Target discovery for IBD has traditionally relied on genetic associations, which lack the cellular resolution needed to identify novel, actionable, cell type-specific disease pathways. Here, we describe an integrated analytical and experimental framework that leverages harmonized single-cell data to systematically discover novel therapeutic strategies for IBD. We used AMICA DBTM, Immunai's harmonized database of single-cell RNA datasets to construct a harmonized 1 million single-cell atlas of the human intestine. We applied a machine learning framework (Immune Patient Representation, IPR) to identify disease-associated transcriptional programs and cell type-specific gene targets. Candidate targets were prioritized using atlas-derived metrics, refined using custom criteria emphasizing translational actionability, and validated across independent clinical cohorts. Select candidates were evaluated in human primary-cell models reflecting the target's cell-type context. The IPR framework identified 85 disease-associated transcriptional programs and ranked 400 cell type-specific target genes across immune and stromal lineages. Disease-associated programs were interpreted using a structured AI-assisted reasoning framework for structured biological reasoning, linking them to IBD-relevant pathways and guiding the identification of novel, promising gene targets. Functional validation of two cell-type-specific candidates, PTGIR in myeloid cells and IL6ST in fibroblasts, confirmed the reduction of inflammatory and fibrotic pathways linked to IBD pathology. Multi-omic profiling and projection of in vitro phenotypes to patient datasets demonstrated the reversal of disease-associated programs via mechanisms distinct from those of existing biologics. Our single-cell anchored, machine-learning framework integrates in silico discovery with experimental validation, revealing new cell type-specific therapeutic opportunities and supporting a scalable approach for precision target discovery in IBD and other immune-mediated diseases. ### Competing Interest Statement All authors declare potential competing interests. Authors affiliated with Immunai and AstraZeneca are employees of their respective organizations and may hold equity or stock options as part of standard compensation. The authors' institutions have filed provisional patent applications related to aspects of the work described in this manuscript. No other financial, professional, or personal conflicts relevant to this manuscript are reported.

Abstract Introduction Despite advancements for Inflammatory Bowel Disease (IBD), high rates of non-response, relapse, and adverse effects underscore the need for novel therapeutic interventions. This necessity is compounded by the inherent complexity of IBD; wherein, multiple resident cellular components receive and generate aberrant signals within tissues and migrating immune cells. Methods To identify novel therapeutic targets, we constructed a harmonized single-cell tissue atlas (IBD atlas) derived from published clinical single-cell datasets. Machine learning-based methods were employed to extract relevant transcriptional signals and their contributing genes, which were then ranked using orthogonal translation metrics to generate a list of candidate targets for in vitro functional validation. Using this approach, we nominated candidates for cell type-specific functional evaluation in macrophages and fibroblasts. We utilized in vitro models optimized to recapitulate specific transcriptional states of these cells in inflamed IBD tissue, and evaluated the impact of target deletion in the presence or absence of appropriate ligands. Results We highlight a specific target for which deletion reduced the inflammatory proteome and induced an anti-inflammatory transcriptional state similar to that of healthy intestinal myeloid cells with a reduction in IBD associated signatures. Comparison to transcriptional shifts induced by standard-of-care therapies showed that this target’s profile is distinct from anti-TNF and anti-integrin while recapitulating anti-inflammatory effects of JAK inhibitors. We contrast this with a second target for which ablation induced a proinflammatory response in macrophages but a favorable, anti-fibrotic response in intestinal fibroblasts highlighting two novel cell type targeted therapeutic approaches. Conclusion In conclusion, our framework leverages a robust single-cell data foundation to nominate disease-relevant targets in specific cell types optimally poised for desirable clinical outcomes. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)

