligo reagents, avoiding the possibility of decreased marker resolution from co-staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE-Seq.

Non-Redundant Mechanisms of Immune Resistance to Radiotherapy Converge on Innate Immunity

Despite evidence of preclinical synergy between radiotherapy (RT) and immune checkpoint blockade (ICB), randomized trials of RT/ICB have demonstrated limited benefit in solid tumors. We performed single-cell RNA sequencing (scRNA-seq) and CITE-seq (cellular indexing of transcriptomes and epitopes) to address the discordance between preclinical and clinical data. We hypothesized that multiple orthogonal inhibitory immune pathways restrain the local and systemic efficacy of RT beyond T-cell oriented immune checkpoints. We used the EO771 syngeneic murine model of breast cancer to characterize the immune tumor microenvironment following RT with or without ICB. RT (16 Gy x 1) was delivered using the X-RAD SmART platform with CT image guidance. Neutralizing antibodies (anti-PD-1/Ly6G/Gr-1/CD47) were delivered by intraperitoneal injections. scRNA-seq analysis were performed by Seurat and BBrowser (BioTuring). We found that adaptive ICB (anti-PD-1) reprogrammed the immune response to RT by promoting an M1-like interferon-primed state (ISG15, CXCL10) in tumor associated macrophages (TAMs) and by increasing the late recruitment of intratumoral neutrophils. Given that neutrophils may drive resistance to RT in other models, we evaluated the effect of intratumoral neutrophil depletion using anti-Ly6G or anti-Gr-1 on the antitumor efficacy of RT/ICB. Both neutrophil depletion strategies led to enhanced tumor control and improved survival in advanced EO771 tumors compared to RT/ICB alone (P<0.001). In parallel to this approach, we found that TAMs upregulated several innate immune checkpoints including SIRPα in response to RT. Disruption of the SIRPα-CD47 interaction by anti-CD47 antibodies similarly enhanced the antitumor efficacy of RT/ICB by improving tumor control and survival (P<0.001). Using scRNA-seq and unbiased clustering, we found that anti-CD47 eliminated an entire cluster of chronically inflamed TAMs, characterized by pro-inflammatory markers (IL1A, NOS2) and chemokines (CCL3, CXCL1/2/3). Anti-CD47 also reduced intratumoral neutrophils by eliminating a cluster of pathologically activated neutrophils, termed myeloid-derived suppressor cells (PMN-MDSCs) that expressed several markers of ferroptosis (TFRC, PTGS2, SLC3A2). Consistent with the potent immunosuppressive capacity of PMN-MDSCs, we found that anti-CD47 increased tumor-infiltrating lymphocytes including central memory TCF7+ T cells and CD19+ B cells. Lastly, by inference and analysis of cell-cell communication (CellChat), we found that anti-CD47 strengthened the interactions between TAMs and CD8+ T cells compared to RT/ICB alone. Our data collectively indicate that resistance to RT/ICB in the EO771 model Is driven by innate immune cells including neutrophils and chronically inflamed TAMs. Targeted disruption of the CD47-SIRPα axis is a promising approach to overcoming immune resistance by reprogramming TAMs and eliminating PMN-MDSCs.

Identification of cell-type-specific genes in multimodal single-cell data using deep neural network algorithm

The emergence of single-cell RNA sequencing (scRNA-seq) technology makes it possible to measure DNA, RNA, and protein in a single cell. Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) is a powerful multimodal single-cell research innovation, allowing researchers to capture RNA and surface protein expression on the same cells. Currently, identification of cell-type-specific genes in CITE-seq data is still challenging. In this study, we obtained a set of CITE-seq datasets from Kaggle database, which included the sequencing dataset of seven cell types during bone marrow stem cell differentiation. We used Student's t-test to analyze these transcription RNAs and pick out 133 significantly differentially expressed genes (DEGs) among all cell types. Functional enrichment revealed that these DEGs were strongly associated with blood-related diseases, providing important insights into the cellular heterogeneity within bone marrow stem cells. The relation between RNA and protein levels was performed by deep neural network (DNN) model and achieved a high prediction score of 0.867. Based on their coefficients in the DNN model, three genes (LGALS1, CENPV, TRIM24) were identified as cell-type-specific genes in erythrocyte progenitor. Our works provide a novel perspective regarding the differentiation of stem cells in the bone marrow and provide valuable insights for further research in this field.

Single-cell multiomics of human fetal hematopoiesis define a developmental-specific population and a fetal signature.

Knowledge of human fetal blood development and how it differs from adult blood is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space, adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single-cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), the molecular profile of established immunophenotype-gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs), such as CD90 and CD49F, were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow data set revealed that the HSC state was less frequent in FL, whereas cells with a lymphomyeloid signature were more abundant. An erythromyeloid-primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature was identified. The core gene set could separate subgroups of acute lymphoblastic leukemia by age, suggesting that a fetal program may be partially retained in specific subgroups of pediatric leukemia. Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, which are of significance for future studies of pediatric leukemia and blood development in general.

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

STREAK: A supervised cell surface receptor abundance estimation strategy for single cell RNA-sequencing data using feature selection and thresholded gene set scoring.

The accurate estimation of cell surface receptor abundance for single cell transcriptomics data is important for the tasks of cell type and phenotype categorization and cell-cell interaction quantification. We previously developed an unsupervised receptor abundance estimation technique named SPECK (Surface Protein abundance Estimation using CKmeans-based clustered thresholding) to address the challenges associated with accurate abundance estimation. In that paper, we concluded that SPECK results in improved concordance with Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) data relative to comparative unsupervised abundance estimation techniques using only single-cell RNA-sequencing (scRNA-seq) data. In this paper, we outline a new supervised receptor abundance estimation method called STREAK (gene Set Testing-based Receptor abundance Estimation using Adjusted distances and cKmeans thresholding) that leverages associations learned from joint scRNA-seq/CITE-seq training data and a thresholded gene set scoring mechanism to estimate receptor abundance for scRNA-seq target data. We evaluate STREAK relative to both unsupervised and supervised receptor abundance estimation techniques using two evaluation approaches on six joint scRNA-seq/CITE-seq datasets that represent four human and mouse tissue types. We conclude that STREAK outperforms other abundance estimation strategies and provides a more biologically interpretable and transparent statistical model.

Unsupervised removal of systematic background noise from droplet-based single-cell experiments using CellBender.

Droplet-based single-cell assays, including single-cell RNA sequencing (scRNA-seq), single-nucleus RNA sequencing (snRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), generate considerable background noise counts, the hallmark of which is nonzero counts in cell-free droplets and off-target gene expression in unexpected cell types. Such systematic background noise can lead to batch effects and spurious differential gene expression results. Here we develop a deep generative model based on the phenomenology of noise generation in droplet-based assays. The proposed model accurately distinguishes cell-containing droplets from cell-free droplets, learns the background noise profile and provides noise-free quantification in an end-to-end fashion. We implement this approach in the scalable and robust open-source software package CellBender. Analysis of simulated data demonstrates that CellBender operates near the theoretically optimal denoising limit. Extensive evaluations using real datasets and experimental benchmarks highlight enhanced concordance between droplet-based single-cell data and established gene expression patterns, while the learned background noise profile provides evidence of degraded or uncaptured cell types.

Meis1 establishes the pre-hemogenic endothelial state prior to Runx1 expression

Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.