Systematic immune cell dysregulation and molecular subtypes revealed by single-cell RNA-seq of subjects with type 1 diabetes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a prototypic endocrine autoimmune disease resulting from an immune-mediated destruction of pancreatic insulin-secreting ß  cells. A comprehensive immune cell phenotype evaluation in T1DM has not been performed thus far at the single-cell level. METHODS: In this cross-sectional analysis, we generated a single-cell transcriptomic dataset of peripheral blood mononuclear cells (PBMCs) from 46 manifest T1DM (stage 3) cases and 31 matched controls. RESULTS: We surprisingly detected profound alterations in circulatory immune cells (1784 dysregulated genes in 13 immune cell types), far exceeding the count in the comparator systemic autoimmune disease SLE. Genes upregulated in T1DM were involved in WNT signaling, interferon signaling and migration of T/NK cells, antigen presentation by B cells, and monocyte activation. A significant fraction of these differentially expressed genes were also altered in T1DM pancreatic islets. We used the single-cell data to construct a T1DM metagene z-score (TMZ score) that distinguished cases and controls and classified patients into molecular subtypes. This score correlated with known prognostic immune markers of T1DM, as well as with drug response in clinical trials. CONCLUSIONS: Our study reveals a surprisingly strong systemic dimension at the level of immune cell network in T1DM, defines disease-relevant molecular subtypes, and has the potential to guide non-invasive test development and patient stratification.

Towards a better preclinical cancer model - human immune aging in humanized mice.

BACKGROUND: Preclinical models are often used for cancer studies and evaluation of novel therapeutics. The relevance of these models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. Nonetheless, cancer is an age-related disease, and studies often overlook the effects of aging. Here we have established a humanized mouse model of human immune aging to investigate the role of this phenomenon on liver tumor dynamics. METHODS: Multiple organs and tissues (blood, thymus, lung, liver, spleen and bone marrow) were harvested from NOD-scid IL2rγ-/- (NIKO) mice reconstituted with human immune cells, over a period of 60 weeks post-birth, for immune profiling. Young and aging immune cells were compared for transcriptomic changes and functional differences. Effect of immune aging was investigated in a liver cancer humanized mouse model. RESULTS: Focusing on the T cell population, which is central to cancer immunosurveillance and immunotherapy, we showed that the proportion of naïve T cells declined while memory subsets and senescent-like cells increased with age. RNA-sequencing revealed that downregulated genes were related to immune responses and processes, and this was corroborated by reduced cytokine production in aging T cells. Finally, we showed faster liver tumor growth in aging than younger humanized mice, which could be attributed to specific pathways of aging T cell exhaustion. CONCLUSION: Our work improves on existing humanized (immune) mouse model and highlights the importance of considering immune aging in liver cancer modeling.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Imaging Memory T-Cells Stratifies Response to Adjuvant Metformin Combined with αPD-1 Therapy.

The low response rates associated with immune checkpoint inhibitor (ICI) use has led to a surge in research investigating adjuvant combination strategies in an attempt to enhance efficacy. Repurposing existing drugs as adjuvants accelerates the pace of cancer immune therapy research; however, many combinations exacerbate the immunogenic response elicited by ICIs and can lead to adverse immune-related events. Metformin, a widely used type 2 diabetes drug is an ideal candidate to repurpose as it has a good safety profile and studies suggest that metformin can modulate the tumour microenvironment, promoting a favourable environment for T cell activation but has no direct action on T cell activation on its own. In the current study we used PET imaging with [18F]AlF-NOTA-KCNA3P, a radiopharmaceutical specifically targeting KV1.3 the potassium channel over-expressed on active effector memory T-cells, to determine whether combining PD1 with metformin leads to an enhanced immunological memory response in a preclinical colorectal cancer model. Flow cytometry was used to assess which immune cell populations infiltrate the tumours in response to the treatment combination. Imaging with [18F]AlF-NOTA-KCNA3P demonstrated that adjuvant metformin significantly improved anti-PD1 efficacy and led to a robust anti-tumour immunological memory response in a syngeneic colon cancer model through changes in tumour infiltrating effector memory T-cells.