Hospitalized COVID-19 patients exhibit diverse immune responses during acute infection, which are associated with a wide range of clinical outcomes. However, understanding these immune heterogeneities and their links to various clinical complications, especially long COVID, remains a challenge. In this study, we performed unsupervised subtyping of longitudinal multi-omics immunophenotyping in over 1,000 hospitalized patients, identifying two critical subtypes linked to mortality or mechanical ventilation with prolonged hospital stay and three severe subtypes associated with timely acute recovery. We confirmed that unresolved systemic inflammation and T-cell dysfunctions were hallmarks of increased severity and further distinguished patients with similar acute respiratory severity by their distinct immune profiles, which correlated with differences in demographic and clinical complications. Notably, one critical subtype (SubF) was uniquely characterized by early excessive inflammation, insufficient anticoagulation, and fatty acid dysregulation, alongside higher incidences of hematologic, cardiac, and renal complications, and an elevated risk of long COVID. Among the severe subtypes, significant differences in viral clearance and early antiviral responses were observed, with one subtype (SubC) showing strong early T-cell cytotoxicity but a poor humoral response, slower viral clearance, and greater risks of chronic organ dysfunction and long COVID. These findings provide crucial insights into the complex and context-dependent nature of COVID-19 immune responses, highlighting the importance of personalized therapeutic strategies to improve both acute and long-term outcomes.

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

Increased levels of matrix metalloproteinase 8, expressed by circulating myeloid cells, may have a role in stress-induced changes in social behaviour in mice.

Supplementary Methods and Data

Correlation of expression of canonical cell type markers across different modalities

Psychosocial stress has profound effects on the body, including the peripheral immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3,4,5, the underlying mechanisms are not well understood. Here we show that a peripheral myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is elevated in serum of subjects with MDD as well as in stress-susceptible (SUS) mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), thereby altering social behaviour. Using a combination of mass cytometry and single-cell RNA-sequencing, we performed high-dimensional phenotyping of immune cells in circulation and brain and demonstrate that peripheral monocytes are strongly affected by stress. Both peripheral and brain-infiltrating monocytes of SUS mice showed increased Mmp8 expression following CSDS. We further demonstrate that peripheral MMP8 directly infiltrates the NAc parenchyma to control the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a novel mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

The development of new immunotherapies to treat the inflammatory mechanisms that sustain atherosclerotic cardiovascular disease (ASCVD) is urgently needed. Herein, we present a path to drug repurposing to identify immunotherapies for ASCVD. The integration of time-of-flight mass cytometry and RNA sequencing identified unique inflammatory signatures in peripheral blood mononuclear cells stimulated with ASCVD plasma. By comparing these inflammatory signatures to large-scale gene expression data from the LINCS L1000 dataset, we identified drugs that could reverse this inflammatory response. Ex vivo screens, using human samples, showed that saracatinib—a phase 2a-ready SRC and ABL inhibitor—reversed the inflammatory responses induced by ASCVD plasma. In Apoe −/− mice, saracatinib reduced atherosclerosis progression by reprogramming reparative macrophages. In a rabbit model of advanced atherosclerosis, saracatinib reduced plaque inflammation measured by [ 18 F]fluorodeoxyglucose positron emission tomography–magnetic resonance imaging. Here we show a systems immunology-driven drug repurposing with a preclinical validation strategy to aid the development of cardiovascular immunotherapies.

