Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity.

Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.

An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion.

Regulatory T (Treg) cells accumulate into tumors, hindering the success of cancer immunotherapy. Yet, therapeutic targeting of Treg cells shows limited efficacy or leads to autoimmunity. The molecular mechanisms that guide Treg cell stability in tumors remain elusive. In the present study, we identify a cell-intrinsic role of the alarmin interleukin (IL)-33 in the functional stability of Treg cells. Specifically, IL-33-deficient Treg cells demonstrated attenuated suppressive properties in vivo and facilitated tumor regression in a suppression of tumorigenicity 2 receptor (ST2) (IL-33 receptor)-independent fashion. On activation, Il33-/- Treg cells exhibited epigenetic re-programming with increased chromatin accessibility of the Ifng locus, leading to elevated interferon (IFN)-γ production in a nuclear factor (NF)-κB-T-bet-dependent manner. IFN-γ was essential for Treg cell defective function because its ablation restored Il33-/- Treg cell-suppressive properties. Importantly, genetic ablation of Il33 potentiated the therapeutic effect of immunotherapy. Our findings reveal a new and therapeutically important intrinsic role of IL-33 in Treg cell stability in cancer.

The role of neutrophils in trained immunity.

The principle of trained immunity represents innate immune memory due to sustained, mainly epigenetic, changes triggered by endogenous or exogenous stimuli in bone marrow (BM) progenitors (central trained immunity) and their innate immune cell progeny, thereby triggering elevated responsiveness against secondary stimuli. BM progenitors can respond to microbial and sterile signals, thereby possibly acquiring trained immunity-mediated long-lasting alterations that may shape the fate and function of their progeny, for example, neutrophils. Neutrophils, the most abundant innate immune cell population, are produced in the BM from committed progenitor cells in a process designated granulopoiesis. Neutrophils are the first responders against infectious or inflammatory challenges and have versatile functions in immunity. Together with other innate immune cells, neutrophils are effectors of peripheral trained immunity. However, given the short lifetime of neutrophils, their ability to acquire immunological memory may lie in the central training of their BM progenitors resulting in generation of reprogrammed, that is, "trained", neutrophils. Although trained immunity may have beneficial effects in infection or cancer, it may also mediate detrimental outcomes in chronic inflammation. Here, we review the emerging research area of trained immunity with a particular emphasis on the role of neutrophils and granulopoiesis.

Cross-species transcriptome analysis for early detection and specific therapeutic targeting of human lupus nephritis.

OBJECTIVES: Patients with lupus nephritis (LN) are in urgent need for early diagnosis and therapeutic interventions targeting aberrant molecular pathways enriched in affected kidneys. METHODS: We used mRNA-sequencing in effector (spleen) and target (kidneys, brain) tissues from lupus and control mice at sequential time points, and in the blood from 367 individuals (261 systemic lupus erythematosus (SLE) patients and 106 healthy individuals). Comparative cross-tissue and cross-species analyses were performed. The human dataset was split into training and validation sets and machine learning was applied to build LN predictive models. RESULTS: In murine SLE, we defined a kidney-specific molecular signature, as well as a molecular signature that underlies transition from preclinical to overt disease and encompasses pathways linked to metabolism, innate immune system and neutrophil degranulation. The murine kidney transcriptome partially mirrors the blood transcriptome of patients with LN with 11 key transcription factors regulating the cross-species active LN molecular signature. Integrated protein-to-protein interaction and drug prediction analyses identified the kinases TRRAP, AKT2, CDK16 and SCYL1 as putative targets of these factors and capable of reversing the LN signature. Using murine kidney-specific genes as disease predictors and machine-learning training of the human RNA-sequencing dataset, we developed and validated a peripheral blood-based algorithm that discriminates LN patients from normal individuals (based on 18 genes) and non-LN SLE patients (based on 20 genes) with excellent sensitivity and specificity (area under the curve range from 0.80 to 0.99). CONCLUSIONS: Machine-learning analysis of a large whole blood RNA-sequencing dataset of SLE patients using human orthologs of mouse kidney-specific genes can be used for early, non-invasive diagnosis and therapeutic targeting of LN. The kidney-specific gene predictors may facilitate prevention and early intervention trials.

De novo-induced self-antigen-specific Foxp3+ regulatory T cells impair the accumulation of inflammatory dendritic cells in draining lymph nodes.

Foxp3(+) regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3(+) Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3(+) Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3(+) Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo-isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3(+) Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3(+) Treg cellular target in unmanipulated mice.

DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.

We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

A novel pathogenic peptide of thyroglobulin (2208-2227) induces autoreactive T-cell and B-cell responses in both high and low responder mouse strains.

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208-2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A(b)), or susceptibility (A(s), E(k)) to EAT. We therefore immunized LR BALB/c (H-2(d)) and C57BL/6 (H-2(b)) strains, as well as HR CBA/J (H-2(k)) and SJL/J (H-2(s)) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1-3), as well as in SJL/J mice (two of six mice, infiltration index 1-2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.

Potent induction of trained immunity by Saccharomyces cerevisiae ß-glucans.

Candida albicans cell wall component ß-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual ß-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this ß-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the ß-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on ß-glucan structures that induce an effective trained immunity response.

Immunological synapse formation between T regulatory cells and cancer-associated fibroblasts promotes tumour development.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA+ CAFs can form immunological synapses with Foxp3+ regulatory T cells (Tregs) in tumours. Notably, α-SMA+ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA+ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA+ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA+ CAFs and Tregs in an autophagy-dependent manner.

Fine epitope mapping within the pathogenic thyroglobulin peptide 2340-2359: minimal epitopes retaining antigenicity across various MHC haplotypes are not necessarily immunogenic.

We have previously reported that the 20-mer peptide p2340 (amino acids 2340-2359), of human thyroglobulin (Tg) has the unique feature that it causes experimental autoimmune thyroiditis (EAT) in mouse strains bearing high-responder (HR) or low-responder (LR) MHC haplotypes in Tg-induced EAT. In this study, we have employed fine epitope mapping to examine whether this property of p2340 is the result of recognition of distinct or shared minimal T-cell epitopes in the context of HR or LR MHC class II molecules. Use of overlapping peptides showed that a core minimal 9-mer epitope (LTWVQTHIR, amino acids 2344-2352) was recognized by p2340-primed T cells from both HR (H2(k,s) ) and LR (H2(b,d) ) strains, whereas a second 9-mer epitope (HIRGFGGDP, amino acids 2350-2358) was antigenic only in H2(s) hosts. Truncation analysis of LTWVQTHIR and HIRGFGGDP peptides delineated them as the minimal epitopes recognized by p2340-primed T cells from the above strains. Subcutaneous challenge of all mouse strains with the 9-mer core peptide LTWVQTHIR in adjuvant elicited specific lymph node cell proliferative responses and mild EAT only in HR hosts, highlighting this sequence as a minimal pathogenic Tg peptide in EAT. The 9-mer peptide HIRGFGGDP was not found to be immunogenic in H2(s) hosts. These data demonstrate that minimal T-cell epitopes, defined as autoantigenic in hosts of various MHC haplotypes, are not intrinsically immunogenic. Activation of naive autoreactive T cells may require contributions from flanking residues within longer peptide sequences encompassing these epitopes.

Ablation of NLRP3 inflammasome rewires MDSC function and promotes tumor regression.

Myeloid-derived suppressor cells (MDSCs) are myeloid precursors that exert potent immunosuppressive properties in cancer. Despite the extensive knowledge on mechanisms implicated in mobilization, recruitment, and function of MDSCs, their therapeutic targeting remains an unmet need in cancer immunotherapy, suggesting that unappreciated mechanisms of MDSC-mediated suppression exist. Herein, we demonstrate an important role of NLRP3 inflammasome in the functional properties of MDSCs in tumor-bearing hosts. Specifically, Nlrp3-deficient mice exhibited reduced tumor growth compared to wild-type animals and induction of robust anti-tumor immunity, accompanied by re-wiring of the MDSC compartment. Interestingly, both monocytic (M-MDSCs) and granulocytic (G-MDSCs) subsets from Nlrp3-/- mice displayed impaired suppressive activity and demonstrated significant transcriptomic alterations supporting the loss-of-function and associated with metabolic re-programming. Finally, therapeutic targeting of NLRP3 inhibited tumor development and re-programmed the MDSC compartment. These findings propose that targeting NLRP3 in MDSCs could overcome tumor-induced tolerance and may provide new checkpoints of cancer immunotherapy.

Regulatory T Cells in Autoimmunity and Cancer: A Duplicitous Lifestyle.