Imaging Effector Memory T-Cells Predicts Response to PD1-Chemotherapy Combinations in Colon Cancer.

Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI-chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [18F]AlF-NOTA-KCNA3P, a peptide radiopharmaceutical that targets the Kv1.3 potassium channel overexpressed on T-effector memory (TEM) cells as a PET imaging biomarker for lasting immunological memory response. The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.

Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response.

Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of [18F]AlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with [18F]AlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8+ effector memory T cells.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model.

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

Granzyme B PET Imaging of Combined Chemotherapy and Immune Checkpoint Inhibitor Therapy in Colon Cancer.

PURPOSE: Chemotherapeutic adjuvants, such as oxaliplatin (OXA) and 5-fluorouracil (5-FU), that enhance the immune system, are being assessed as strategies to improve durable response rates when used in combination with immune checkpoint inhibitor (ICI) monotherapy in cancer patients. In this study, we explored granzyme B (GZB), released by tumor-associated immune cells, as a PET imaging-based stratification marker for successful combination therapy using a fluorine-18 (18F)-labelled GZB peptide ([18F]AlF-mNOTA-GZP). METHODS: Using the immunocompetent CT26 syngeneic mouse model of colon cancer, we assessed the potential for [18F]AlF-mNOTA-GZP to stratify OXA/5-FU and ICI combination therapy response via GZB PET. In vivo tumor uptake of [18F]AlF-mNOTA-GZP in different treatment arms was quantified by PET, and linked to differences in tumor-associated immune cell populations defined by using multicolour flow cytometry. RESULTS: [18F]AlF-mNOTA-GZP tumor uptake was able to clearly differentiate treatment responders from non-responders when stratified based on changes in tumor volume. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumor-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells and GZB+ NK+ cells. CONCLUSIONS: [18F]AlF-mNOTA-GZP tumor uptake, driven by changes in immune cell populations expressing GZB, is able to stratify tumor response to chemotherapeutics combined with ICIs. Our results show that, while the immunomodulatory mode of action of the chemotherapies may be different, the ultimate mechanism of tumor lysis through release of Granzyme B is an accurate biomarker for treatment response.

Granzyme B PET Imaging Stratifies Immune Checkpoint Inhibitor Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a notoriously difficult cancer to treat. The recent development of immune checkpoint inhibitors has revolutionised HCC therapy; however, successful response is only observed in a small percentage of patients. Biomarkers typically used to predict treatment response in other tumour types are ineffective in HCC, which arises in an immune-suppressive environment. However, imaging markers that measure changes in tumour infiltrating immune cells may supply information that can be used to determine which patients are responding to therapy posttreatment. We have evaluated [18F]AlF-mNOTA-GZP, a radiolabeled peptide targeting granzyme B, to stratify response to ICIs in a HEPA 1-tumours, a syngeneic model of HCC. Posttherapy, in vivo tumour retention of [18F]AlF-mNOTA-GZP was correlated to changes in tumour volume and tumour-infiltrating immune cells. [18F]AlF-mNOTA-GZP successfully stratified response to immune checkpoint inhibition in the syngeneic HEPA 1-6 model. FACS indicated significant changes in the immune environment including a decrease in immune suppressive CD4+ T regulatory cells and increases in tumour-associated GZB+ NK+ cells, which correlated well with tumour radiopharmaceutical uptake. While the immune response to ICI therapies differs in HCC compared to many other cancers, [18F]AlF-mNOTA-GZP retention is able to stratify response to ICI therapy associated with tumour infiltrating GZB+ NK+ cells in this complex tumour microenvironment.

The untwining of immunosenescence and aging.