Supplementary figures for Cohen et al. CIR-18-0198R2

Introduction Multimodal single cell technologies allow us to dissect the mechanisms of therapeutic resistance by integrating transcriptomics and proteomics through the combination of antibody labeling and next-generation sequencing. Using CITE-seq, mass cytometry (CyTOF) and quantitative multiplexed proteomics (Olink), we sought to understand the determinants of chimeric antigen receptor (CAR) T cell response and the overall survival of patients with relapsed or refractory multiple myeloma (RRMM) receiving ciltacabtagene autoleucel (Cilta-Cel) cellular therapy. Methods Twenty five patients received Cilta-Cel and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and after CAR T infusion. We isolated 262,520 BM cells and 241,657 PB cells, which were later sequenced using CITE-seq. Additionally, we submitted PB samples for CyTOF analysis and acquired 10,162,426 cells used for downstream analysis. We analyzed 92 cytokines using Olink immuno-oncology panel in PB samples. Downstream analysis was performed using the R packages Seurat, CATALYST, FlowSOM and Olink Analyze. Results Our cohort had a median progression-free survival (PFS) of 732 days. To focus our correlative analyses on patients experiencing early relapse, the initial cohort was divided into 2 groups: PFS <18 months (n = 10) and PFS >18 months (n = 15). CAR-T cell expansion was observed in week 2 post-infusion and continued up to week 4. The percentage of CD4 and CD8 CAR-T cells significantly increased between weeks 1-3 and weeks 4-6 weeks (p<0.001) and significantly decreased after week seven (p<0.05). We detected novel and significant differences among four cell populations associated with PFS longer than 18 months. These patients had a higher percentage of activated CD4 Central Memory (CM) and CD4 cytotoxic cells (p<0.05) relative to the total percentage of CAR-T cells in weeks 4-6, suggesting a key role for CD4 cells in cross priming or direct cytotoxicity in this context. In patients with a PFS longer than 18 months, the CD8 CM CART cell population had a significantly higher percentage in weeks 1-3 and 4-6 (p<0.05) when compared to their counterparts, suggesting that these were the cardinal effector population in these patients. Myeloid-derived suppressor cells (MDSCs) have been shown to be a central component of the tumor microenvironment in myeloma, with subsets being capable to mount potent suppressive activity at the tumor site. In the BM CITE-seq myeloid compartment, MDSCs were significantly increased in month 1 (p<0.05) in patients with a shorter PFS. Our PB CyTOF data confirmed this finding as the percentage of MDSCs was significantly higher in weeks 1-3 (p<0.05) in the shorter PFS group when compared to their counterparts. Using a mixed linear regression model on Olink data, we detected 26 cytokines significantly different (p<0.05) between the shorter and longer PFS groups. A pseudobulk analysis of the BM CITE-seq samples for differentially expressed genes encoding the 26 cytokines revealed 22 genes differentially expressed (p<0.05) between patients with a PFS shorter than 18 months and patients with a PFS longer than 18 months in CAR-T, T-cell, NK cell and myeloid cell populations. In the shorter PFS group, VEGFA was significantly higher in CD8 TEMRA CAR-T cells when compared to their counterparts. In the longer PFS group, we observed significantly higher levels of genes involved in T cell activation, such as CD27 and CD28, and pro-inflammatory cytokines such as TNF and IL-15 in the T cell and Myeloid cell compartments. This pattern suggests that higher production of cytotoxic and pro-inflammatory cytokines, combined with enhanced T cell activation, plays an important role in prolonging the response to CAR-T therapy. Conclusions Single cell immune profiling and transcriptomic sequencing identified subpopulations of CD4 and CD8 cells which in concert may influence long term CAR-T outcomes. Our findings demonstrate an early expansion of CART, with very few CART cells surviving after 3 months, suggesting that the efficacy of this therapy is related to early dynamics of these populations. We also provide additional evidence associating immunosuppressive MDSC populations in BM and PB patients with a shorter PFS. Ongoing studies will further analyze the role of the immune microenvironment and clonal T cell dynamics in relation to patient outcomes.

The role of the immune microenvironment in maintaining disease remission in patients with multiple myeloma (MM) is not well understood. In this study, we comprehensively profile the immune system in patients with newly diagnosed MM receiving continuous lenalidomide maintenance therapy with the aim of discovering correlates of long-term treatment response. Leveraging single-cell RNA sequencing and T cell receptor β sequencing of the peripheral blood and CyTOF mass cytometry of the bone marrow, we longitudinally characterize the immune landscape in 23 patients before and one year after lenalidomide exposure. We compare patients achieving sustained minimal residual disease (MRD) negativity to patients who never achieved or were unable to maintain MRD negativity. We observe that the composition of the immune microenvironment in both the blood and the marrow varied substantially according to both MRD negative status and history of autologous stem cell transplant, supporting the hypothesis that the immune microenvironment influences the depth and duration of treatment response.

