Abstract Introduction: Immune checkpoint inhibitor (ICI)-related myocarditis (irMyocarditis) is a potentially lethal complication of ICI use that is characterized by the presence of clonally expanded T cells in the heart. The antigens recognized by these expanded intracardiac T-cell clones and their relationships to T-cell clones in the blood and tumors of irMyocarditis patients are poorly understood. Methods: Paired single-cell RNA sequencing (scRNA-seq) and T-cell receptor (TCR) sequencing was performed on heart tissue (n = 13) and peripheral blood from patients with irMyocarditis (n = 25) and ICI-treated controls (n = 28) using the 10X Chromium (10X Genomics) system. TCR-β chain sequencing was performed (Adaptive Biotechnologies) on four autopsy cases of patients with available scRNA-seq data and irMyocarditis heart, tumor, and histologically normal tissue. An established high-throughput protocol was used to screen expanded intracardiac TCRs against candidate antigens (Oliveira G, et al. Nature 2021). Briefly, full-length TCRs were cloned, transduced into donor T cells, and screened against autologous or major histocompatibility complex (MHC) Class I-matched antigen presenting cell lines pulsed with peptide pools that covered the full length of the ⍺-myosin, troponin-I, and troponin-T proteins alongside pools of common viral antigens and appropriate controls. Results: scRNA-seq data showed that TCRs shared between heart and blood are predominantly found in circulating CD8 T cell subsets as compared to circulating CD4 T cell subsets. The gene expression patterns of these shared T-cell clones in circulation appear distinct in fatal and non-fatal irMyocarditis patients, with shared T-cell clones in fatal cases expressing cycling markers (MKI67, STMN1) and the chemokine receptor CXCR3. TCR-β chain sequences most enriched in irMyocarditis tissue relative to control tissues were distinct from those enriched in tumor tissues, and their full-length TCR sequences could be recovered from scRNA-seq data. In total, 52 cardiac-expanded TCRs across eight donors were screened against candidate antigens. None of the screened TCRs recognized the putative cardiac autoantigens. Conclusions: T-cell clones expanded in irMyocarditis are shared in circulation, where the gene expression of these clones may help to distinguish fatal from non-fatal irMyocarditis. TCRs enriched in irMyocarditis appear to be largely distinct from those enriched in tumor and likely recognize currently unknown cardiac autoantigens. Citation Format: Steven Blum, Daniel A. Zlotoff, Neal P. Smith, Isabela J. Kernin, Swetha Ramesh, Giacomo Oliveira, Leyre Zubiri, Joshua Caplin, Nandini Samanta, Sidney Martin, Mike Wang, Alice Tirard, Pritha Sen, Yuhui Song, Katherine Xu, Jaimie L. Barth, Kamil Slowikowski, Mazen Nasrallah, Jessica Tantivit, Kasidet Manakongtreecheep, Benjamin Y. Arnold, John McGuire, Alexander B. Afeyan, Christopher J. Pinto, Daniel McLoughlin, Monica Jackson, PuiYee Chan, Aleigha Lawless, William A. Michaud, Tatyana Sharova, Linda T. Nieman, Justin F. Gainor, Dejan Juric, Mari Mino-Kenudsen, Ryan J. Sullivn, Genevieve M. Boland, James R. Stone, Catherine J. Wu, Molly F. Thomas, Tomas G. Neilan, Kerry L. Reynolds, Alexandra-Chloé Villani. T-cell responses across heart, blood, and tumor in patients with immune checkpoint inhibitor-related myocarditis (irMyocarditis) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7474.

Immune checkpoint inhibitors (ICIs) are widely used anti-cancer therapies that can cause morbid and potentially fatal immune-related adverse events (irAEs). ICI-related myocarditis (irMyocarditis) is uncommon but has the highest mortality of any irAE. The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood during irMyocarditis and identify biomarkers of clinical severity by leveraging single-cell (sc)RNA-seq coupled with T cell receptor (TCR) sequencing, microscopy, and proteomics analysis of 28 irMyocarditis patients and 23 controls. Our analysis of 284,360 cells from heart and blood specimens identified cytotoxic T cells, inflammatory macrophages, conventional dendritic cells (cDCs), and fibroblasts enriched in irMyocarditis heart tissue. Additionally, potentially targetable, pro-inflammatory transcriptional programs were upregulated across multiple cell types. TCR clones enriched in heart and paired tumor tissue were largely non-overlapping, suggesting distinct T cell responses within these tissues. We also identify the presence of cardiac-expanded TCRs in a circulating, cycling CD8 T cell population as a novel peripheral biomarker of fatality. Collectively, these findings highlight critical biology driving irMyocarditis and putative biomarkers for therapeutic intervention.