Regulatory T (Treg) cells, possess a strategic role in the maintenance of immune homeostasis, and their function has been closely linked to development of diverse pathologies including autoimmunity and cancer. Comprehensive studies in various disease contexts revealed an increased plasticity as a characteristic of Treg cells. Although Treg cell plasticity comes in various flavors, the major categories enclose the loss of Foxp3 expression, which is the master regulator of Treg cell lineage, giving rise to "ex-Treg" cells and the "fragile" Treg cells in which FOXP3 expression is retained but accompanied by the engagement of an inflammatory program and attenuation of the suppressive activity. Treg cell plasticity possess a tremendous therapeutic potential either by inducing Treg cell de-stabilization to promote anti-tumor immunity, or re-enforcing Treg cell stability to attenuate chronic inflammation. Herein, we review the literature on the Treg cell plasticity with lessons learned in autoimmunity and cancer and discuss challenges and open questions with potential therapeutic implications.

Measuring Suppressive Activity and Autophagy in Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are potent suppressor cells that accumulate in tumor microenvironment and inhibit anti-tumor responses. Assessment of cell-autonomous MDSC responses allows the precise characterization of MDSCs in various disease settings and elucidates the underlying mechanisms of MDSC-mediated immune suppression. Here we describe a protocol for the isolation of tumor infiltrating or splenic MDSC, as well as their subpopulations, from melanoma-inoculated mice using Fluorescent Activated Cell Sorting (FACS). We further provide protocols for investigation of the autophagy pathway and ex vivo assessment of MDSC suppressive function using lymph node responder cells. These assays allow a comprehensive characterization of MDSC in murine experimental models.

Regulatory T-cell Transcriptomic Reprogramming Characterizes Adverse Events by Checkpoint Inhibitors in Solid Tumors.

Immune checkpoint inhibitors (ICI), which target immune regulatory pathways to unleash antitumor responses, have revolutionized cancer immunotherapy. Despite the remarkable success of ICI immunotherapy, a significant proportion of patients whose tumors respond to these treatments develop immune-related adverse events (irAE) resembling autoimmune diseases. Although the clinical spectrum of irAEs is well characterized, their successful management remains empiric. This is in part because the pathogenic mechanisms involved in the breakdown of peripheral tolerance and induction of irAEs remain elusive. Herein, we focused on regulatory T cells (Treg) in individuals with irAEs because these cells are vital for maintenance of peripheral tolerance, appear expanded in the peripheral blood of individuals with cancer, and abundantly express checkpoint molecules, hence representing direct targets of ICI immunotherapy. Our data demonstrate an intense transcriptomic reprogramming of CD4+CD25+CD127- Tregs in the blood of individuals with advanced metastatic melanoma who develop irAEs following ICI immunotherapy, with a characteristic inflammatory, apoptotic, and metabolic signature. This inflammatory signature was shared by Tregs from individuals with different types of cancer developing irAEs and individuals with autoimmune diseases. Our findings suggest that inflammatory Treg reprogramming is a feature of immunotherapy-induced irAEs, and this may facilitate translational approaches aiming to induce robust antitumor immunity without disturbing peripheral tolerance.

Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.

Intratumoral accumulation of podoplanin-expressing lymph node stromal cells promote tumor growth through elimination of CD4+ tumor-infiltrating lymphocytes.

The beneficial effects of checkpoint blockade in tumor immunotherapy are limited to patients with increased tumor-infiltrating lymphocytes (TILs). Delineation of the regulatory networks that orchestrate the presence of TILs holds great promise for the design of effective immunotherapies. Podoplanin/gp38 (PDPN)-expressing lymph node stromal cells (LNSCs) are present in tumor stroma; however, their effect in the regulation of TILs remains elusive. Herein we demonstrate that intratumor injection of ex-vivo-isolated PDPN+ LNSCs into melanoma-bearing mice induces elimination of TILs and promotes tumor growth. In support, PDPN+ LNSCs exert their function through direct inhibition of CD4+ T cell proliferation in a cell-to-cell contact independent fashion. Mechanistically, we demonstrate that PDPN+ LNSCs mediate T cell growth arrest and induction of apoptosis to activated CD69+CD4+ T cells. Importantly, LTbR-Ig-mediated blockade of PDPN+ LNSCs expansion and function significantly attenuates melanoma tumor growth and enhances the infiltration and proliferation of CD4+ TILs. Overall, our findings decipher a novel role of PDPN-expressing LNSCs in the elimination of CD4+ TILs and propose a new target for tumor immunotherapy.

Pathogenicity of a human thyroglobulin peptide (2340-2359) in mice with high or low genetic susceptibility to thyroiditis.

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.