supplementary table 1 shows cell type annotation markers, clinical information of patients, cross-technique comparison of cell subset abundance and expression of cell type marker genes in CITE-seq and CyTOF

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.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13 + CH25H + IL-21 + PD-1 + CD4 + T helper cells (“CXCL13 + T H ”) and Granzyme K + PD-1 + effector-like CD8 + T cells, whereas terminally exhausted CD39 hi TOX hi PD-1 hi CD8 + T cells dominated in nonresponders. CD4 + and CD8 + T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1 + TCF-1 + (Progenitor-exhausted) CD8 + T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8 + T cell differentiation occurs upon ICB. We found that these Progenitor CD8 + T cells interact with CXCL13 + T H within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or “mregDC”. These results suggest that discrete intratumoral niches that include mregDC and CXCL13 + T H control the differentiation of tumor-specific Progenitor exhasuted CD8 + T cells following ICB.

Despite advancements in understanding the pathophysiology of Multiple Myeloma (MM), the cause of rapid progressing disease in a subset of patients is still unclear. MM’s progression is facilitated by complex interactions with the surrounding bone marrow (BM) cells, forming a microenvironment that supports tumor growth and drug resistance. Understanding the immune microenvironment is key to identifying factors that promote rapid progression of MM. To accomplish this, we performed a multi-center single-cell RNA sequencing (scRNA-seq) study on 102,207 cells from 48 CD138 - BM samples collected at the time of disease diagnosis from 18 patients with either rapid progressing (progression-free survival (PFS) < 18 months) or non-progressing (PFS > 4 years) disease. Comparative analysis of data from three centers demonstrated similar transcriptome profiles and cell type distributions, indicating subtle technical variation in scRNA-seq, opening avenues for an expanded multicenter trial. Rapid progressors depicted significantly higher enrichment of GZMK + and TIGIT + exhausted CD8 + T-cells ( P = 0.022) along with decreased expression of cytolytic markers ( PRF1, GZMB, GNLY ). We also observed a significantly higher enrichment of M2 tolerogenic macrophages in rapid progressors and activation of pro-proliferative signaling pathways, such as BAFF, CCL, and IL16. On the other hand, non-progressive patients depicted higher enrichment for immature B Cells (i.e., Pre/Pro B cells), with elevated expression for markers of B cell development ( IGLL1 , SOX4 , DNTT ). This multi-center study identifies the enrichment of various pro-tumorigenic cell populations and pathways in those with rapid progressing disease and further validates the robustness of scRNA-seq data generated at different study centers.

T lymphocytes migrate to barrier sites after exposure to pathogens, providing localized immunity and long-term protection. Here, we obtained blood and tissues from human organ donors to examine T cells across major barrier sites (skin, lung, jejunum), associated lymph nodes, lymphoid organs (spleen, bone marrow), and in circulation. By integrating single-cell protein and transcriptome profiling, we demonstrate that human barrier sites contain tissue-resident memory T (T-RM) cells that exhibit site-adapted profiles for residency, homing and function distinct from circulating memory T cells. Incorporating T cell receptor and transcriptome analysis, we show that circulating memory T cells are highly expanded, display extensive overlap between sites and exhibit effector and cytolytic functional profiles, while T-RM clones exhibit site-specific expansions and distinct functional capacities. Together, our findings indicate that circulating T cells are more disseminated and differentiated, while T-RM cells exhibit tissue-specific adaptation and clonal segregation, suggesting that strategies to promote barrier immunity require tissue targeting. Farber and colleagues examine the phenotypic, transcriptomic, clonal, and functional differences between tissue-resident T cells in various barrier tissue sites relative to T cells in lymphoid organs and circulation in humans.