Pregnancy is a risk factor for increased severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory infections, but the mechanisms underlying this risk are poorly understood. To gain insight into the role of pregnancy in modulating immune responses at baseline and upon SARS-CoV-2 infection, we collected peripheral blood mononuclear cells and plasma from 226 women, including 152 pregnant individuals and 74 non-pregnant women. We find that SARS-CoV-2 infection is associated with altered T cell responses in pregnant women, including a clonal expansion of CD4-expressing CD8+ T cells, diminished interferon responses, and profound suppression of monocyte function. We also identify shifts in cytokine and chemokine levels in the sera of pregnant individuals, including a robust increase of interleukin-27, known to drive T cell exhaustion. Our findings reveal nuanced pregnancy-associated immune responses, which may contribute to the increased susceptibility of pregnant individuals to viral respiratory infection.

Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAE(Hi) CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2(Hi) CD8 T cells. Cytotoxic GNLY(Hi) CD4 T cells, recirculating ITGB2(Hi) CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.

Background Immune checkpoint inhibitor-induced diabetes mellitus (ICI-DM) is a rare adverse effect of ICI therapy, presumably caused by immune-mediated destruction of insulin-producing pancreatic β-cells. ICI-DM carries significant mortality risk and markedly disrupts patients' quality of life. Importantly, ICI-DM is a valuable model to examine how ICI toxicities relate to spontaneous autoimmunity at the clinical, genetic, and immunological level, and to dissect how distinct clinical endotypes of an ICI-toxicity may differ mechanistically. Methods We combined: (1)retrospective analysis of data from 14,440 ICI-treated patients, (2)genetic analysis from >1000 of these ICI-treated patients, and (3)high-definition multimodal analysis of circulating immune cells at ICI-DM diagnosis from 11 patients, to comprehensively characterize ICI-DM. In (1), we identified 65 cases of ICI-DM among 14,440 patients treated with ICI in 2010–2022 within our multi-centered academic hospital system, which we further stratified by β-cell function (i.e., insulin production) and by the presence of type 1 diabetes (T1D)-specific autoantibodies for further clinical phenotyping. In (2), we imputed germline genotype using the OncoPanel data from patient tumors and calculated a composite T1D polygenic risk score for patients with and without ICI-DM. Finally in (3), we performed single-cell multi-omics analyses on >150,000 circulating immune cells isolated from these ICI-DM patients and compared these results with patients with newly diagnosed T1D. Results Risk factors: The incidence of ICI-DM in our cohort was 0.45%, significantly increased in patients with pre-existing T2D (OR 5.8) or treated with combination ICI (OR 2.5). Patients with ICI-DM had a significantly higher T1D composite genetic risk compared to ICI-treated patients without ICI-DM (OR 6.4). Clinical endotypes: We identified three distinct ICI-DM groups, including patients with (1)preserved insulin production (14% of ICI-DM, referred to as β+), (2, 3)loss of insulin production (β-) with elevated (40%, Ab+β-) or absent (45%, Ab-β-) islet-specific autoantibodies (40%, Ab+β-) islet-specific autoantibodies. Among these groups, Ab+β- presented with fulminant ICI-DM, with high rates of life-threatening complications (63%), whereas presentation of both β+ and Ab-β- were more protracted. Immunological phenotyping: We will present our paired single-cell RNA sequencing and T-cell receptor analysis examining distinct circulating T cell subsets associated with ICI-DM pathogenesis and heterogeneity. Conclusions Our analysis identified several risk factors for ICI-DM that may identify patients at higher risk for ICI-DM. The results from our multi-modal strategy – combining clinical, genetic and translational approaches – to dissecting ICI-DM pathogenesis provides a roadmap on how to dissect the mechanistic determinants of ICI toxicities more broadly. Ethics Approval This study was approved through the institutional review board of Mass General Brigham, protocol numbers: 2017P000501, 13–416 and 11–181. Participants who provided blood or serum gave their informed consent before taking part; for patients included exclusively in retrospective chart review, the IRB determined that individual informed consent was not needed.

Lyme disease is caused by the bacterial pathogen Borrelia burgdorferi, which can be readily modeled in laboratory mice. In order to understand the cellular and transcriptional changes that occur during B. burgdorferi infection, we conducted single-cell RNA sequencing (scRNA-seq) of ankle joints of infected C57BL/6 mice over time. We found that macrophages/monocytes, T cells, synoviocytes and fibroblasts all showed significant differences in gene expression of both inflammatory and non-inflammatory genes that peaked early and returned to baseline before the typical resolution of arthritis. Predictions of cellular interactions showed that macrophages appear to communicate extensively between different clusters of macrophages as well as with fibroblasts and synoviocytes. Our data give unique insights into the interactions between B. burgdorferi and the murine immune system over time and allow for a better understanding of mechanisms by which the dysregulation of the immune response may lead to prolonged symptoms in some patients.