From a holistic point of view, aging results from the cumulative erosion of the various systems. Among these, the immune system is interconnected to the rest as immune cells are present in all organs and recirculate through bloodstream. Immunosenescence is the term used to define the remodelling of immune changes during aging. Because immune cells-and particularly lymphocytes-can further differentiate after their maturation in response to pathogen recognition, it is therefore unclear when senescence is induced in these cells. Additionally, it is also unclear which signals triggers senescence in immune cells (i) aging per se, (ii) specific response to pathogens, (iii) underlying conditions, or (iv) inflammaging. In this review, we will cover the current knowledge and concepts linked to immunosenescence and we focus this review on lymphocytes and T cells, which represent the typical model for replicative senescence. With the evidence presented, we propose to disentangle the senescence of immune cells from chronological aging.

Plasmacytoid dendritic cells develop from Ly6D+ lymphoid progenitors distinct from the myeloid lineage.

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.

RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types.

The molecular characterization of immune subsets is important for designing effective strategies to understand and treat diseases. We characterized 29 immune cell types within the peripheral blood mononuclear cell (PBMC) fraction of healthy donors using RNA-seq (RNA sequencing) and flow cytometry. Our dataset was used, first, to identify sets of genes that are specific, are co-expressed, and have housekeeping roles across the 29 cell types. Then, we examined differences in mRNA heterogeneity and mRNA abundance revealing cell type specificity. Last, we performed absolute deconvolution on a suitable set of immune cell types using transcriptomics signatures normalized by mRNA abundance. Absolute deconvolution is ready to use for PBMC transcriptomic data using our Shiny app (https://github.com/giannimonaco/ABIS). We benchmarked different deconvolution and normalization methods and validated the resources in independent cohorts. Our work has research, clinical, and diagnostic value by making it possible to effectively associate observations in bulk transcriptomics data to specific immune subsets.

Group 2 innate lymphoid cells license dendritic cells to potentiate memory TH2 cell responses.

Rapid activation of memory CD4(+) T helper 2 (TH2) cells during allergic inflammation requires their recruitment into the affected tissue. Here we demonstrate that group 2 innate lymphoid (ILC2) cells have a crucial role in memory TH2 cell responses, with targeted depletion of ILC2 cells profoundly impairing TH2 cell localization to the lungs and skin of sensitized mice after allergen re-challenge. ILC2-derived interleukin 13 (IL-13) is critical for eliciting production of the TH2 cell-attracting chemokine CCL17 by IRF4(+)CD11b(+)CD103(-) dendritic cells (DCs). Consequently, the sentinel function of DCs is contingent on ILC2 cells for the generation of an efficient memory TH2 cell response. These results elucidate a key innate mechanism in the regulation of the immune memory response to allergens.

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.

Innate lymphoid cells in immunity and disease.

The family of innate lymphoid cells (ILCs) comprises of natural killer (NK) cells, Rorγt-dependent ILCs (lymphoid tissue inducer (LTi) cells, ILC22, and ILC17), and type 2 ILCs. Apart from a common requirement for inhibitor of DNA binding 2 (Id2) expression and common γ-chain (γc) signaling, the differentiation of ILC populations is regulated by distinct transcription factors. ILCs play fundamental roles in processes such as cytotoxicity, lymphoid organogenesis, intestinal homeostasis, immunity against infections, and wound healing. However, the dysregulation of ILCs has been implicated in autoimmune and inflammatory diseases. Here, we will review the recent advances in ILC development and their roles in immunity and disease, with a primary focus on type 2 ILCs.

Transcription factor RORα is critical for nuocyte development.

Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are distinct, previously unknown members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signaling for development in vitro. Pro-T cell progenitors at double-negative stage 1 (DN1) and DN2 maintained nuocyte potential in vitro, although the thymus was not essential for nuocyte development. Notably, the transcription factor RORα was critical for the development of nuocytes and their role in the expulsion of parasitic worms.