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR<0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR <0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR <0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR <0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Background Antibodies targeting immune checkpoint inhibitors (ICIs) CTLA-4 and PD-1/PD-L1 have revolutionized the treatment of metastatic solid tumors. However, their use is limited by a high incidence of immune-related adverse events. The colon is a frequent target of this immune attack seen in up to 45% of patients on dual PD-1 and CTLA-4 blockade. We leveraged a multi-omics strategy to further our understanding of the cellular and molecular drivers giving rise to ICI-associated colitis and nominate treatment solutions that spare anti-tumor immune response. Methods We collected paired endoscopic colon mucosal biopsies and blood specimens from 13 irColitis patients, 8 healthy individuals, and 8 controls on ICIs, and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis. Results Analyses of over 300,000 single epithelial, mesenchymal, and immune single cells revealed that patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs. We also identified two circulating ITGB2+ CD8 T cell populations associated with irColitis – a CX3CR1Hi population predicted to be intravascular and an EOMESHi KLRG1Hi population. Comparison of dual anti-PD-1/CTLA-4 versus anti-PD1 monotherapy revealed expansion of those two circulating ITGB2Hi CD8 T cell populations, as well as a cytotoxic GNLYHi CD4 T cell subset, and endothelial cells associated with hypoxia gene programs. Cell-cell communication analysis predicted crucial roles for ICAM and CXCR3 ligand-mediated recruitment and retention of these two circulating T cell populations by epithelial, endothelial and myeloid cells during active colitis. In irColitis, we also observed significant epithelial turnover marked by fewer LGR5+ stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Transcriptional programs associated with irColitis were distinct from those in the tumor microenvironment, which may have important therapeutic implications. Finally, by examining many drugs in clinical trials for inflammatory bowel disease, we expand the putative therapeutic options for treating irColitis reported to-date. Conclusions This multi-omics approach nominates novel irColitis therapeutic targets and redefines irColitis as a disease not simply marked by the aberrant expansion of CD8 T cells but rather altered global interactions between immune cells and the colon mucosal epithelial or mesenchymal cells. Ethics Approval Informed consent was obtained from all patients in accordance with protocols obtained from the Mass General Brigham and/or DANA- Farber/Harvard Cancer Center Institutional Review Boards (DFCI/HCC 11-181 and 13-416, Mass General Brigham 2015P001333).

Introduction: Myocarditis due to immune checkpoint inhibitors (ICIs), a type of cancer immunotherapy, is associated with high morbidity and mortality. The cellular and molecular pathogenesis of ICI myocarditis remains largely unknown. Identification of circulating factors associated with intracardiac pathology may aid new clinical approaches. Hypothesis: We hypothesized that ICI myocarditis is associated with increased abundance of intracardiac immune cells and upregulated inflammatory genes in the heart and serum. Methods: Heart tissue from 13 patients with ICI myocarditis was acquired by endomyocardial biopsy or autopsy; peripheral blood mononuclear cells (PBMCs) were also collected from ten of these patients and eight additional ICI myocarditis patients. Control heart tissue was derived from six hearts declined for transplantation (non-ICI-exposed) and from the biopsy and autopsy of one ICI-treated patient without myocarditis. The 10x Genomics Chromium system was used to generate single-cell RNA sequencing (scRNAseq) data from heart and PBMC specimens. Serum proteins were measured by core lab assay (for troponin T) or by multiplexed Luminex immunoassay. Results: Unbiased clustering of scRNAseq data from 77,071 cells recovered from heart samples revealed 37 cell subsets across 10 cell lineages. Myocarditis heart tissue demonstrated enrichment of T/NK cells (odds ratio 8.3, p=0.0006), B/plasma cells (OR 5.1, p=0.01), and dendritic cells (DCs) (OR 9.2, p=0.006) relative to controls. Circulating DC abundance was decreased in fatal (n=3) versus nonfatal (n=15) myocarditis cases (p=0.008), while intracardiac DC abundance was directly associated with serum troponin T values (p=0.02). The levels of five immunomodulatory factors - IL-15, CXCL9, CCL3, TNFα, and CCL21 - were found to be significantly upregulated transcriptionally in at least one intracardiac cell subset and at the protein level in the serum of myocarditis cases. Conclusions: Fatality status and troponin level associated with intracardiac DC abundance and multiple immunomodulatory genes were upregulated in both the heart and serum in ICI myocarditis. These observations may guide novel diagnostic and therapeutic strategies.

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (T RM ) ITGAE -expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 T RM and effector CD4 T cells upregulated type 17 immune programs ( IL17A, IL26 ) and Tfh-like programs ( CXCL13, PDCD1 ). We also identified for the first time an increased abundance of two KLRG1 and ITGB2 -expressing CD8 T cell populations with circulatory cell markers, including a GZMK T RM -like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5 -expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 T RM homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.